Harpers

a LANGE medical bookHarper’sIllustratedBiochemistrytwenty-sixth editionRobert K. Murray, MD, PhDProfessor (Emeritus) of BiochemistryUniversity of TorontoToronto, OntarioDaryl K. Granner, MDJoe C. Davis Professor of Biomedical ScienceDirector, Vanderbilt Diabetes CenterProfessor of Molecular Physiology and Biophysicsand of MedicineVanderbilt UniversityNashville, TennesseePeter A. Mayes, PhD, DScEmeritus Professor of Veterinary BiochemistryRoyal Veterinary CollegeUniversity of LondonLondonVictor W. Rodwell, PhDProfessor of BiochemistryPurdue UniversityWest Lafayette, IndianaLange Medical Books/McGraw-HillMedical Publishing DivisionNew York Chicago San Francisco Lisbon London Madrid Mexico CityMilan New Delhi San Juan Seoul Singapore Sydney Toronto

Harper’s Illustrated Biochemistry, Twenty-Sixth EditionCopyright © 2003 by The McGraw-Hill Companies, Inc. All rights reserved. Printed in the United States of America. Except aspermitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in anyform or by any means, or stored in a data base or retrieval system, without the prior written permission of the publisher.Previous editions copyright © 2000, 1996, 1993, 1990 by Appleton & Lange; copyright © 1988 by Lange Medical Publications.234567890DOC/DOC 09876543ISBN 0-07-138901-6ISSN 1043-9811NoticeMedicine is an ever-changing science. As new research and clinical experience broaden our knowledge, changes in treatmentand drug therapy are required. The authors and the publisher of this work have checked with sources believed to bereliable in their efforts to provide information that is complete and generally in accord with the standards accepted at thetime of publication. However, in view of the possibility of human error or changes in medical sciences, neither the authorsnor the publisher nor any other party who has been involved in the preparation or publication of this work warrants thatthe information contained herein is in every respect accurate or complete, and they disclaim all responsibility for any errorsor omissions or for the results obtained from use of the information contained in this work. Readers are encouragedto confirm the information contained herein with other sources. For example and in particular, readers are advised tocheck the product information sheet included in the package of each drug they plan to administer to be certain that theinformation contained in this work is accurate and that changes have not been made in the recommended dose or in thecontraindications for administration. This recommendation is of particular importance in connection with new or infrequentlyused drugs.This book was set in Garamond by Pine Tree CompositionThe editors were Janet Foltin, Jim Ransom, and Janene Matragrano Oransky.The production supervisor was Phil Galea.The illustration manager was Charissa Baker.The text designer was Eve Siegel.The cover designer was Mary McKeon.The index was prepared by Kathy Pitcoff.RR Donnelley was printer and binder.This book is printed on acid-free paper.ISBN-0-07-121766-5 (International Edition)Copyright © 2003. Exclusive rights by the McGraw-Hill Companies, Inc., formanufacture and export. This book cannot be re-exported from the country to which itis consigned by McGraw-Hill. The International Edition is not available in North America.

AuthorsDavid A. Bender, PhDSub-Dean Royal Free and University College MedicalSchool, Assistant Faculty Tutor and Tutor to MedicalStudents, Senior Lecturer in Biochemistry, Departmentof Biochemistry and Molecular Biology,University College LondonKathleen M. Botham, PhD, DScReader in Biochemistry, Royal Veterinary College,University of LondonDaryl K. Granner, MDJoe C. Davis Professor of Biomedical Science, Director,Vanderbilt Diabetes Center, Professor of MolecularPhysiology and Biophysics and of Medicine, VanderbiltUniversity, Nashville, TennesseeFrederick W. Keeley, PhDAssociate Director and Senior Scientist, Research Institute,Hospital for Sick Children, Toronto, and Professor,Department of Biochemistry, University ofTorontoPeter J. Kennelly, PhDProfessor of Biochemistry, Virginia Polytechnic Instituteand State University, Blacksburg, VirginiaPeter A. Mayes, PhD, DScEmeritus Professor of Veterinary Biochemistry, RoyalVeterinary College, University of LondonRobert K. Murray, MD, PhDProfessor (Emeritus) of Biochemistry, University ofTorontoMargaret L. Rand, PhDScientist, Research Institute, Hospital for Sick Children,Toronto, and Associate Professor, Departmentsof Laboratory Medicine and Pathobiologyand Department of Biochemistry, University ofTorontoVictor W. Rodwell, PhDProfessor of Biochemistry, Purdue University, WestLafayette, IndianaP. Anthony Weil, PhDProfessor of Molecular Physiology and Biophysics,Vanderbilt University School of Medicine, Nashville,Tennesseevii

PrefaceThe authors and publisher are pleased to present the twenty-sixth edition of Harper’s Illustrated Biochemistry. Reviewof Physiological Chemistry was first published in 1939 and revised in 1944, and it quickly gained a wide readership. In1951, the third edition appeared with Harold A. Harper, University of California School of Medicine at San Francisco,as author. Dr. Harper remained the sole author until the ninth edition and co-authored eight subsequent editions.Peter Mayes and Victor Rodwell have been authors since the tenth edition, Daryl Granner since the twentiethedition, and Rob Murray since the twenty-first edition. Because of the increasing complexity of biochemical knowledge,they have added co-authors in recent editions.Fred Keeley and Margaret Rand have each co-authored one chapter with Rob Murray for this and previous editions.Peter Kennelly joined as a co-author in the twenty-fifth edition, and in the present edition has co-authoredwith Victor Rodwell all of the chapters dealing with the structure and function of proteins and enzymes. The followingadditional co-authors are very warmly welcomed in this edition: Kathleen Botham has co-authored, with PeterMayes, the chapters on bioenergetics, biologic oxidation, oxidative phosphorylation, and lipid metabolism. DavidBender has co-authored, also with Peter Mayes, the chapters dealing with carbohydrate metabolism, nutrition, digestion,and vitamins and minerals. P. Anthony Weil has co-authored chapters dealing with various aspects of DNA, ofRNA, and of gene expression with Daryl Granner. We are all very grateful to our co-authors for bringing their expertiseand fresh perspectives to the text.CHANGES IN THE TWENTY-SIXTH EDITIONA major goal of the authors continues to be to provide both medical and other students of the health sciences with abook that both describes the basics of biochemistry and is user-friendly and interesting. A second major ongoinggoal is to reflect the most significant advances in biochemistry that are important to medicine. However, a thirdmajor goal of this edition was to achieve a substantial reduction in size, as feedback indicated that many readers prefershorter texts.To achieve this goal, all of the chapters were rigorously edited, involving their amalgamation, division, or deletion,and many were reduced to approximately one-half to two-thirds of their previous size. This has been effectedwithout loss of crucial information but with gain in conciseness and clarity.Despite the reduction in size, there are many new features in the twenty-sixth edition. These include:• A new chapter on amino acids and peptides, which emphasizes the manner in which the properties of biologicpeptides derive from the individual amino acids of which they are comprised.• A new chapter on the primary structure of proteins, which provides coverage of both classic and newly emerging“proteomic” and “genomic” methods for identifying proteins. A new section on the application of mass spectrometryto the analysis of protein structure has been added, including comments on the identification of covalent modifications.• The chapter on the mechanisms of action of enzymes has been revised to provide a comprehensive description ofthe various physical mechanisms by which enzymes carry out their catalytic functions.• The chapters on integration of metabolism, nutrition, digestion and absorption, and vitamins and minerals havebeen completely re-written.• Among important additions to the various chapters on metabolism are the following: update of the informationon oxidative phosphorylation, including a description of the rotary ATP synthase; new insights into the role ofGTP in gluconeogenesis; additional information on the regulation of acetyl-CoA carboxylase; new information onreceptors involved in lipoprotein metabolism and reverse cholesterol transport; discussion of the role of leptin infat storage; and new information on bile acid regulation, including the role of the farnesoid X receptor (FXR).• The chapter on membrane biochemistry in the previous edition has been split into two, yielding two new chapterson the structure and function of membranes and intracellular traffic and sorting of proteins.• Considerable new material has been added on RNA synthesis, protein synthesis, gene regulation, and various aspectsof molecular genetics.• Much of the material on individual endocrine glands present in the twenty-fifth edition has been replaced withnew chapters dealing with the diversity of the endocrine system, with molecular mechanisms of hormone action,and with signal transduction.ix

x / PREFACE• The chapter on plasma proteins, immunoglobulins, and blood coagulation in the previous edition has been splitinto two new chapters on plasma proteins and immunoglobulins and on hemostasis and thrombosis.• New information has been added in appropriate chapters on lipid rafts and caveolae, aquaporins, connexins, disordersdue to mutations in genes encoding proteins involved in intracellular membrane transport, absorption ofiron, and conformational diseases and pharmacogenomics.• A new and final chapter on “The Human Genome Project” (HGP) has been added, which builds on the materialcovered in Chapters 35 through 40. Because of the impact of the results of the HGP on the future of biology andmedicine, it appeared appropriate to conclude the text with a summary of its major findings and their implicationsfor future work.• As initiated in the previous edition, references to useful Web sites have been included in a brief Appendix at theend of the text.ORGANIZATION OF THE BOOKThe text is divided into two introductory chapters (“Biochemistry & Medicine” and “Water & pH”) followed by sixmain sections.Section I deals with the structures and functions of proteins and enzymes, the workhorses of the body. Becausealmost all of the reactions in cells are catalyzed by enzymes, it is vital to understand the properties of enzymes beforeconsidering other topics.Section II explains how various cellular reactions either utilize or release energy, and it traces the pathways bywhich carbohydrates and lipids are synthesized and degraded. It also describes the many functions of these twoclasses of molecules.Section III deals with the amino acids and their many fates and also describes certain key features of protein catabolism.Section IV describes the structures and functions of the nucleotides and nucleic acids, and covers many majortopics such as DNA replication and repair, RNA synthesis and modification, and protein synthesis. It also discussesnew findings on how genes are regulated and presents the principles of recombinant DNA technology.Section V deals with aspects of extracellular and intracellular communication. Topics covered include membranestructure and function, the molecular bases of the actions of hormones, and the key field of signal transduction.Section VI consists of discussions of eleven special topics: nutrition, digestion, and absorption; vitamins andminerals; intracellular traffic and sorting of proteins; glycoproteins; the extracellular matrix; muscle and the cytoskeleton;plasma proteins and immunoglobulins; hemostasis and thrombosis; red and white blood cells; the metabolismof xenobiotics; and the Human Genome Project.ACKNOWLEDGMENTSThe authors thank Janet Foltin for her thoroughly professional approach. Her constant interest and input have had asignificant impact on the final structure of this text. We are again immensely grateful to Jim Ransom for his excellenteditorial work; it has been a pleasure to work with an individual who constantly offered wise and informed alternativesto the sometimes primitive text transmitted by the authors. The superb editorial skills of Janene MatragranoOransky and Harriet Lebowitz are warmly acknowledged, as is the excellent artwork of Charissa Baker and her colleagues.The authors are very grateful to Kathy Pitcoff for her thoughtful and meticulous work in preparing theIndex. Suggestions from students and colleagues around the world have been most helpful in the formulation of thisedition. We look forward to receiving similar input in the future.Toronto, OntarioNashville, TennesseeLondonWest Lafayette, IndianaMarch 2003Robert K. Murray, MD, PhDDaryl K. Granner, MDPeter A. Mayes, PhD, DScVictor W. Rodwell, PhD

ContentsAuthors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viiPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix1. Biochemistry & MedicineRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12. Water & pHVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5SECTION I. STRUCTURES & FUNCTIONS OF PROTEINS & ENZYMES . . . . . . . . . . . . . . . . . . . 143. Amino Acids & PeptidesVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144. Proteins: Determination of Primary StructureVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215. Proteins: Higher Orders of StructureVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306. Proteins: Myoglobin & HemoglobinVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 407. Enzymes: Mechanism of ActionVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498. Enzymes: KineticsVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609. Enzymes: Regulation of ActivitiesVictor W. Rodwell, PhD, & Peter J. Kennelly, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72SECTION II. BIOENERGETICS & THE METABOLISM OF CARBOHYDRATES& LIPIDS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8010. Bioenergetics: The Role of ATPPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8011. Biologic OxidationPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8612. The Respiratory Chain & Oxidative PhosphorylationPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9213. Carbohydrates of Physiologic SignificancePeter A. Mayes, PhD, DSc, & David A. Bender, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102iii

iv / CONTENTS14. Lipids of Physiologic SignificancePeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11115. Overview of MetabolismPeter A. Mayes, PhD, DSc, & David A. Bender, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12216. The Citric Acid Cycle: The Catabolism of Acetyl-CoAPeter A. Mayes, PhD, DSc, & David A. Bender, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13017. Glycolysis & the Oxidation of PyruvatePeter A. Mayes, PhD, DSc, & David A. Bender, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13618. Metabolism of GlycogenPeter A. Mayes, PhD, DSc, & David A. Bender, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14519. Gluconeogenesis & Control of the Blood GlucosePeter A. Mayes, PhD, DSc, & David A. Bender, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15320. The Pentose Phosphate Pathway & Other Pathways of Hexose MetabolismPeter A. Mayes, PhD, DSc, & David A. Bender, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16321. Biosynthesis of Fatty AcidsPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17322. Oxidation of Fatty Acids: KetogenesisPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18023. Metabolism of Unsaturated Fatty Acids & EicosanoidsPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19024. Metabolism of Acylglycerols & SphingolipidsPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19725. Lipid Transport & StoragePeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20526. Cholesterol Synthesis, Transport, & ExcretionPeter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21927. Integration of Metabolism—the Provision of Metabolic FuelsDavid A. Bender, PhD, & Peter A. Mayes, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231SECTION III. METABOLISM OF PROTEINS & AMINO ACIDS . . . . . . . . . . . . . . . . . . . . . . . . . 23728. Biosynthesis of the Nutritionally Nonessential Amino AcidsVictor W. Rodwell, PhD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23729. Catabolism of Proteins & of Amino Acid NitrogenVictor W. Rodwell, PhD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242

CONTENTS / v30. Catabolism of the Carbon Skeletons of Amino AcidsVictor W. Rodwell, PhD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24931. Conversion of Amino Acids to Specialized ProductsVictor W. Rodwell, PhD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26432. Porphyrins & Bile PigmentsRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 270SECTION IV. STRUCTURE, FUNCTION, & REPLICATIONOF INFORMATIONAL MACROMOLECULES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28633. NucleotidesVictor W. Rodwell, PhD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28634. Metabolism of Purine & Pyrimidine NucleotidesVictor W. Rodwell, PhD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29335. Nucleic Acid Structure & FunctionDaryl K. Granner, MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30336. DNA Organization, Replication, & RepairDaryl K. Granner, MD, & P. Anthony Weil, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31437. RNA Synthesis, Processing, & ModificationDaryl K. Granner, MD, & P. Anthony Weil, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34138. Protein Synthesis & the Genetic CodeDaryl K. Granner, MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35839. Regulation of Gene ExpressionDaryl K. Granner, MD, & P. Anthony Weil, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37440. Molecular Genetics, Recombinant DNA, & Genomic TechnologyDaryl K. Granner, MD, & P. Anthony Weil, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396SECTION V. BIOCHEMISTRY OF EXTRACELLULAR& INTRACELLULAR COMMUNICATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41541. Membranes: Structure & FunctionRobert K. Murray, MD, PhD, & Daryl K. Granner, MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41542. The Diversity of the Endocrine SystemDaryl K. Granner, MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43443. Hormone Action & Signal TransductionDaryl K. Granner, MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456

vi / CONTENTSSECTION VI. SPECIAL TOPICS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47444. Nutrition, Digestion, & AbsorptionDavid A. Bender, PhD, & Peter A. Mayes, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47445. Vitamins & MineralsDavid A. Bender, PhD, & Peter A. Mayes, PhD, DSc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48146. Intracellular Traffic & Sorting of ProteinsRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49847. GlycoproteinsRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51448. The Extracellular MatrixRobert K. Murray, MD, PhD, & Frederick W. Keeley, PhD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53549. Muscle & the CytoskeletonRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55650. Plasma Proteins & ImmunoglobulinsRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58051. Hemostasis & ThrombosisMargaret L. Rand, PhD, & Robert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59852. Red & White Blood CellsRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60953. Metabolism of XenobioticsRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62654. The Human Genome ProjectRobert K. Murray, MD, PhD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633Appendix . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 639Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643

2 / CHAPTER 1Table 1–1. The principal methods andpreparations used in biochemical laboratories.Methods for Separating and Purifying Biomolecules 1Salt fractionation (eg, precipitation of proteins with ammoniumsulfate)Chromatography: Paper; ion exchange; affinity; thin-layer;gas-liquid; high-pressure liquid; gel filtrationElectrophoresis: Paper; high-voltage; agarose; celluloseacetate; starch gel; polyacrylamide gel; SDS-polyacrylamidegelUltracentrifugationMethods for Determining Biomolecular StructuresElemental analysisUV, visible, infrared, and NMR spectroscopyUse of acid or alkaline hydrolysis to degrade the biomoleculeunder study into its basic constituentsUse of a battery of enzymes of known specificity to degradethe biomolecule under study (eg, proteases, nucleases,glycosidases)Mass spectrometrySpecific sequencing methods (eg, for proteins and nucleicacids)X-ray crystallographyPreparations for Studying Biochemical ProcessesWhole animal (includes transgenic animals and animalswith gene knockouts)Isolated perfused organTissue sliceWhole cellsHomogenateIsolated cell organellesSubfractionation of organellesPurified metabolites and enzymesIsolated genes (including polymerase chain reaction andsite-directed mutagenesis)1 Most of these methods are suitable for analyzing the componentspresent in cell homogenates and other biochemical preparations.The sequential use of several techniques will generallypermit purification of most biomolecules. The reader is referredto texts on methods of biochemical research for details.disease can open up areas of cell function for basic biochemicalresearch.The relationship between medicine and biochemistryhas important implications for the former. As longas medical treatment is firmly grounded in a knowledgeof biochemistry and other basic sciences, the practice ofmedicine will have a rational basis that can be adaptedto accommodate new knowledge. This contrasts withunorthodox health cults and at least some “alternativemedicine” practices, which are often founded on littlemore than myth and wishful thinking and generallylack any intellectual basis.NORMAL BIOCHEMICAL PROCESSES ARETHE BASIS OF HEALTHThe World Health Organization (WHO) defineshealth as a state of “complete physical, mental and socialwell-being and not merely the absence of diseaseand infirmity.” From a strictly biochemical viewpoint,health may be considered that situation in which all ofthe many thousands of intra- and extracellular reactionsthat occur in the body are proceeding at rates commensuratewith the organism’s maximal survival in thephysiologic state. However, this is an extremely reductionistview, and it should be apparent that caring forthe health of patients requires not only a wide knowledgeof biologic principles but also of psychologic andsocial principles.Biochemical Research Has Impact onNutrition & Preventive MedicineOne major prerequisite for the maintenance of health isthat there be optimal dietary intake of a number ofchemicals; the chief of these are vitamins, certainamino acids, certain fatty acids, various minerals, andwater. Because much of the subject matter of both biochemistryand nutrition is concerned with the study ofvarious aspects of these chemicals, there is a close relationshipbetween these two sciences. Moreover, moreemphasis is being placed on systematic attempts tomaintain health and forestall disease, ie, on preventivemedicine. Thus, nutritional approaches to—for example—theprevention of atherosclerosis and cancer arereceiving increased emphasis. Understanding nutritiondepends to a great extent on a knowledge of biochemistry.Most & Perhaps All Disease Hasa Biochemical BasisWe believe that most if not all diseases are manifestationsof abnormalities of molecules, chemical reactions,or biochemical processes. The major factors responsiblefor causing diseases in animals and humans are listed inTable 1–2. All of them affect one or more criticalchemical reactions or molecules in the body. Numerousexamples of the biochemical bases of diseases will be encounteredin this text; the majority of them are due tocauses 5, 7, and 8. In most of these conditions, biochemicalstudies contribute to both the diagnosis andtreatment. Some major uses of biochemical investigationsand of laboratory tests in relation to diseases aresummarized in Table 1–3.Additional examples of many of these uses are presentedin various sections of this text.

4 / CHAPTER 1in early 2001. It is anticipated that the entire sequencewill be completed by 2003. The implications of thiswork for biochemistry, all of biology, and for medicineare tremendous, and only a few points are mentionedhere. Many previously unknown genes have been revealed;their protein products await characterization.New light has been thrown on human evolution, andprocedures for tracking disease genes have been greatlyrefined. The results are having major effects on areassuch as proteomics, bioinformatics, biotechnology, andpharmacogenomics. Reference to the human genomewill be made in various sections of this text. TheHuman Genome Project is discussed in more detail inChapter 54.SUMMARY• Biochemistry is the science concerned with studyingthe various molecules that occur in living cells andorganisms and with their chemical reactions. Becauselife depends on biochemical reactions, biochemistryhas become the basic language of all biologic sciences.• Biochemistry is concerned with the entire spectrumof life forms, from relatively simple viruses and bacteriato complex human beings.• Biochemistry and medicine are intimately related.Health depends on a harmonious balance of biochemicalreactions occurring in the body, and diseasereflects abnormalities in biomolecules, biochemicalreactions, or biochemical processes.• Advances in biochemical knowledge have illuminatedmany areas of medicine. Conversely, the studyof diseases has often revealed previously unsuspectedaspects of biochemistry. The determination of the sequenceof the human genome, nearly complete, willhave a great impact on all areas of biology, includingbiochemistry, bioinformatics, and biotechnology.• Biochemical approaches are often fundamental in illuminatingthe causes of diseases and in designingappropriate therapies.• The judicious use of various biochemical laboratorytests is an integral component of diagnosis and monitoringof treatment.• A sound knowledge of biochemistry and of other relatedbasic disciplines is essential for the rationalpractice of medical and related health sciences.REFERENCESFruton JS: Proteins, Enzymes, Genes: The Interplay of Chemistry andBiology. Yale Univ Press, 1999. (Provides the historical backgroundfor much of today’s biochemical research.)Garrod AE: Inborn errors of metabolism. (Croonian Lectures.)Lancet 1908;2:1, 73, 142, 214.International Human Genome Sequencing Consortium. Initial sequencingand analysis of the human genome. Nature2001:409;860. (The issue [15 February] consists of articlesdedicated to analyses of the human genome.)Kornberg A: Basic research: The lifeline of medicine. FASEB J1992;6:3143.Kornberg A: Centenary of the birth of modern biochemistry.FASEB J 1997;11:1209.McKusick VA: Mendelian Inheritance in Man. Catalogs of HumanGenes and Genetic Disorders, 12th ed. Johns Hopkins UnivPress, 1998. [Abbreviated MIM]Online Mendelian Inheritance in Man (OMIM): Center for MedicalGenetics, Johns Hopkins University and National Centerfor Biotechnology Information, National Library of Medicine,1997. http://www.ncbi.nlm.nih.gov/omim/(The numbers assigned to the entries in MIM and OMIM will becited in selected chapters of this work. Consulting this extensivecollection of diseases and other relevant entries—specificproteins, enzymes, etc—will greatly expand the reader’sknowledge and understanding of various topics referred toand discussed in this text. The online version is updated almostdaily.)Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Venter JC et al: The Sequence of the Human Genome. Science2001;291:1304. (The issue [16 February] contains the Celeradraft version and other articles dedicated to analyses of thehuman genome.)Williams DL, Marks V: Scientific Foundations of Biochemistry inClinical Practice, 2nd ed. Butterworth-Heinemann, 1994.

Water & pH 2Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEWater is the predominant chemical component of livingorganisms. Its unique physical properties, which includethe ability to solvate a wide range of organic andinorganic molecules, derive from water’s dipolar structureand exceptional capacity for forming hydrogenbonds. The manner in which water interacts with a solvatedbiomolecule influences the structure of each. Anexcellent nucleophile, water is a reactant or product inmany metabolic reactions. Water has a slight propensityto dissociate into hydroxide ions and protons. Theacidity of aqueous solutions is generally reported usingthe logarithmic pH scale. Bicarbonate and other buffersnormally maintain the pH of extracellular fluid between7.35 and 7.45. Suspected disturbances of acidbasebalance are verified by measuring the pH of arterialblood and the CO 2 content of venous blood. Causesof acidosis (blood pH < 7.35) include diabetic ketosisand lactic acidosis. Alkalosis (pH > 7.45) may, for example,follow vomiting of acidic gastric contents. Regulationof water balance depends upon hypothalamicmechanisms that control thirst, on antidiuretic hormone(ADH), on retention or excretion of water by thekidneys, and on evaporative loss. Nephrogenic diabetesinsipidus, which involves the inability to concentrateurine or adjust to subtle changes in extracellular fluidosmolarity, results from the unresponsiveness of renaltubular osmoreceptors to ADH.WATER IS AN IDEAL BIOLOGIC SOLVENTWater Molecules Form DipolesA water molecule is an irregular, slightly skewed tetrahedronwith oxygen at its center (Figure 2–1). The twohydrogens and the unshared electrons of the remainingtwo sp 3 -hybridized orbitals occupy the corners of thetetrahedron. The 105-degree angle between the hydrogensdiffers slightly from the ideal tetrahedral angle,109.5 degrees. Ammonia is also tetrahedral, with a 107-degree angle between its hydrogens. Water is a dipole,a molecule with electrical charge distributed asymmetricallyabout its structure. The strongly electronegative5oxygen atom pulls electrons away from the hydrogennuclei, leaving them with a partial positive charge,while its two unshared electron pairs constitute a regionof local negative charge.Water, a strong dipole, has a high dielectric constant.As described quantitatively by Coulomb’s law,the strength of interaction F between oppositelycharged particles is inversely proportionate to the dielectricconstant ε of the surrounding medium. The dielectricconstant for a vacuum is unity; for hexane it is1.9; for ethanol it is 24.3; and for water it is 78.5.Water therefore greatly decreases the force of attractionbetween charged and polar species relative to water-freeenvironments with lower dielectric constants. Its strongdipole and high dielectric constant enable water to dissolvelarge quantities of charged compounds such assalts.Water Molecules Form Hydrogen BondsAn unshielded hydrogen nucleus covalently bound toan electron-withdrawing oxygen or nitrogen atom caninteract with an unshared electron pair on another oxygenor nitrogen atom to form a hydrogen bond. Sincewater molecules contain both of these features, hydrogenbonding favors the self-association of water moleculesinto ordered arrays (Figure 2–2). Hydrogen bondingprofoundly influences the physical properties ofwater and accounts for its exceptionally high viscosity,surface tension, and boiling point. On average, eachmolecule in liquid water associates through hydrogenbonds with 3.5 others. These bonds are both relativelyweak and transient, with a half-life of about one microsecond.Rupture of a hydrogen bond in liquid waterrequires only about 4.5 kcal/mol, less than 5% of theenergy required to rupture a covalent O ⎯H bond.Hydrogen bonding enables water to dissolve manyorganic biomolecules that contain functional groupswhich can participate in hydrogen bonding. The oxygenatoms of aldehydes, ketones, and amides providepairs of electrons that can serve as hydrogen acceptors.Alcohols and amines can serve both as hydrogen acceptorsand as donors of unshielded hydrogen atoms forformation of hydrogen bonds (Figure 2–3).

6 / CHAPTER 2H2eCH 3CH 2OHOHFigure 2–1.geometry.HHOHOHH105°HH2eThe water molecule has tetrahedralINTERACTION WITH WATER INFLUENCESTHE STRUCTURE OF BIOMOLECULESCovalent & Noncovalent Bonds StabilizeBiologic MoleculesThe covalent bond is the strongest force that holdsmolecules together (Table 2–1). Noncovalent forces,while of lesser magnitude, make significant contributionsto the structure, stability, and functional competenceof macromolecules in living cells. These forces,which can be either attractive or repulsive, involve interactionsboth within the biomolecule and between itand the water that forms the principal component ofthe surrounding environment.Biomolecules Fold to Position Polar &Charged Groups on Their SurfacesMost biomolecules are amphipathic; that is, they possessregions rich in charged or polar functional groupsas well as regions with hydrophobic character. Proteinstend to fold with the R-groups of amino acids with hydrophobicside chains in the interior. Amino acids withcharged or polar amino acid side chains (eg, arginine,glutamate, serine) generally are present on the surfacein contact with water. A similar pattern prevails in aphospholipid bilayer, where the charged head groups ofHH HOHH H OOO H HFigure 2–2. Left: Association of two dipolar watermolecules by a hydrogen bond (dotted line). Right:Hydrogen-bonded cluster of four water molecules.Note that water can serve simultaneously both as a hydrogendonor and as a hydrogen acceptor.OHCH 3CH2RCR IOOHHHONCH 2 CH 3IIRIIIRFigure 2–3. Additional polar groups participate inhydrogen bonding. Shown are hydrogen bonds formedbetween an alcohol and water, between two moleculesof ethanol, and between the peptide carbonyl oxygenand the peptide nitrogen hydrogen of an adjacentamino acid.phosphatidyl serine or phosphatidyl ethanolamine contactwater while their hydrophobic fatty acyl side chainscluster together, excluding water. This pattern maximizesthe opportunities for the formation of energeticallyfavorable charge-dipole, dipole-dipole, and hydrogenbonding interactions between polar groups on thebiomolecule and water. It also minimizes energeticallyunfavorable contact between water and hydrophobicgroups.Hydrophobic InteractionsHydrophobic interaction refers to the tendency of nonpolarcompounds to self-associate in an aqueous environment.This self-association is driven neither by mutualattraction nor by what are sometimes incorrectlyreferred to as “hydrophobic bonds.” Self-associationarises from the need to minimize energetically unfavorableinteractions between nonpolar groups and water.Table 2–1. Bond energies for atoms of biologicsignificance.Bond Energy Bond EnergyType (kcal/mol) Type (kcal/mol)O—O 34 O==O 96S—S 51 C—H 99C—N 70 C==S 108S—H 81 O—H 110C—C 82 C==C 147C—O 84 C==N 147N—H 94 C==O 164

8 / CHAPTER 2of hydrolytic reactions when needed. Proteases catalyzethe hydrolysis of proteins into their component aminoacids, while nucleases catalyze the hydrolysis of thephosphoester bonds in DNA and RNA. Careful controlof the activities of these enzymes is required to ensurethat they act only on appropriate target molecules.Many Metabolic Reactions InvolveGroup TransferIn group transfer reactions, a group G is transferredfrom a donor D to an acceptor A, forming an acceptorgroup complex A–G:D− G + A = A− G + DThe hydrolysis and phosphorolysis of glycogen representgroup transfer reactions in which glucosyl groupsare transferred to water or to orthophosphate. Theequilibrium constant for the hydrolysis of covalentbonds strongly favors the formation of split products.The biosynthesis of macromolecules also involves grouptransfer reactions in which the thermodynamically unfavoredsynthesis of covalent bonds is coupled to favoredreactions so that the overall change in free energyfavors biopolymer synthesis. Given the nucleophiliccharacter of water and its high concentration in cells,why are biopolymers such as proteins and DNA relativelystable? And how can synthesis of biopolymersoccur in an apparently aqueous environment? Centralto both questions are the properties of enzymes. In theabsence of enzymic catalysis, even thermodynamicallyhighly favored reactions do not necessarily take placerapidly. Precise and differential control of enzyme activityand the sequestration of enzymes in specific organellesdetermine under what physiologic conditions agiven biopolymer will be synthesized or degraded.Newly synthesized polymers are not immediately hydrolyzed,in part because the active sites of biosyntheticenzymes sequester substrates in an environment fromwhich water can be excluded.Water Molecules Exhibit a Slight butImportant Tendency to DissociateThe ability of water to ionize, while slight, is of centralimportance for life. Since water can act both as an acidand as a base, its ionization may be represented as anintermolecular proton transfer that forms a hydroniumion (H 3 O + ) and a hydroxide ion (OH − ):+ −HO 2 + HO 2 = HO 3 + OHThe transferred proton is actually associated with acluster of water molecules. Protons exist in solution notonly as H 3 O + , but also as multimers such as H 5 O 2 + andH 7 O 3 + . The proton is nevertheless routinely representedas H + , even though it is in fact highly hydrated.Since hydronium and hydroxide ions continuouslyrecombine to form water molecules, an individual hydrogenor oxygen cannot be stated to be present as anion or as part of a water molecule. At one instant it isan ion. An instant later it is part of a molecule. Individualions or molecules are therefore not considered. Werefer instead to the probability that at any instant intime a hydrogen will be present as an ion or as part of awater molecule. Since 1 g of water contains 3.46 × 10 22molecules, the ionization of water can be described statistically.To state that the probability that a hydrogenexists as an ion is 0.01 means that a hydrogen atom hasone chance in 100 of being an ion and 99 chances outof 100 of being part of a water molecule. The actualprobability of a hydrogen atom in pure water existing asa hydrogen ion is approximately 1.8 × 10 −9 . The probabilityof its being part of a molecule thus is almostunity. Stated another way, for every hydrogen ion andhydroxyl ion in pure water there are 1.8 billion or 1.8 ×10 9 water molecules. Hydrogen ions and hydroxyl ionsnevertheless contribute significantly to the properties ofwater.For dissociation of water,+[ H ][ OH− ]K =[ HO 2 ]where brackets represent molar concentrations (strictlyspeaking, molar activities) and K is the dissociationconstant. Since one mole (mol) of water weighs 18 g,one liter (L) (1000 g) of water contains 1000 × 18 =55.56 mol. Pure water thus is 55.56 molar. Since theprobability that a hydrogen in pure water will exist as ahydrogen ion is 1.8 × 10 −9 , the molar concentration ofH + ions (or of OH − ions) in pure water is the productof the probability, 1.8 × 10 −9 , times the molar concentrationof water, 55.56 mol/L. The result is 1.0 × 10 −7mol/L.We can now calculate K for water:+ − −7 −7[ H ][ OH ] [ 10 ][ 10 ]K = =[ H2O][ 55. 56]−14 −16= 0. 018 × 10 = 1. 8 × 10 mol / LThe molar concentration of water, 55.56 mol/L, istoo great to be significantly affected by dissociation. Ittherefore is considered to be essentially constant. Thisconstant may then be incorporated into the dissociationconstant K to provide a useful new constant K w termedthe ion product for water. The relationship betweenK w and K is shown below:

WATER & pH / 9+ −[ H ][ OH ]−16K = = 18 . × 10 mol / L[ H2O]+ −Kw= ( K)[ H2O] = [ H ][ OH ]−16= ( 1. 8 × 10 mol / L) ( 55. 56 mol / L)−14 2= 100 . × 10 ( mol / L)Note that the dimensions of K are moles per liter andthose of K w are moles 2 per liter 2 . As its name suggests,the ion product K w is numerically equal to the productof the molar concentrations of H + and OH − :+ −K w = [ H ][ OH ]At 25 °C, K w = (10 −7 ) 2 , or 10 −14 (mol/L) 2 . At temperaturesbelow 25 °C, K w is somewhat less than 10 −14 ; andat temperatures above 25 °C it is somewhat greater than10 −14 . Within the stated limitations of the effect of temperature,K w equals 10 -14 (mol/L) 2 for all aqueous solutions,even solutions of acids or bases. We shall useK w to calculate the pH of acidic and basic solutions.termediates, whose phosphoryl group contains two dissociableprotons, the first of which is strongly acidic.The following examples illustrate how to calculatethe pH of acidic and basic solutions.Example 1: What is the pH of a solution whose hydrogenion concentration is 3.2 × 10 − 4 mol/L?Example 2: What is the pH of a solution whose hydroxideion concentration is 4.0 × 10 − 4 mol/L? We firstdefine a quantity pOH that is equal to −log [OH − ] andthat may be derived from the definition of K w :Therefore:+pH = −log [ H ]−4= −log ( 32 . × 10 )−4= −log ( 32 . ) −log ( 10 )= −05 . + 40 .= 35 .+ − −1K w = [ H ][ OH ] = 10 4pH IS THE NEGATIVE LOG OF THEHYDROGEN ION CONCENTRATIONThe term pH was introduced in 1909 by Sörensen,who defined pH as the negative log of the hydrogen ionconcentration:+pH = −log [ H ]This definition, while not rigorous, suffices for manybiochemical purposes. To calculate the pH of a solution:1. Calculate hydrogen ion concentration [H + ].2. Calculate the base 10 logarithm of [H + ].3. pH is the negative of the value found in step 2.For example, for pure water at 25°C,or+ −log [ H ] + log [ OH ] = log 10 −14pH + pOH =14To solve the problem by this approach:−−4[ OH ] = 40 . × 10−pOH = −log [ OH ]−4= −log ( 40 . × 10 )−4= −log ( 40 . ) − log ( 10 )= − 060 . + 40 .= 34 .Low pH values correspond to high concentrations ofH + and high pH values correspond to low concentrationsof H + .Acids are proton donors and bases are proton acceptors.Strong acids (eg, HCl or H 2 SO 4 ) completelydissociate into anions and cations even in strongly acidicsolutions (low pH). Weak acids dissociate only partiallyin acidic solutions. Similarly, strong bases (eg, KOH orNaOH)—but not weak bases (eg, Ca[OH] 2 )—arecompletely dissociated at high pH. Many biochemicalsare weak acids. Exceptions include phosphorylated in-+−7pH = −log [ H ] = −log 10 = −(−7) = 7.0Now:pH = 14 −pOH= 14 − 3.4= 10.6Example 3: What are the pH values of (a) 2.0 × 10 −2mol/L KOH and of (b) 2.0 × 10 −6 mol/L KOH? TheOH − arises from two sources, KOH and water. SincepH is determined by the total [H + ] (and pOH by thetotal [OH − ]), both sources must be considered. In thefirst case (a), the contribution of water to the total[OH − ] is negligible. The same cannot be said for thesecond case (b):

10 / CHAPTER 2Concentration (mol/L)(a)(b)Molarity of KOH 2.0 × 10 −2 2.0 × 10 −6[OH − ] from KOH 2.0 × 10 −2 2.0 × 10 −6[OH − ] from water 1.0 × 10 −7 1.0 × 10 −7Total [OH − ] 2.00001 × 10 −2 2.1 × 10 −6Once a decision has been reached about the significanceof the contribution by water, pH may be calculated asabove.The above examples assume that the strong baseKOH is completely dissociated in solution and that theconcentration of OH − ions was thus equal to that of theKOH. This assumption is valid for dilute solutions ofstrong bases or acids but not for weak bases or acids.Since weak electrolytes dissociate only slightly in solution,we must use the dissociation constant to calculatethe concentration of [H + ] (or [OH − ]) produced bya given molarity of a weak acid (or base) before calculatingtotal [H + ] (or total [OH − ]) and subsequently pH.Functional Groups That Are Weak AcidsHave Great Physiologic SignificanceMany biochemicals possess functional groups that areweak acids or bases. Carboxyl groups, amino groups,and the second phosphate dissociation of phosphate estersare present in proteins and nucleic acids, mostcoenzymes, and most intermediary metabolites. Knowledgeof the dissociation of weak acids and bases thus isbasic to understanding the influence of intracellular pHon structure and biologic activity. Charge-based separationssuch as electrophoresis and ion exchange chromatographyalso are best understood in terms of thedissociation behavior of functional groups.We term the protonated species (eg, HA orR⎯NH + 3 ) the acid and the unprotonated species (eg,A − or R⎯NH 2 ) its conjugate base. Similarly, we mayrefer to a base (eg, A − or R⎯NH 2 ) and its conjugateacid (eg, HA or R⎯NH + 3 ). Representative weak acids(left), their conjugate bases (center), and the pK a values(right) include the following:−R — CH2— COOH R — CH 2 — COO pKa= 4−5+R — CH 2— NH3 R — CH 2—NH2pKa= 9−10−HCO 2 3HCO 3pKa= 64 .−−2HPO 2 4HPO 4pKa= 72 .We express the relative strengths of weak acids andbases in terms of their dissociation constants. Shownbelow are the expressions for the dissociation constant(K a ) for two representative weak acids, R⎯COOH andR⎯NH 3 + .Since the numeric values of K a for weak acids are negativeexponential numbers, we express K a as pK a , whereNote that pK a is related to K a as pH is to [H + ]. Thestronger the acid, the lower its pK a value.pK a is used to express the relative strengths of bothacids and bases. For any weak acid, its conjugate is astrong base. Similarly, the conjugate of a strong base isa weak acid. The relative strengths of bases are expressedin terms of the pK a of their conjugate acids. Forpolyproteic compounds containing more than one dissociableproton, a numerical subscript is assigned toeach in order of relative acidity. For a dissociation ofthe typethe pK a is the pH at which the concentration of theacid R⎯NH 3 + equals that of the base R⎯NH 2 .From the above equations that relate K a to [H + ] andto the concentrations of undissociated acid and its conjugatebase, whenor whenthen− +R — COOH = R — COO + H− +[ R — COO ][ H ]Ka=[ R — COOH]+ +R — NH3 = R — NH2+H+[ R— NH ][ H ]Ka=2+[ R— NH3]pKa= − log K+R— NH 3 → R — NH2−[ R — COO ] = [ R — COOH][ R— NH2] = [ R—NH +3 ]+K a = [ H ]Thus, when the associated (protonated) and dissociated(conjugate base) species are present at equal concentrations,the prevailing hydrogen ion concentration [H + ]is numerically equal to the dissociation constant, K a . Ifthe logarithms of both sides of the above equation are

12 / CHAPTER 2meq of alkali added per meq of acid1.00.80.60.40.202 3 4 5 6 7pHFigure 2–4. Titration curve for an acid of the typeHA. The heavy dot in the center of the curve indicatesthe pK a 5.0.in pH. For experiments using tissue extracts or enzymes,constant pH is maintained by the addition ofbuffers such as MES ([2-N-morpholino]ethanesulfonicacid, pK a 6.1), inorganic orthophosphate (pK a2 7.2),HEPES (N-hydroxyethylpiperazine-N9-2-ethanesulfonicacid, pK a 6.8), or Tris (tris[hydroxymethyl] aminomethane,pK a 8.3). The value of pK a relative to the desiredpH is the major determinant of which buffer is selected.Buffering can be observed by using a pH meterwhile titrating a weak acid or base (Figure 2–4). Wecan also calculate the pH shift that accompanies additionof acid or base to a buffered solution. In the example,the buffered solution (a weak acid, pK a = 5.0, andits conjugate base) is initially at one of four pH values.We will calculate the pH shift that results when 0.1meq of KOH is added to 1 meq of each solution:81.00.80.60.40.20Net chargeInitial pH 5.00 5.37 5.60 5.86[A − ] initial 0.50 0.70 0.80 0.88[HA] initial 0.50 0.30 0.20 0.12([A − ]/[HA]) initial 1.00 2.33 4.00 7.33Addition of 0.1 meq of KOH produces[A − ] final 0.60 0.80 0.90 0.98[HA] final 0.40 0.20 0.10 0.02([A − ]/[HA]) final 1.50 4.00 9.00 49.0log ([A − ]/[HA]) final 0.176 0.602 0.95 1.69Final pH 5.18 5.60 5.95 6.69∆pH 0.18 0.60 0.95 1.69Notice that the change in pH per milliequivalent ofOH − added depends on the initial pH. The solution resistschanges in pH most effectively at pH values closeto the pK a . A solution of a weak acid and its conjugatebase buffers most effectively in the pH range pK a ± 1.0pH unit.Figure 2–4 also illustrates the net charge on onemolecule of the acid as a function of pH. A fractionalcharge of −0.5 does not mean that an individual moleculebears a fractional charge, but the probability that agiven molecule has a unit negative charge is 0.5. Considerationof the net charge on macromolecules as afunction of pH provides the basis for separatory techniquessuch as ion exchange chromatography and electrophoresis.Acid Strength Depends onMolecular StructureMany acids of biologic interest possess more than onedissociating group. The presence of adjacent negativecharge hinders the release of a proton from a nearbygroup, raising its pK a . This is apparent from the pK avalues for the three dissociating groups of phosphoricacid and citric acid (Table 2–2). The effect of adjacentcharge decreases with distance. The second pK a for succinicacid, which has two methylene groups between itscarboxyl groups, is 5.6, whereas the second pK a for glu-Table 2–2. Relative strengths of selected acids ofbiologic significance. Tabulated values are the pK avalues (−log of the dissociation constant) ofselected monoprotic, diprotic, and triprotic acids.Monoprotic AcidsFormic pK 3.75Lactic pK 3.86Acetic pK 4.76Ammonium ion pK 9.25Diprotic AcidsCarbonic pK 1 6.37pK 2 10.25Succinic pK 1 4.21pK 2 5.64Glutaric pK 1 4.34pK 2 5.41Triprotic AcidsPhosphoric pK 1 2.15pK 2 6.82pK 3 12.38Citric pK 1 3.08pK 2 4.74pK 3 5.40

WATER & pH / 13taric acid, which has one additional methylene group,is 5.4.pK a Values Depend on the Propertiesof the MediumThe pK a of a functional group is also profoundly influencedby the surrounding medium. The medium mayeither raise or lower the pK a depending on whether theundissociated acid or its conjugate base is the chargedspecies. The effect of dielectric constant on pK a may beobserved by adding ethanol to water. The pK a of a carboxylicacid increases, whereas that of an amine decreasesbecause ethanol decreases the ability of water to solvatea charged species. The pK a values of dissociating groupsin the interiors of proteins thus are profoundly affectedby their local environment, including the presence orabsence of water.SUMMARY• Water forms hydrogen-bonded clusters with itself andwith other proton donors or acceptors. Hydrogenbonds account for the surface tension, viscosity, liquidstate at room temperature, and solvent power of water.• Compounds that contain O, N, or S can serve as hydrogenbond donors or acceptors.• Macromolecules exchange internal surface hydrogenbonds for hydrogen bonds to water. Entropic forcesdictate that macromolecules expose polar regions toan aqueous interface and bury nonpolar regions.• Salt bonds, hydrophobic interactions, and van derWaals forces participate in maintaining molecularstructure.• pH is the negative log of [H + ]. A low pH characterizesan acidic solution, and a high pH denotes a basicsolution.• The strength of weak acids is expressed by pK a , thenegative log of the acid dissociation constant. Strongacids have low pK a values and weak acids have highpK a values.• Buffers resist a change in pH when protons are producedor consumed. Maximum buffering capacityoccurs ± 1 pH unit on either side of pK a . Physiologicbuffers include bicarbonate, orthophosphate, andproteins.REFERENCESSegel IM: Biochemical Calculations. Wiley, 1968.Wiggins PM: Role of water in some biological processes. MicrobiolRev 1990;54:432.

SECTION IStructures & Functionsof Proteins & EnzymesAmino Acids & Peptides 3Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEIn addition to providing the monomer units from whichthe long polypeptide chains of proteins are synthesized,the L-α-amino acids and their derivatives participate incellular functions as diverse as nerve transmission andthe biosynthesis of porphyrins, purines, pyrimidines,and urea. Short polymers of amino acids called peptidesperform prominent roles in the neuroendocrine systemas hormones, hormone-releasing factors, neuromodulators,or neurotransmitters. While proteins contain onlyL-α-amino acids, microorganisms elaborate peptidesthat contain both D- and L-α-amino acids. Several ofthese peptides are of therapeutic value, including the antibioticsbacitracin and gramicidin A and the antitumoragent bleomycin. Certain other microbial peptides aretoxic. The cyanobacterial peptides microcystin andnodularin are lethal in large doses, while small quantitiespromote the formation of hepatic tumors. Neither humansnor any other higher animals can synthesize 10 ofthe 20 common L-α-amino acids in amounts adequateto support infant growth or to maintain health in adults.Consequently, the human diet must contain adequatequantities of these nutritionally essential amino acids.PROPERTIES OF AMINO ACIDSThe Genetic Code Specifies20 L--Amino AcidsOf the over 300 naturally occurring amino acids, 20 constitutethe monomer units of proteins. While a nonredundantthree-letter genetic code could accommodate14more than 20 amino acids, its redundancy limits theavailable codons to the 20 L-α-amino acids listed inTable 3–1, classified according to the polarity of their Rgroups. Both one- and three-letter abbreviations for eachamino acid can be used to represent the amino acids inpeptides (Table 3–1). Some proteins contain additionalamino acids that arise by modification of an amino acidalready present in a peptide. Examples include conversionof peptidyl proline and lysine to 4-hydroxyprolineand 5-hydroxylysine; the conversion of peptidyl glutamateto γ-carboxyglutamate; and the methylation,formylation, acetylation, prenylation, and phosphorylationof certain aminoacyl residues. These modificationsextend the biologic diversity of proteins by altering theirsolubility, stability, and interaction with other proteins.Only L--Amino Acids Occur in ProteinsWith the sole exception of glycine, the α-carbon ofamino acids is chiral. Although some protein aminoacids are dextrorotatory and some levorotatory, all sharethe absolute configuration of L-glyceraldehyde and thusare L-α-amino acids. Several free L-α-amino acids fulfillimportant roles in metabolic processes. Examples includeornithine, citrulline, and argininosuccinate thatparticipate in urea synthesis; tyrosine in formation ofthyroid hormones; and glutamate in neurotransmitterbiosynthesis. D-Amino acids that occur naturally includefree D-serine and D-aspartate in brain tissue,D-alanine and D-glutamate in the cell walls of grampositivebacteria, and D-amino acids in some nonmammalianpeptides and certain antibiotics.

Table 3–1.L- α-Amino acids present in proteins.Name Symbol Structural Formula pK 1 pK 2 pK 3With Aliphatic Side Chains -COOH -NH + 3 R GroupGlycine Gly [G]H CH COO –2.4 9.8+NH 3Alanine Ala [A]CH 3 CH COO –2.4 9.9+NH 3H 3 CValine Val [V]CH CH COO –H 3 C+NH 32.2 9.7H 3 CLeucine Leu [L]CH CH COO –2 CHH 3 C+NH 32.3 9.7CH 3CH 2Isoleucine Ile [I] CH CH COO –2.3 9.8CH 3+NH 3With Side Chains Containing Hydroxylic (OH) GroupsSerine Ser [S]CH 2 CH COO –2.2 9.2 about 13OH+NH 3Threonine Thr [T]CH 3 CH CH COO –2.1 9.1 about 13OH+NH 3Tyrosine Tyr [Y] See below.With Side Chains Containing Sulfur AtomsCysteine Cys [C] CH 2 CH COO –1.9 10.8 8.3SH+NH 3Methionine Met [M] CH CH COO –2 CH 22.1 9.3+S CH 3 NH 3With Side Chains Containing Acidic Groups or Their AmidesAspartic acid Asp [D] – OOC CH 2 CH COO –2.0 9.9 3.9+NH 3Asparagine Asn [N]H 2 N C CH 2 CH COO –2.1 8.8O+NH 3Glutamic acid Glu [E] – OOC CH 2 CH 2 CH COO –2.1 9.5 4.1+NH 3Glutamine Gln [Q]C CH 2CH COO –2.2 9.1O+NH 3H 2 N CH 215(continued)

16 / CHAPTER 3Table 3–1.L-α-Amino acids present in proteins. (continued)Name Symbol Structural Formula pK 1 pK 2 pK 3With Side Chains Containing Basic Groups -COOH -NH + 3 R GroupArginine Arg [R]H N CH 2 CH 2 CH 2 CH COO –1.8 9.0 12.5C+NH 3NH 2NH 2+Lysine Lys [K]CH 2 CH 2 CH 2 CH 2 CH COO –2.2 9.2 10.8+NH 3+NH 3CH CH COOHistidine His [H] –21.8 9.3 6.0HN N+NH 3Containing Aromatic RingsHistidine His [H] See above.Phenylalanine Phe [F] CH 2 CH COO –2.2 9.2+NH 3Tyrosine Tyr [Y] 2.2 9.1 10.1HO CH 2 CH COO –+NH 3Tryptophan Trp [W] 2.4 9.4CH 2 CH COO –N+NH 3HImino AcidProline Pro [P] 2.0 10.6Amino Acids May Have Positive, Negative,or Zero Net ChargeCharged and uncharged forms of the ionizable⎯COOH and ⎯NH + 3 weak acid groups exist in solutionin protonic equilibrium:R — COOH = R — COO − ++ H+ +R— NH3 = R — NH2+HWhile both R⎯COOH and R⎯NH + 3 are weak acids,R⎯COOH is a far stronger acid than R⎯NH + 3 . Atphysiologic pH (pH 7.4), carboxyl groups exist almostentirely as R⎯COO − and amino groups predominantlyas R⎯NH + 3 . Figure 3–1 illustrates the effect ofpH on the charged state of aspartic acid.+NH 2COO – Molecules that contain an equal number of ionizablegroups of opposite charge and that therefore bearno net charge are termed zwitterions. Amino acids inblood and most tissues thus should be represented as inA, below.RNH 3+OAO –NH 2OBStructure B cannot exist in aqueous solution because atany pH low enough to protonate the carboxyl groupthe amino group would also be protonated. Similarly,at any pH sufficiently high for an uncharged aminoROH

AMINO ACIDS & PEPTIDES / 17OOHH +OOHH +OO –H +OO –HONH 3+pK 1 = 2.09(α-COOH)– ONH 3+pK 2 = 3.86(β-COOH)– ONH 3+pK 3 = 9.82(— NH 3 + )– ONH 2OOOOAIn strong acid(below pH 1);net charge = +1BAround pH 3;net charge = 0CAround pH 6–8;net charge = –1DIn strong alkali(above pH 11);net charge = –2Figure 3–1.Protonic equilibria of aspartic acid.group to predominate, a carboxyl group will be presentas R⎯COO − . The uncharged representation B (above)is, however, often used for reactions that do not involveprotonic equilibria.pK a Values Express the Strengthsof Weak AcidsThe acid strengths of weak acids are expressed as theirpK a (Table 3–1). The imidazole group of histidine andthe guanidino group of arginine exist as resonance hybridswith positive charge distributed between both nitrogens(histidine) or all three nitrogens (arginine) (Figure3–2). The net charge on an amino acid—thealgebraic sum of all the positively and negativelycharged groups present—depends upon the pK a valuesof its functional groups and on the pH of the surroundingmedium. Altering the charge on amino acids andtheir derivatives by varying the pH facilitates the physicalseparation of amino acids, peptides, and proteins(see Chapter 4).RNHNHC NH 2NH 2RNHRNHC NH 2NH 2NHRNHRNHC NH 2NH 2Figure 3–2. Resonance hybrids of the protonatedforms of the R groups of histidine and arginine.At Its Isoelectric pH (pI), an Amino AcidBears No Net ChargeThe isoelectric species is the form of a molecule thathas an equal number of positive and negative chargesand thus is electrically neutral. The isoelectric pH, alsocalled the pI, is the pH midway between pK a values oneither side of the isoelectric species. For an amino acidsuch as alanine that has only two dissociating groups,there is no ambiguity. The first pK a (R⎯ COOH) is2.35 and the second pK a (R⎯NH + 3 ) is 9.69. The isoelectricpH (pI) of alanine thus ispKpl =For polyfunctional acids, pI is also the pH midway betweenthe pK a values on either side of the isoionicspecies. For example, the pI for aspartic acid ispKpl =1+pK221+pK22For lysine, pI is calculated from:235 . + 969 .== 602 .2209 . + 396 .== 302 .2pK2 + pK3pl =2Similar considerations apply to all polyprotic acids (eg,proteins), regardless of the number of dissociatinggroups present. In the clinical laboratory, knowledge ofthe pI guides selection of conditions for electrophoreticseparations. For example, electrophoresis at pH 7.0 willseparate two molecules with pI values of 6.0 and 8.0because at pH 8.0 the molecule with a pI of 6.0 willhave a net positive charge, and that with pI of 8.0 a netnegative charge. Similar considerations apply to understandingchromatographic separations on ionic supportssuch as DEAE cellulose (see Chapter 4).

18 / CHAPTER 3pK a Values Vary With the EnvironmentThe environment of a dissociable group affects its pK a .The pK a values of the R groups of free amino acids inaqueous solution (Table 3–1) thus provide only an approximateguide to the pK a values of the same aminoacids when present in proteins. A polar environmentfavors the charged form (R⎯ COO − or R⎯NH 3 + ),and a nonpolar environment favors the uncharged form(R⎯COOH or R⎯NH 2 ). A nonpolar environmentthus raises the pK a of a carboxyl group (making it aweaker acid) but lowers that of an amino group (makingit a stronger acid). The presence of adjacent chargedgroups can reinforce or counteract solvent effects. ThepK a of a functional group thus will depend upon its locationwithin a given protein. Variations in pK a can encompasswhole pH units (Table 3–2). pK a values thatdiverge from those listed by as much as three pH unitsare common at the active sites of enzymes. An extremeexample, a buried aspartic acid of thioredoxin, has apK a above 9—a shift of over six pH units!The Solubility and Melting Pointsof Amino Acids ReflectTheir Ionic CharacterThe charged functional groups of amino acids ensurethat they are readily solvated by—and thus soluble in—polar solvents such as water and ethanol but insolublein nonpolar solvents such as benzene, hexane, or ether.Similarly, the high amount of energy required to disruptthe ionic forces that stabilize the crystal latticeaccount for the high melting points of amino acids(> 200 °C).Amino acids do not absorb visible light and thus arecolorless. However, tyrosine, phenylalanine, and especiallytryptophan absorb high-wavelength (250–290nm) ultraviolet light. Tryptophan therefore makes themajor contribution to the ability of most proteins toabsorb light in the region of 280 nm.Table 3–2. Typical range of pK a values forionizable groups in proteins.Dissociating GrouppK a Rangeα-Carboxyl 3.5–4.0Non-α COOH of Asp or Glu 4.0–4.8Imidazole of His 6.5–7.4SH of Cys 8.5–9.0OH of Tyr 9.5–10.5α-Amino 8.0–9.0ε-Amino of Lys 9.8–10.4Guanidinium of Arg ~12.0THE -R GROUPS DETERMINE THEPROPERTIES OF AMINO ACIDSSince glycine, the smallest amino acid, can be accommodatedin places inaccessible to other amino acids, it oftenoccurs where peptides bend sharply. The hydrophobic Rgroups of alanine, valine, leucine, and isoleucine and thearomatic R groups of phenylalanine, tyrosine, and tryptophantypically occur primarily in the interior of cytosolicproteins. The charged R groups of basic andacidic amino acids stabilize specific protein conformationsvia ionic interactions, or salt bonds. These bondsalso function in “charge relay” systems during enzymaticcatalysis and electron transport in respiring mitochondria.Histidine plays unique roles in enzymatic catalysis.The pK a of its imidazole proton permits it to function atneutral pH as either a base or an acid catalyst. The primaryalcohol group of serine and the primary thioalcohol(⎯SH) group of cysteine are excellent nucleophilesand can function as such during enzymatic catalysis.However, the secondary alcohol group of threonine,while a good nucleophile, does not fulfill an analogousrole in catalysis. The ⎯OH groups of serine, tyrosine,and threonine also participate in regulation of the activityof enzymes whose catalytic activity depends on thephosphorylation state of these residues.FUNCTIONAL GROUPS DICTATE THECHEMICAL REACTIONS OF AMINO ACIDSEach functional group of an amino acid exhibits all ofits characteristic chemical reactions. For carboxylic acidgroups, these reactions include the formation of esters,amides, and acid anhydrides; for amino groups, acylation,amidation, and esterification; and for ⎯ OH and⎯ SH groups, oxidation and esterification. The mostimportant reaction of amino acids is the formation of apeptide bond (shaded blue).+ H3 NOAlanylHNOSHAmino Acid Sequence DeterminesPrimary StructureThe number and order of all of the amino acid residuesin a polypeptide constitute its primary structure.Amino acids present in peptides are called aminoacylresidues and are named by replacing the -ate or -ine suffixesof free amino acids with -yl (eg, alanyl, aspartyl, ty-NHCysteinylOO –Valine

AMINO ACIDS & PEPTIDES / 19rosyl). Peptides are then named as derivatives of thecarboxyl terminal aminoacyl residue. For example, Lys-Leu-Tyr-Gln is called lysyl-leucyl-tyrosyl-glutamine.The -ine ending on glutamine indicates that its α-carboxylgroup is not involved in peptide bond formation.CH 2OCNSHCH 2 HCH NC CH 2Peptide Structures Are Easy to DrawPrefixes like tri- or octa- denote peptides with three oreight residues, respectively, not those with three oreight peptide bonds. By convention, peptides are writtenwith the residue that bears the free α-amino groupat the left. To draw a peptide, use a zigzag to representthe main chain or backbone. Add the main chain atoms,which occur in the repeating order: α-nitrogen, α-carbon,carbonyl carbon. Now add a hydrogen atom toeach α-carbon and to each peptide nitrogen, and anoxygen to the carbonyl carbon. Finally, add the appropriateR groups (shaded) to each α-carbon atom.N C C α N CC α N C C αO H 3 C H+ H3 N C CC N CHHCH 2O– OOCHN COO –CH CH 2Three-letter abbreviations linked by straight linesrepresent an unambiguous primary structure. Lines areomitted for single-letter abbreviations.Glu - Ala - Lys - Gly - Tyr - AlaE A K G Y AWhere there is uncertainty about the order of a portionof a polypeptide, the questionable residues are enclosedin brackets and separated by commas.Glu -Lys -( Ala, Gly, Tyr)-His -AlaSome Peptides Contain UnusualAmino AcidsIn mammals, peptide hormones typically contain onlythe α-amino acids of proteins linked by standard peptidebonds. Other peptides may, however, contain nonproteinamino acids, derivatives of the protein aminoacids, or amino acids linked by an atypical peptidebond. For example, the amino terminal glutamate ofglutathione, which participates in protein folding andin the metabolism of xenobiotics (Chapter 53), islinked to cysteine by a non-α peptide bond (Figure3–3). The amino terminal glutamate of thyrotropin-OHHCH H2O COO –+C NH 3COO –Figure 3–3. Glutathione (γ-glutamyl-cysteinylglycine).Note the non-α peptide bond that linksGlu to Cys.releasing hormone (TRH) is cyclized to pyroglutamicacid, and the carboxyl group of the carboxyl terminalprolyl residue is amidated. Peptides elaborated by fungi,bacteria, and lower animals can contain nonproteinamino acids. The antibiotics tyrocidin and gramicidin Sare cyclic polypeptides that contain D-phenylalanineand ornithine. The heptapeptide opioids dermorphinand deltophorin in the skin of South American treefrogs contain D-tyrosine and D-alanine.Peptides Are PolyelectrolytesThe peptide bond is uncharged at any pH of physiologicinterest. Formation of peptides from amino acids istherefore accompanied by a net loss of one positive andone negative charge per peptide bond formed. Peptidesnevertheless are charged at physiologic pH owing to theircarboxyl and amino terminal groups and, where present,their acidic or basic R groups. As for amino acids, the netcharge on a peptide depends on the pH of its environmentand on the pK a values of its dissociating groups.The Peptide Bond Has PartialDouble-Bond CharacterAlthough peptides are written as if a single bond linkedthe α-carboxyl and α-nitrogen atoms, this bond in factexhibits partial double-bond character:OCNHThere thus is no freedom of rotation about the bondthat connects the carbonyl carbon and the nitrogen of apeptide bond. Consequently, all four of the coloredatoms of Figure 3–4 are coplanar. The imposed semirigidityof the peptide bond has important conse-O –CN +H

20 / CHAPTER 3121°OC117°122°120°120°NHR′ H HC110°120°CNCO H R′′0.36 nm0.147 nm0.123 nmOC0.153 nm0.132 nmFigure 3–4. Dimensions of a fully extended polypeptidechain. The four atoms of the peptide bond(colored blue) are coplanar. The unshaded atoms arethe α-carbon atom, the α-hydrogen atom, and the α-Rgroup of the particular amino acid. Free rotation canoccur about the bonds that connect the α-carbon withthe α-nitrogen and with the α-carbonyl carbon (bluearrows). The extended polypeptide chain is thus a semirigidstructure with two-thirds of the atoms of the backboneheld in a fixed planar relationship one to another.The distance between adjacent α-carbon atoms is 0.36nm (3.6 Å). The interatomic distances and bond angles,which are not equivalent, are also shown. (Redrawn andreproduced, with permission, from Pauling L, Corey LP,Branson HR: The structure of proteins: Two hydrogenbondedhelical configurations of the polypeptide chain.Proc Natl Acad Sci U S A 1951;37:205.)quences for higher orders of protein structure. Encirclingarrows (Figure 3 – 4) indicate free rotation aboutthe remaining bonds of the polypeptide backbone.Noncovalent Forces Constrain PeptideConformationsFolding of a peptide probably occurs coincident withits biosynthesis (see Chapter 38). The physiologicallyactive conformation reflects the amino acid sequence,steric hindrance, and noncovalent interactions (eg, hydrogenbonding, hydrophobic interactions) betweenresidues. Common conformations include α-helicesand β-pleated sheets (see Chapter 5).ANALYSIS OF THE AMINO ACIDCONTENT OF BIOLOGIC MATERIALSIn order to determine the identity and quantity of eachamino acid in a sample of biologic material, it is first necessaryto hydrolyze the peptide bonds that link the aminoacids together by treatment with hot HCl. The resulting0.1 nmNHmixture of free amino acids is then treated with 6-amino-N-hydroxysuccinimidyl carbamate, which reacts withtheir α-amino groups, forming fluorescent derivativesthat are then separated and identified using high-pressureliquid chromatography (see Chapter 5). Ninhydrin, alsowidely used for detecting amino acids, forms a purpleproduct with α-amino acids and a yellow adduct withthe imine groups of proline and hydroxyproline.SUMMARY• Both D-amino acids and non-α-amino acids occurin nature, but only L-α-amino acids are present inproteins.• All amino acids possess at least two weakly acidicfunctional groups, R⎯NH+ 3 and R⎯ COOH.Many also possess additional weakly acidic functionalgroups such as ⎯ OH, ⎯SH, guanidino, or imidazolegroups.• The pK a values of all functional groups of an aminoacid dictate its net charge at a given pH. pI is the pHat which an amino acid bears no net charge and thusdoes not move in a direct current electrical field.• Of the biochemical reactions of amino acids, themost important is the formation of peptide bonds.• The R groups of amino acids determine their uniquebiochemical functions. Amino acids are classified asbasic, acidic, aromatic, aliphatic, or sulfur-containingbased on the properties of their R groups.• Peptides are named for the number of amino acidresidues present, and as derivatives of the carboxylterminal residue. The primary structure of a peptideis its amino acid sequence, starting from the aminoterminalresidue.• The partial double-bond character of the bond thatlinks the carbonyl carbon and the nitrogen of a peptiderenders four atoms of the peptide bond coplanarand restricts the number of possible peptide conformations.REFERENCESDoolittle RF: Reconstructing history with amino acid sequences.Protein Sci 1992;1:191.Kreil G: D-Amino acids in animal peptides. Annu Rev Biochem1997;66:337.Nokihara K, Gerhardt J: Development of an improved automatedgas-chromatographic chiral analysis system: application tonon-natural amino acids and natural protein hydrolysates.Chirality 2001;13:431.Sanger F: Sequences, sequences, and sequences. Annu Rev Biochem1988;57:1.Wilson NA et al: Aspartic acid 26 in reduced Escherichia coli thioredoxinhas a pK a greater than 9. Biochemistry 1995;34:8931.

Proteins: Determination ofPrimary Structure 4Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEProteins perform multiple critically important roles. Aninternal protein network, the cytoskeleton (Chapter49), maintains cellular shape and physical integrity.Actin and myosin filaments form the contractile machineryof muscle (Chapter 49). Hemoglobin transportsoxygen (Chapter 6), while circulating antibodiessearch out foreign invaders (Chapter 50). Enzymes catalyzereactions that generate energy, synthesize and degradebiomolecules, replicate and transcribe genes,process mRNAs, etc (Chapter 7). Receptors enable cellsto sense and respond to hormones and other environmentalcues (Chapters 42 and 43). An important goalof molecular medicine is the identification of proteinswhose presence, absence, or deficiency is associatedwith specific physiologic states or diseases. The primarysequence of a protein provides both a molecular fingerprintfor its identification and information that can beused to identify and clone the gene or genes that encodeit.PROTEINS & PEPTIDES MUST BEPURIFIED PRIOR TO ANALYSISHighly purified protein is essential for determination ofits amino acid sequence. Cells contain thousands of differentproteins, each in widely varying amounts. Theisolation of a specific protein in quantities sufficient foranalysis thus presents a formidable challenge that mayrequire multiple successive purification techniques.Classic approaches exploit differences in relative solubilityof individual proteins as a function of pH (isoelectricprecipitation), polarity (precipitation withethanol or acetone), or salt concentration (salting outwith ammonium sulfate). Chromatographic separationspartition molecules between two phases, one mobileand the other stationary. For separation of amino acidsor sugars, the stationary phase, or matrix, may be asheet of filter paper (paper chromatography) or a thinlayer of cellulose, silica, or alumina (thin-layer chromatography;TLC).Column ChromatographyColumn chromatography of proteins employs as thestationary phase a column containing small sphericalbeads of modified cellulose, acrylamide, or silica whosesurface typically has been coated with chemical functionalgroups. These stationary phase matrices interactwith proteins based on their charge, hydrophobicity,and ligand-binding properties. A protein mixture is appliedto the column and the liquid mobile phase is percolatedthrough it. Small portions of the mobile phaseor eluant are collected as they emerge (Figure 4–1).Partition ChromatographyColumn chromatographic separations depend on therelative affinity of different proteins for a given stationaryphase and for the mobile phase. Association betweeneach protein and the matrix is weak and transient.Proteins that interact more strongly with thestationary phase are retained longer. The length of timethat a protein is associated with the stationary phase is afunction of the composition of both the stationary andmobile phases. Optimal separation of the protein of interestfrom other proteins thus can be achieved by carefulmanipulation of the composition of the two phases.Size Exclusion ChromatographySize exclusion—or gel filtration—chromatography separatesproteins based on their Stokes radius, the diameterof the sphere they occupy as they tumble in solution.The Stokes radius is a function of molecular massand shape. A tumbling elongated protein occupies alarger volume than a spherical protein of the same mass.Size exclusion chromatography employs porous beads(Figure 4–2). The pores are analogous to indentationsin a river bank. As objects move downstream, those thatenter an indentation are retarded until they drift backinto the main current. Similarly, proteins with Stokesradii too large to enter the pores (excluded proteins) remainin the flowing mobile phase and emerge beforeproteins that can enter the pores (included proteins).21

22 / CHAPTER 4RCFigure 4–1. Components of a simple liquid chromatography apparatus. R: Reservoirof mobile phase liquid, delivered either by gravity or using a pump. C: Glass orplastic column containing stationary phase. F: Fraction collector for collecting portions,called fractions, of the eluant liquid in separate test tubes.FProteins thus emerge from a gel filtration column in descendingorder of their Stokes radii.Absorption ChromatographyFor absorption chromatography, the protein mixture isapplied to a column under conditions where the proteinof interest associates with the stationary phase sotightly that its partition coefficient is essentially unity.Nonadhering molecules are first eluted and discarded.Proteins are then sequentially released by disrupting theforces that stabilize the protein-stationary phase complex,most often by using a gradient of increasing saltconcentration. The composition of the mobile phase isaltered gradually so that molecules are selectively releasedin descending order of their affinity for the stationaryphase.Ion Exchange ChromatographyIn ion exchange chromatography, proteins interact withthe stationary phase by charge-charge interactions. Proteinswith a net positive charge at a given pH adhere tobeads with negatively charged functional groups such ascarboxylates or sulfates (cation exchangers). Similarly,proteins with a net negative charge adhere to beads withpositively charged functional groups, typically tertiary orquaternary amines (anion exchangers). Proteins, whichare polyanions, compete against monovalent ions forbinding to the support—thus the term “ion exchange.”For example, proteins bind to diethylaminoethyl(DEAE) cellulose by replacing the counter-ions (generallyCl − or CH 3 COO − ) that neutralize the protonatedamine. Bound proteins are selectively displaced by graduallyraising the concentration of monovalent ions in

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE / 23A B CFigure 4–2. Size-exclusion chromatography. A: A mixture of large molecules(diamonds) and small molecules (circles) are applied to the top of a gel filtrationcolumn. B: Upon entering the column, the small molecules enter pores in the stationaryphase matrix from which the large molecules are excluded. C: As the mobilephase flows down the column, the large, excluded molecules flow with itwhile the small molecules, which are temporarily sheltered from the flow when insidethe pores, lag farther and farther behind.the mobile phase. Proteins elute in inverse order of thestrength of their interactions with the stationary phase.Since the net charge on a protein is determined bythe pH (see Chapter 3), sequential elution of proteinsmay be achieved by changing the pH of the mobilephase. Alternatively, a protein can be subjected to consecutiverounds of ion exchange chromatography, eachat a different pH, such that proteins that co-elute at onepH elute at different salt concentrations at another pH.Hydrophobic Interaction ChromatographyHydrophobic interaction chromatography separatesproteins based on their tendency to associate with a stationaryphase matrix coated with hydrophobic groups(eg, phenyl Sepharose, octyl Sepharose). Proteins withexposed hydrophobic surfaces adhere to the matrix viahydrophobic interactions that are enhanced by a mobilephase of high ionic strength. Nonadherent proteins arefirst washed away. The polarity of the mobile phase isthen decreased by gradually lowering the salt concentration.If the interaction between protein and stationaryphase is particularly strong, ethanol or glycerol may beadded to the mobile phase to decrease its polarity andfurther weaken hydrophobic interactions.Affinity ChromatographyAffinity chromatography exploits the high selectivity ofmost proteins for their ligands. Enzymes may be purifiedby affinity chromatography using immobilized substrates,products, coenzymes, or inhibitors. In theory,only proteins that interact with the immobilized ligandadhere. Bound proteins are then eluted either by competitionwith soluble ligand or, less selectively, by disruptingprotein-ligand interactions using urea, guanidinehydrochloride, mildly acidic pH, or high salt concentrations.Stationary phase matrices available commerciallycontain ligands such as NAD + or ATP analogs. Amongthe most powerful and widely applicable affinity matricesare those used for the purification of suitably modifiedrecombinant proteins. These include a Ni 2+ matrixthat binds proteins with an attached polyhistidine “tag”and a glutathione matrix that binds a recombinant proteinlinked to glutathione S-transferase.Peptides Are Purified by Reversed-PhaseHigh-Pressure ChromatographyThe stationary phase matrices used in classic columnchromatography are spongy materials whose compressibilitylimits flow of the mobile phase. High-pressure liquidchromatography (HPLC) employs incompressiblesilica or alumina microbeads as the stationary phase andpressures of up to a few thousand psi. Incompressiblematrices permit both high flow rates and enhanced resolution.HPLC can resolve complex mixtures of lipids orpeptides whose properties differ only slightly. ReversedphaseHPLC exploits a hydrophobic stationary phase of

O24 / CHAPTER 4aliphatic polymers 3–18 carbon atoms in length. Peptidemixtures are eluted using a gradient of a water-miscibleorganic solvent such as acetonitrile or methanol.Protein Purity Is Assessed byPolyacrylamide Gel Electrophoresis(PAGE)The most widely used method for determining the purityof a protein is SDS-PAGE—polyacrylamide gelelectrophoresis (PAGE) in the presence of the anionicdetergent sodium dodecyl sulfate (SDS). Electrophoresisseparates charged biomolecules based on the rates atwhich they migrate in an applied electrical field. ForSDS-PAGE, acrylamide is polymerized and crosslinkedto form a porous matrix. SDS denatures andbinds to proteins at a ratio of one molecule of SDS pertwo peptide bonds. When used in conjunction with 2-mercaptoethanol or dithiothreitol to reduce and breakdisulfide bonds (Figure 4–3), SDS separates the componentpolypeptides of multimeric proteins. The largenumber of anionic SDS molecules, each bearing acharge of −1, on each polypeptide overwhelms thecharge contributions of the amino acid functionalgroups. Since the charge-to-mass ratio of each SDSpolypeptidecomplex is approximately equal, the physicalresistance each peptide encounters as it movesthrough the acrylamide matrix determines the rate ofmigration. Since large complexes encounter greater resistance,polypeptides separate based on their relativemolecular mass (M r ). Individual polypeptides trappedin the acrylamide gel are visualized by staining withdyes such as Coomassie blue (Figure 4–4).Isoelectric Focusing (IEF)Ionic buffers called ampholytes and an applied electricfield are used to generate a pH gradient within a polyacrylamidematrix. Applied proteins migrate until theyreach the region of the matrix where the pH matchestheir isoelectric point (pI), the pH at which a peptide’snet charge is zero. IEF is used in conjunction with SDS-PAGE for two-dimensional electrophoresis, which separatespolypeptides based on pI in one dimension andbased on M r in the second (Figure 4–5). Two-dimensionalelectrophoresis is particularly well suited for separatingthe components of complex mixtures of proteins.SANGER WAS THE FIRST TO DETERMINETHE SEQUENCE OF A POLYPEPTIDEMature insulin consists of the 21-residue A chain andthe 30-residue B chain linked by disulfide bonds. FrederickSanger reduced the disulfide bonds (Figure 4–3),ONHHHNOSSHNHNHOOSHHCOOHC 2 H 5OHOHNNHHSO − 2HSOHNNHOOFigure 4–3. Oxidative cleavage of adjacent polypeptidechains linked by disulfide bonds (shaded) by performicacid (left) or reductive cleavage by β-mercaptoethanol(right) forms two peptides that containcysteic acid residues or cysteinyl residues, respectively.HFigure 4–4. Use of SDS-PAGE to observe successivepurification of a recombinant protein. The gel wasstained with Coomassie blue. Shown are protein standards(lane S) of the indicated mass, crude cell extract(E), high-speed supernatant liquid (H), and the DEAE-Sepharose fraction (D). The recombinant protein has amass of about 45 kDa.

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE / 25pH = 3 pH = 10IEFFigure 4–5. Two-dimensional IEF-SDS-PAGE. Thegel was stained with Coomassie blue. A crude bacterialextract was first subjected to isoelectric focusing(IEF) in a pH 3–10 gradient. The IEF gel was thenplaced horizontally on the top of an SDS gel, and theproteins then further resolved by SDS-PAGE. Noticethe greatly improved resolution of distinct polypeptidesrelative to ordinary SDS-PAGE gel (Figure 4–4).SDSPAGEseparated the A and B chains, and cleaved each chaininto smaller peptides using trypsin, chymotrypsin, andpepsin. The resulting peptides were then isolated andtreated with acid to hydrolyze peptide bonds and generatepeptides with as few as two or three amino acids.Each peptide was reacted with 1-fluoro-2,4-dinitrobenzene(Sanger’s reagent), which derivatizes the exposedα-amino group of amino terminal residues. The aminoacid content of each peptide was then determined.While the ε-amino group of lysine also reacts withSanger’s reagent, amino-terminal lysines can be distinguishedfrom those at other positions because they reactwith 2 mol of Sanger’s reagent. Working backwards tolarger fragments enabled Sanger to determine the completesequence of insulin, an accomplishment for whichhe received a Nobel Prize in 1958.THE EDMAN REACTION ENABLESPEPTIDES & PROTEINSTO BE SEQUENCEDPehr Edman introduced phenylisothiocyanate (Edman’sreagent) to selectively label the amino-terminal residueof a peptide. In contrast to Sanger’s reagent, thephenylthiohydantoin (PTH) derivative can be removedunder mild conditions to generate a new amino terminalresidue (Figure 4–6). Successive rounds of derivatizationwith Edman’s reagent can therefore be used to sequencemany residues of a single sample of peptide. Edman sequencinghas been automated, using a thin film or solidmatrix to immobilize the peptide and HPLC to identifyPTH amino acids. Modern gas-phase sequencers cananalyze as little as a few picomoles of peptide.Large Polypeptides Are First Cleaved IntoSmaller SegmentsWhile the first 20–30 residues of a peptide can readilybe determined by the Edman method, most polypeptidescontain several hundred amino acids. Consequently,most polypeptides must first be cleaved intosmaller peptides prior to Edman sequencing. Cleavagealso may be necessary to circumvent posttranslationalmodifications that render a protein’s α-amino group“blocked”, or unreactive with the Edman reagent.It usually is necessary to generate several peptidesusing more than one method of cleavage. This reflectsboth inconsistency in the spacing of chemically or enzymaticallysusceptible cleavage sites and the need for setsof peptides whose sequences overlap so one can inferthe sequence of the polypeptide from which they derive(Figure 4–7). Reagents for the chemical or enzymaticcleavage of proteins include cyanogen bromide (CNBr),trypsin, and Staphylococcus aureus V8 protease (Table4–1). Following cleavage, the resulting peptides are purifiedby reversed-phase HPLC—or occasionally bySDS-PAGE—and sequenced.MOLECULAR BIOLOGY HASREVOLUTIONIZED THE DETERMINATIONOF PRIMARY STRUCTUREKnowledge of DNA sequences permits deduction ofthe primary structures of polypeptides. DNA sequencingrequires only minute amounts of DNA and canreadily yield the sequence of hundreds of nucleotides.To clone and sequence the DNA that encodes a partic-

26 / CHAPTER 4NHOR′HNONH 2Phenylisothiocyanate (Edman reagent)and a peptideNSC+RPeptide XCarboxyl terminalportion ofpeptide XPeptide ZPeptide YAmino terminalportion ofpeptide YFigure 4–7. The overlapping peptide Z is used to deducethat peptides X and Y are present in the originalprotein in the order X → Y, not Y ← X.ONNHSORR′NHHNSONHA phenylthiohydantoic acidH + , nitromethaneNH+H 2 ONHORRNH 2sequence can be determined and the genetic code usedto infer the primary structure of the encoded polypeptide.The hybrid approach enhances the speed and efficiencyof primary structure analysis and the range ofproteins that can be sequenced. It also circumvents obstaclessuch as the presence of an amino-terminal blockinggroup or the lack of a key overlap peptide. Only afew segments of primary structure must be determinedby Edman analysis.DNA sequencing reveals the order in which aminoacids are added to the nascent polypeptide chain as it issynthesized on the ribosomes. However, it provides noinformation about posttranslational modifications suchas proteolytic processing, methylation, glycosylation,phosphorylation, hydroxylation of proline and lysine,and disulfide bond formation that accompany maturation.While Edman sequencing can detect the presenceof most posttranslational events, technical limitationsoften prevent identification of a specific modification.A phenylthiohydantoin and a peptideshorter by one residueFigure 4–6. The Edman reaction. Phenylisothiocyanatederivatizes the amino-terminal residue of apeptide as a phenylthiohydantoic acid. Treatment withacid in a nonhydroxylic solvent releases a phenylthiohydantoin,which is subsequently identified by its chromatographicmobility, and a peptide one residueshorter. The process is then repeated.ular protein, some means of identifying the correctclone—eg, knowledge of a portion of its nucleotide sequence—isessential. A hybrid approach thus hasemerged. Edman sequencing is used to provide a partialamino acid sequence. Oligonucleotide primers modeledon this partial sequence can then be used to identifyclones or to amplify the appropriate gene by the polymerasechain reaction (PCR) (see Chapter 40). Once anauthentic DNA clone is obtained, its oligonucleotideTable 4–1. Methods for cleaving polypeptides.CNBrTrypsinMethodChymotrypsinEndoproteinase Lys-CEndoproteinase Arg-CEndoproteinase Asp-NV8 proteaseHydroxylamineo-IodosobenzeneMild acidMet-XLys-X and Arg-XBond CleavedHydrophobic amino acid-XLys-XArg-XX-AspGlu-X, particularly where X is hydrophobicAsn-GlyTrp-XAsp-Pro

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE / 27Table 4–2. Mass increases resulting fromcommon posttranslational modifications.ModificationMass Increase (Da)Phosphorylation 80Hydroxylation 16Methylation 14Acetylation 42Myristylation 210Palmitoylation 238Glycosylation 162SAFigure 4–8. Basic components of a simple massspectrometer. A mixture of molecules is vaporized in anionized state in the sample chamber S. These moleculesare then accelerated down the flight tube by anelectrical potential applied to accelerator grid A. An adjustableelectromagnet, E, applies a magnetic field thatdeflects the flight of the individual ions until they strikethe detector, D. The greater the mass of the ion, thehigher the magnetic field required to focus it onto thedetector.MASS SPECTROMETRY DETECTSCOVALENT MODIFICATIONSMass spectrometry, which discriminates moleculesbased solely on their mass, is ideal for detecting thephosphate, hydroxyl, and other groups on posttranslationallymodified amino acids. Each adds a specific andreadily identified increment of mass to the modifiedamino acid (Table 4–2). For analysis by mass spectrometry,a sample in a vacuum is vaporized underconditions where protonation can occur, impartingpositive charge. An electrical field then propels thecations through a magnetic field which deflects themat a right angle to their original direction of flight andfocuses them onto a detector (Figure 4–8). The magneticforce required to deflect the path of each ionicspecies onto the detector, measured as the current appliedto the electromagnet, is recorded. For ions ofidentical net charge, this force is proportionate to theirmass. In a time-of-flight mass spectrometer, a brieflyapplied electric field accelerates the ions towards a detectorthat records the time at which each ion arrives.For molecules of identical charge, the velocity to whichthey are accelerated—and hence the time required toreach the detector—will be inversely proportionate totheir mass.Conventional mass spectrometers generally are usedto determine the masses of molecules of 1000 Da orless, whereas time-of-flight mass spectrometers aresuited for determining the large masses of proteins.The analysis of peptides and proteins by mass spectometryinitially was hindered by difficulties involatilizing large organic molecules. However, matrixassistedlaser-desorption (MALDI) and electrospraydispersion (eg, nanospray) permit the masses of evenlarge polypeptides (> 100,000 Da) to be determinedwith extraordinary accuracy (± 1 Da). Using electrospraydispersion, peptides eluting from a reversedphaseHPLC column are introduced directly into themass spectrometer for immediate determination oftheir masses.Peptides inside the mass spectrometer are brokendown into smaller units by collisions with neutral heliumatoms (collision-induced dissociation), and themasses of the individual fragments are determined.Since peptide bonds are much more labile than carboncarbonbonds, the most abundant fragments will differfrom one another by units equivalent to one or twoamino acids. Since—with the exception of leucine andisoleucine—the molecular mass of each amino acid isunique, the sequence of the peptide can be reconstructedfrom the masses of its fragments.Tandem Mass SpectrometryComplex peptide mixtures can now be analyzed withoutprior purification by tandem mass spectrometry,which employs the equivalent of two mass spectrometerslinked in series. The first spectrometer separates individualpeptides based upon their differences in mass.By adjusting the field strength of the first magnet, a singlepeptide can be directed into the second mass spectrometer,where fragments are generated and theirmasses determined. As the sensitivity and versatility ofmass spectrometry continue to increase, it is displacingEdman sequencers for the direct analysis of protein primarystructure.ED

28 / CHAPTER 4GENOMICS ENABLES PROTEINS TO BEIDENTIFIED FROM SMALL AMOUNTSOF SEQUENCE DATAPrimary structure analysis has been revolutionized bygenomics, the application of automated oligonucleotidesequencing and computerized data retrieval and analysisto sequence an organism’s entire genetic complement.The first genome sequenced was that of Haemophilusinfluenzae, in 1995. By mid 2001, the completegenome sequences for over 50 organisms had been determined.These include the human genome and thoseof several bacterial pathogens; the results and significanceof the Human Genome Project are discussed inChapter 54. Where genome sequence is known, thetask of determining a protein’s DNA-derived primarysequence is materially simplified. In essence, the secondhalf of the hybrid approach has already been completed.All that remains is to acquire sufficient informationto permit the open reading frame (ORF) thatencodes the protein to be retrieved from an Internetaccessiblegenome database and identified. In somecases, a segment of amino acid sequence only four orfive residues in length may be sufficient to identify thecorrect ORF.Computerized search algorithms assist the identificationof the gene encoding a given protein and clarifyuncertainties that arise from Edman sequencing andmass spectrometry. By exploiting computers to solvecomplex puzzles, the spectrum of information suitablefor identification of the ORF that encodes a particularpolypeptide is greatly expanded. In peptide mass profiling,for example, a peptide digest is introduced into themass spectrometer and the sizes of the peptides are determined.A computer is then used to find an ORFwhose predicted protein product would, if brokendown into peptides by the cleavage method selected,produce a set of peptides whose masses match those observedby mass spectrometry.PROTEOMICS & THE PROTEOMEThe Goal of Proteomics Is to Identify theEntire Complement of Proteins Elaboratedby a Cell Under Diverse ConditionsWhile the sequence of the human genome is known,the picture provided by genomics alone is both staticand incomplete. Proteomics aims to identify the entirecomplement of proteins elaborated by a cell under diverseconditions. As genes are switched on and off, proteinsare synthesized in particular cell types at specifictimes of growth or differentiation and in response toexternal stimuli. Muscle cells express proteins not expressedby neural cells, and the type of subunits presentin the hemoglobin tetramer undergo change pre- andpostpartum. Many proteins undergo posttranslationalmodifications during maturation into functionallycompetent forms or as a means of regulating their properties.Knowledge of the human genome therefore representsonly the beginning of the task of describing livingorganisms in molecular detail and understandingthe dynamics of processes such as growth, aging, anddisease. As the human body contains thousands of celltypes, each containing thousands of proteins, the proteome—theset of all the proteins expressed by an individualcell at a particular time—represents a movingtarget of formidable dimensions.Two-Dimensional Electrophoresis &Gene Array Chips Are Used to SurveyProtein ExpressionOne goal of proteomics is the identification of proteinswhose levels of expression correlate with medically significantevents. The presumption is that proteins whoseappearance or disappearance is associated with a specificphysiologic condition or disease will provide insightsinto root causes and mechanisms. Determination of theproteomes characteristic of each cell type requires theutmost efficiency in the isolation and identification ofindividual proteins. The contemporary approach utilizesrobotic automation to speed sample preparationand large two-dimensional gels to resolve cellular proteins.Individual polypeptides are then extracted andanalyzed by Edman sequencing or mass spectroscopy.While only about 1000 proteins can be resolved on asingle gel, two-dimensional electrophoresis has a majoradvantage in that it examines the proteins themselves.An alternative and complementary approach employsgene arrays, sometimes called DNA chips, to detect theexpression of the mRNAs which encode proteins.While changes in the expression of the mRNA encodinga protein do not necessarily reflect comparablechanges in the level of the corresponding protein, genearrays are more sensitive probes than two-dimensionalgels and thus can examine more gene products.Bioinformatics Assists Identificationof Protein FunctionsThe functions of a large proportion of the proteins encodedby the human genome are presently unknown.Recent advances in bioinformatics permit researchers tocompare amino acid sequences to discover clues to potentialproperties, physiologic roles, and mechanisms ofaction of proteins. Algorithms exploit the tendency ofnature to employ variations of a structural theme toperform similar functions in several proteins (eg, theRossmann nucleotide binding fold to bind NAD(P)H,

PROTEINS: DETERMINATION OF PRIMARY STRUCTURE / 29nuclear targeting sequences, and EF hands to bindCa 2+ ). These domains generally are detected in the primarystructure by conservation of particular aminoacids at key positions. Insights into the properties andphysiologic role of a newly discovered protein thus maybe inferred by comparing its primary structure withthat of known proteins.SUMMARY• Long amino acid polymers or polypeptides constitutethe basic structural unit of proteins, and the structureof a protein provides insight into how it fulfills itsfunctions.• The Edman reaction enabled amino acid sequenceanalysis to be automated. Mass spectrometry providesa sensitive and versatile tool for determiningprimary structure and for the identification of posttranslationalmodifications.• DNA cloning and molecular biology coupled withprotein chemistry provide a hybrid approach thatgreatly increases the speed and efficiency for determinationof primary structures of proteins.• Genomics—the analysis of the entire oligonucleotidesequence of an organism’s complete genetic material—hasprovided further enhancements.• Computer algorithms facilitate identification of theopen reading frames that encode a given protein byusing partial sequences and peptide mass profiling tosearch sequence databases.• Scientists are now trying to determine the primarysequence and functional role of every protein expressedin a living cell, known as its proteome.• A major goal is the identification of proteins whoseappearance or disappearance correlates with physiologicphenomena, aging, or specific diseases.REFERENCESDeutscher MP (editor): Guide to Protein Purification. Methods Enzymol1990;182. (Entire volume.)Geveart K, Vandekerckhove J: Protein identification methods inproteomics. Electrophoresis 2000;21:1145.Helmuth L: Genome research: map of the human genome 3.0. Science2001;293:583.Khan J et al: DNA microarray technology: the anticipated impacton the study of human disease. Biochim Biophys Acta1999;1423:M17.McLafferty FW et al: Biomolecule mass spectrometry. Science1999;284:1289.Patnaik SK, Blumenfeld OO: Use of on-line tools and databases forroutine sequence analyses. Anal Biochem 2001;289:1.Schena M et al: Quantitative monitoring of gene expression patternswith a complementary DNA microarray. Science1995;270:467.Semsarian C, Seidman CE: Molecular medicine in the 21st century.Intern Med J 2001;31:53.Temple LK et al: Essays on science and society: defining disease inthe genomics era. Science 2001;293:807.Wilkins MR et al: High-throughput mass spectrometric discoveryof protein post-translational modifications. J Mol Biol1999;289:645.

Proteins: Higher Orders of Structure 5Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEProteins catalyze metabolic reactions, power cellularmotion, and form macromolecular rods and cables thatprovide structural integrity to hair, bones, tendons, andteeth. In nature, form follows function. The structuralvariety of human proteins therefore reflects the sophisticationand diversity of their biologic roles. Maturationof a newly synthesized polypeptide into a biologicallyfunctional protein requires that it be folded into a specificthree-dimensional arrangement, or conformation.During maturation, posttranslational modificationsmay add new chemical groups or remove transientlyneeded peptide segments. Genetic or nutritional deficienciesthat impede protein maturation are deleteriousto health. Examples of the former include Creutzfeldt-Jakob disease, scrapie, Alzheimer’s disease, and bovinespongiform encephalopathy (mad cow disease). Scurvyrepresents a nutritional deficiency that impairs proteinmaturation.CONFORMATION VERSUSCONFIGURATIONThe terms configuration and conformation are oftenconfused. Configuration refers to the geometric relationshipbetween a given set of atoms, for example,those that distinguish L- from D-amino acids. Interconversionof configurational alternatives requires breakingcovalent bonds. Conformation refers to the spatial relationshipof every atom in a molecule. Interconversionbetween conformers occurs without covalent bond rupture,with retention of configuration, and typically viarotation about single bonds.PROTEINS WERE INITIALLY CLASSIFIEDBY THEIR GROSS CHARACTERISTICSScientists initially approached structure-function relationshipsin proteins by separating them into classesbased upon properties such as solubility, shape, or thepresence of nonprotein groups. For example, the proteinsthat can be extracted from cells using solutions atphysiologic pH and ionic strength are classified as soluble.Extraction of integral membrane proteins requiresdissolution of the membrane with detergents.30Globular proteins are compact, are roughly sphericalor ovoid in shape, and have axial ratios (the ratio oftheir shortest to longest dimensions) of not over 3.Most enzymes are globular proteins, whose large internalvolume provides ample space in which to constructcavities of the specific shape, charge, and hydrophobicityor hydrophilicity required to bindsubstrates and promote catalysis. By contrast, manystructural proteins adopt highly extended conformations.These fibrous proteins possess axial ratios of 10or more.Lipoproteins and glycoproteins contain covalentlybound lipid and carbohydrate, respectively. Myoglobin,hemoglobin, cytochromes, and many other proteinscontain tightly associated metal ions and are termedmetalloproteins. With the development and applicationof techniques for determining the amino acid sequencesof proteins (Chapter 4), more precise classificationschemes have emerged based upon similarity, orhomology, in amino acid sequence and structure.However, many early classification terms remain incommon use.PROTEINS ARE CONSTRUCTED USINGMODULAR PRINCIPLESProteins perform complex physical and catalytic functionsby positioning specific chemical groups in a precisethree-dimensional arrangement. The polypeptidescaffold containing these groups must adopt a conformationthat is both functionally efficient and physicallystrong. At first glance, the biosynthesis ofpolypeptides comprised of tens of thousands of individualatoms would appear to be extremely challenging.When one considers that a typical polypeptidecan adopt ≥ 10 50 distinct conformations, folding intothe conformation appropriate to their biologic functionwould appear to be even more difficult. As describedin Chapters 3 and 4, synthesis of the polypeptidebackbones of proteins employs a small set ofcommon building blocks or modules, the amino acids,joined by a common linkage, the peptide bond. Astepwise modular pathway simplifies the folding andprocessing of newly synthesized polypeptides into matureproteins.

PROTEINS: HIGHER ORDERS OF STRUCTURE / 31THE FOUR ORDERS OFPROTEIN STRUCTUREThe modular nature of protein synthesis and foldingare embodied in the concept of orders of protein structure:primary structure, the sequence of the aminoacids in a polypeptide chain; secondary structure, thefolding of short (3- to 30-residue), contiguous segmentsof polypeptide into geometrically ordered units; tertiarystructure, the three-dimensional assembly of secondarystructural units to form larger functional unitssuch as the mature polypeptide and its component domains;and quaternary structure, the number andtypes of polypeptide units of oligomeric proteins andtheir spatial arrangement.SECONDARY STRUCTUREPeptide Bonds Restrict PossibleSecondary ConformationsFree rotation is possible about only two of the three covalentbonds of the polypeptide backbone: the α-carbon(Cα) to the carbonyl carbon (Co) bond and theCα to nitrogen bond (Figure 3–4). The partial doublebondcharacter of the peptide bond that links Co to theα-nitrogen requires that the carbonyl carbon, carbonyloxygen, and α-nitrogen remain coplanar, thus preventingrotation. The angle about the Cα⎯N bond istermed the phi (Φ) angle, and that about the Co⎯Cαbond the psi (Ψ) angle. For amino acids other thanglycine, most combinations of phi and psi angles aredisallowed because of steric hindrance (Figure 5–1).The conformations of proline are even more restricteddue to the absence of free rotation of the N⎯Cα bond.Regions of ordered secondary structure arise when aseries of aminoacyl residues adopt similar phi and psiangles. Extended segments of polypeptide (eg, loops)can possess a variety of such angles. The angles that definethe two most common types of secondary structure,the helix and the sheet, fall within the lowerand upper left-hand quadrants of a Ramachandranplot, respectively (Figure 5–1).The Alpha HelixThe polypeptide backbone of an α helix is twisted byan equal amount about each α-carbon with a phi angleof approximately −57 degrees and a psi angle of approximately−47 degrees. A complete turn of the helix containsan average of 3.6 aminoacyl residues, and the distanceit rises per turn (its pitch) is 0.54 nm (Figure5–2). The R groups of each aminoacyl residue in an αhelix face outward (Figure 5–3). Proteins contain onlyL-amino acids, for which a right-handed α helix is byfar the more stable, and only right-handed α helicesψ900– 90– 90 090φFigure 5–1. Ramachandran plot of the main chainphi (Φ) and psi (Ψ) angles for approximately 1000nonglycine residues in eight proteins whose structureswere solved at high resolution. The dots represent allowablecombinations and the spaces prohibited combinationsof phi and psi angles. (Reproduced, with permission,from Richardson JS: The anatomy and taxonomyof protein structures. Adv Protein Chem 1981;34:167.)occur in nature. Schematic diagrams of proteins representα helices as cylinders.The stability of an α helix arises primarily from hydrogenbonds formed between the oxygen of the peptidebond carbonyl and the hydrogen atom of the peptidebond nitrogen of the fourth residue down thepolypeptide chain (Figure 5–4). The ability to form themaximum number of hydrogen bonds, supplementedby van der Waals interactions in the core of this tightlypacked structure, provides the thermodynamic drivingforce for the formation of an α helix. Since the peptidebond nitrogen of proline lacks a hydrogen atom to contributeto a hydrogen bond, proline can only be stablyaccommodated within the first turn of an α helix.When present elsewhere, proline disrupts the conformationof the helix, producing a bend. Because of itssmall size, glycine also often induces bends in α helices.Many α helices have predominantly hydrophobic Rgroups on one side of the axis of the helix and predominantlyhydrophilic ones on the other. These amphipathichelices are well adapted to the formation of interfacesbetween polar and nonpolar regions such as thehydrophobic interior of a protein and its aqueous envi-

32 / CHAPTER 50.54-nm pitch(3.6 residues)NCCNCNCCCCCCNNNCNCCCCNCNCNCCCCNC0.15 nmFigure 5–2. Orientation of the main chain atoms of apeptide about the axis of an α helix.ronment. Clusters of amphipathic helices can create achannel, or pore, that permits specific polar moleculesto pass through hydrophobic cell membranes.The Beta SheetThe second (hence “beta”) recognizable regular secondarystructure in proteins is the β sheet. The aminoacid residues of a β sheet, when viewed edge-on, form azigzag or pleated pattern in which the R groups of adjacentresidues point in opposite directions. Unlike thecompact backbone of the α helix, the peptide backboneof the β sheet is highly extended. But like the α helix,β sheets derive much of their stability from hydrogenbonds between the carbonyl oxygens and amide hydrogensof peptide bonds. However, in contrast to the αhelix, these bonds are formed with adjacent segments ofβ sheet (Figure 5–5).Interacting β sheets can be arranged either to form aparallel β sheet, in which the adjacent segments of theRRRRFigure 5–3. View down the axis of an α helix. Theside chains (R) are on the outside of the helix. The vander Waals radii of the atoms are larger than shown here;hence, there is almost no free space inside the helix.(Slightly modified and reproduced, with permission, fromStryer L: Biochemistry, 3rd ed. Freeman, 1995. Copyright© 1995 by W.H. Freeman and Co.)polypeptide chain proceed in the same direction aminoto carboxyl, or an antiparallel sheet, in which they proceedin opposite directions (Figure 5–5). Either configurationpermits the maximum number of hydrogenbonds between segments, or strands, of the sheet. Mostβ sheets are not perfectly flat but tend to have a righthandedtwist. Clusters of twisted strands of β sheetform the core of many globular proteins (Figure 5–6).Schematic diagrams represent β sheets as arrows thatpoint in the amino to carboxyl terminal direction.Loops & BendsRoughly half of the residues in a “typical” globular proteinreside in α helices and β sheets and half in loops,turns, bends, and other extended conformational features.Turns and bends refer to short segments ofamino acids that join two units of secondary structure,such as two adjacent strands of an antiparallel β sheet.A β turn involves four aminoacyl residues, in which thefirst residue is hydrogen-bonded to the fourth, resultingin a tight 180-degree turn (Figure 5–7). Proline andglycine often are present in β turns.Loops are regions that contain residues beyond theminimum number necessary to connect adjacent regionsof secondary structure. Irregular in conformation,loops nevertheless serve key biologic roles. For manyenzymes, the loops that bridge domains responsible forbinding substrates often contain aminoacyl residuesRRRRR

PROTEINS: HIGHER ORDERS OF STRUCTURE / 33RCCCCNNNRCCCNNRCRNCRCCRCRCNR CCRCCNNN O CCRCNC RCFigure 5–4. Hydrogen bonds (dotted lines) formedbetween H and O atoms stabilize a polypeptide in anα-helical conformation. (Reprinted, with permission,from Haggis GH et al: Introduction to Molecular Biology.Wiley, 1964.)Figure 5–5. Spacing and bond angles of the hydrogenbonds of antiparallel and parallel pleated β sheets.Arrows indicate the direction of each strand. The hydrogen-donatingα-nitrogen atoms are shown as blue circles.Hydrogen bonds are indicated by dotted lines. Forclarity in presentation, R groups and hydrogens areomitted. Top: Antiparallel β sheet. Pairs of hydrogenbonds alternate between being close together andwide apart and are oriented approximately perpendicularto the polypeptide backbone. Bottom: Parallel βsheet. The hydrogen bonds are evenly spaced but slantin alternate directions.that participate in catalysis. Helix-loop-helix motifsprovide the oligonucleotide-binding portion of DNAbindingproteins such as repressors and transcriptionfactors. Structural motifs such as the helix-loop-helixmotif that are intermediate between secondary and tertiarystructures are often termed supersecondary structures.Since many loops and bends reside on the surfaceof proteins and are thus exposed to solvent, they constitutereadily accessible sites, or epitopes, for recognitionand binding of antibodies.While loops lack apparent structural regularity, theyexist in a specific conformation stabilized through hydrogenbonding, salt bridges, and hydrophobic interactionswith other portions of the protein. However, notall portions of proteins are necessarily ordered. Proteinsmay contain “disordered” regions, often at the extremeamino or carboxyl terminal, characterized by high conformationalflexibility. In many instances, these disorderedregions assume an ordered conformation uponbinding of a ligand. This structural flexibility enablessuch regions to act as ligand-controlled switches that affectprotein structure and function.Tertiary & Quaternary StructureThe term “tertiary structure” refers to the entire threedimensionalconformation of a polypeptide. It indicates,in three-dimensional space, how secondary structuralfeatures—helices, sheets, bends, turns, and loops—assemble to form domains and how these domains relatespatially to one another. A domain is a section ofprotein structure sufficient to perform a particularchemical or physical task such as binding of a substrate

34 / CHAPTER 5COOHHHC αCHNHHNOC αC αCCH 2CH 2 OHOCH 3COHNHC αHFigure 5–7. A β-turn that links two segments of antiparallelβ sheet. The dotted line indicates the hydrogenbond between the first and fourth amino acids ofthe four-residue segment Ala-Gly-Asp-Ser.1452053015018555N50345 80 7033028090350C230377245310320110220260120125170Figure 5–6. Examples of tertiary structure of proteins.Top: The enzyme triose phosphate isomerase.Note the elegant and symmetrical arrangement of alternatingβ sheets and α helices. (Courtesy of J Richardson.)Bottom: Two-domain structure of the subunit of ahomodimeric enzyme, a bacterial class II HMG-CoA reductase.As indicated by the numbered residues, thesingle polypeptide begins in the large domain, entersthe small domain, and ends in the large domain. (Courtesyof C Lawrence, V Rodwell, and C Stauffacher, PurdueUniversity.)30015258or other ligand. Other domains may anchor a protein toa membrane or interact with a regulatory molecule thatmodulates its function. A small polypeptide such astriose phosphate isomerase (Figure 5–6) or myoglobin(Chapter 6) may consist of a single domain. By contrast,protein kinases contain two domains. Protein kinasescatalyze the transfer of a phosphoryl group from ATP toa peptide or protein. The amino terminal portion of thepolypeptide, which is rich in β sheet, binds ATP, whilethe carboxyl terminal domain, which is rich in α helix,binds the peptide or protein substrate (Figure 5–8). Thegroups that catalyze phosphoryl transfer reside in a looppositioned at the interface of the two domains.In some cases, proteins are assembled from morethan one polypeptide, or protomer. Quaternary structuredefines the polypeptide composition of a proteinand, for an oligomeric protein, the spatial relationshipsbetween its subunits or protomers. Monomeric proteinsconsist of a single polypeptide chain. Dimericproteins contain two polypeptide chains. Homodimerscontain two copies of the same polypeptide chain,while in a heterodimer the polypeptides differ. Greekletters (α, β, γ etc) are used to distinguish different subunitsof a heterooligomeric protein, and subscripts indicatethe number of each subunit type. For example, α 4designates a homotetrameric protein, and α 2 β 2 γ a proteinwith five subunits of three different types.Since even small proteins contain many thousandsof atoms, depictions of protein structure that indicatethe position of every atom are generally too complex tobe readily interpreted. Simplified schematic diagramsthus are used to depict key features of a protein’s ter-

PROTEINS: HIGHER ORDERS OF STRUCTURE / 35from water. Other significant contributors include hydrogenbonds and salt bridges between the carboxylatesof aspartic and glutamic acid and the oppositelycharged side chains of protonated lysyl, argininyl, andhistidyl residues. While individually weak relative to atypical covalent bond of 80–120 kcal/mol, collectivelythese numerous interactions confer a high degree of stabilityto the biologically functional conformation of aprotein, just as a Velcro fastener harnesses the cumulativestrength of multiple plastic loops and hooks.Some proteins contain covalent disulfide (S⎯S)bonds that link the sulfhydryl groups of cysteinylresidues. Formation of disulfide bonds involves oxidationof the cysteinyl sulfhydryl groups and requires oxygen.Intrapolypeptide disulfide bonds further enhancethe stability of the folded conformation of a peptide,while interpolypeptide disulfide bonds stabilize thequaternary structure of certain oligomeric proteins.Figure 5–8. Domain structure. Protein kinases containtwo domains. The upper, amino terminal domainbinds the phosphoryl donor ATP (light blue). The lower,carboxyl terminal domain is shown binding a syntheticpeptide substrate (dark blue).tiary and quaternary structure. Ribbon diagrams (Figures5–6 and 5–8) trace the conformation of thepolypeptide backbone, with cylinders and arrows indicatingregions of α helix and β sheet, respectively. In aneven simpler representation, line segments that link theα carbons indicate the path of the polypeptide backbone.These schematic diagrams often include the sidechains of selected amino acids that emphasize specificstructure-function relationships.MULTIPLE FACTORS STABILIZETERTIARY & QUATERNARY STRUCTUREHigher orders of protein structure are stabilized primarily—andoften exclusively—by noncovalent interactions.Principal among these are hydrophobic interactionsthat drive most hydrophobic amino acid sidechains into the interior of the protein, shielding themTHREE-DIMENSIONAL STRUCTUREIS DETERMINED BY X-RAYCRYSTALLOGRAPHY OR BYNMR SPECTROSCOPYX-Ray CrystallographySince the determination of the three-dimensional structureof myoglobin over 40 years ago, the three-dimensionalstructures of thousands of proteins have been determinedby x-ray crystallography. The key to x-raycrystallography is the precipitation of a protein underconditions in which it forms ordered crystals that diffractx-rays. This is generally accomplished by exposingsmall drops of the protein solution to various combinationsof pH and precipitating agents such as salts andorganic solutes such as polyethylene glycol. A detailedthree-dimensional structure of a protein can be constructedfrom its primary structure using the pattern bywhich it diffracts a beam of monochromatic x-rays.While the development of increasingly capable computer-basedtools has rendered the analysis of complexx-ray diffraction patterns increasingly facile, a majorstumbling block remains the requirement of inducinghighly purified samples of the protein of interest tocrystallize. Several lines of evidence, including the abilityof some crystallized enzymes to catalyze chemical reactions,indicate that the vast majority of the structuresdetermined by crystallography faithfully represent thestructures of proteins in free solution.Nuclear Magnetic ResonanceSpectroscopyNuclear magnetic resonance (NMR) spectroscopy, apowerful complement to x-ray crystallography, mea-

36 / CHAPTER 5sures the absorbance of radio frequency electromagneticenergy by certain atomic nuclei. “NMR-active” isotopesof biologically relevant atoms include 1 H, 13 C, 15 N, and31 P. The frequency, or chemical shift, at which a particularnucleus absorbs energy is a function of both thefunctional group within which it resides and the proximityof other NMR-active nuclei. Two-dimensionalNMR spectroscopy permits a three-dimensional representationof a protein to be constructed by determiningthe proximity of these nuclei to one another. NMRspectroscopy analyzes proteins in aqueous solution, obviatingthe need to form crystals. It thus is possible toobserve changes in conformation that accompany ligandbinding or catalysis using NMR spectroscopy.However, only the spectra of relatively small proteins,≤ 20 kDa in size, can be analyzed with current technology.Molecular ModelingAn increasingly useful adjunct to the empirical determinationof the three-dimensional structure of proteins isthe use of computer technology for molecular modeling.The types of models created take two forms. In thefirst, the known three-dimensional structure of a proteinis used as a template to build a model of the probablestructure of a homologous protein. In the second,computer software is used to manipulate the staticmodel provided by crystallography to explore how aprotein’s conformation might change when ligands arebound or when temperature, pH, or ionic strength isaltered. Scientists also are examining the library ofavailable protein structures in an attempt to devisecomputer programs that can predict the three-dimensionalconformation of a protein directly from its primarysequence.PROTEIN FOLDINGThe Native Conformation of a ProteinIs Thermodynamically FavoredThe number of distinct combinations of phi and psiangles specifying potential conformations of even a relativelysmall—15-kDa—polypeptide is unbelievablyvast. Proteins are guided through this vast labyrinth ofpossibilities by thermodynamics. Since the biologicallyrelevant—or native—conformation of a protein generallyis that which is most energetically favored, knowledgeof the native conformation is specified in the primarysequence. However, if one were to wait for apolypeptide to find its native conformation by randomexploration of all possible conformations, the processwould require billions of years to complete. Clearly,protein folding in cells takes place in a more orderlyand guided fashion.Folding Is ModularProtein folding generally occurs via a stepwise process.In the first stage, the newly synthesized polypeptideemerges from ribosomes, and short segments fold intosecondary structural units that provide local regions oforganized structure. Folding is now reduced to the selectionof an appropriate arrangement of this relativelysmall number of secondary structural elements. In thesecond stage, the forces that drive hydrophobic regionsinto the interior of the protein away from solvent drivethe partially folded polypeptide into a “molten globule”in which the modules of secondary structure rearrangeto arrive at the mature conformation of the protein.This process is orderly but not rigid. Considerable flexibilityexists in the ways and in the order in which elementsof secondary structure can be rearranged. In general,each element of secondary or supersecondarystructure facilitates proper folding by directing the foldingprocess toward the native conformation and awayfrom unproductive alternatives. For oligomeric proteins,individual protomers tend to fold before they associatewith other subunits.Auxiliary Proteins Assist FoldingUnder appropriate conditions, many proteins willspontaneously refold after being previously denatured(ie, unfolded) by treatment with acid or base,chaotropic agents, or detergents. However, unlike thefolding process in vivo, refolding under laboratory conditionsis a far slower process. Moreover, some proteinsfail to spontaneously refold in vitro, often forming insolubleaggregates, disordered complexes of unfoldedor partially folded polypeptides held together by hydrophobicinteractions. Aggregates represent unproductivedead ends in the folding process. Cells employ auxiliaryproteins to speed the process of folding and toguide it toward a productive conclusion.ChaperonesChaperone proteins participate in the folding of overhalf of mammalian proteins. The hsp70 (70-kDa heatshock protein) family of chaperones binds short sequencesof hydrophobic amino acids in newly synthesizedpolypeptides, shielding them from solvent.Chaperones prevent aggregation, thus providing an opportunityfor the formation of appropriate secondarystructural elements and their subsequent coalescenceinto a molten globule. The hsp60 family of chaperones,sometimes called chaperonins, differ in sequence andstructure from hsp70 and its homologs. Hsp60 actslater in the folding process, often together with anhsp70 chaperone. The central cavity of the donut-

PROTEINS: HIGHER ORDERS OF STRUCTURE / 37shaped hsp60 chaperone provides a sheltered environmentin which a polypeptide can fold until all hydrophobicregions are buried in its interior, eliminatingaggregation. Chaperone proteins can also “rescue” proteinsthat have become thermodynamically trapped in amisfolded dead end by unfolding hydrophobic regionsand providing a second chance to fold productively.Protein Disulfide IsomeraseDisulfide bonds between and within polypeptides stabilizetertiary and quaternary structure. However, disulfidebond formation is nonspecific. Under oxidizingconditions, a given cysteine can form a disulfide bondwith the ⎯SH of any accessible cysteinyl residue. Bycatalyzing disulfide exchange, the rupture of an S⎯Sbond and its reformation with a different partner cysteine,protein disulfide isomerase facilitates the formationof disulfide bonds that stabilize their native conformation.Proline-cis,trans-IsomeraseAll X-Pro peptide bonds—where X represents anyresidue—are synthesized in the trans configuration.However, of the X-Pro bonds of mature proteins, approximately6% are cis. The cis configuration is particularlycommon in β-turns. Isomerization from trans tocis is catalyzed by the enzyme proline-cis,trans-isomerase(Figure 5–9).SEVERAL NEUROLOGIC DISEASESRESULT FROM ALTERED PROTEINCONFORMATIONPrionsThe transmissible spongiform encephalopathies, orprion diseases, are fatal neurodegenerative diseasescharacterized by spongiform changes, astrocytic gliomas,and neuronal loss resulting from the depositionof insoluble protein aggregates in neural cells. They includeCreutzfeldt-Jakob disease in humans, scrapie inOO OHH α 1 ′NNα 1 Nα 1 NR 1 α 1 ′R 1OFigure 5–9. Isomerization of the N-α 1 prolyl peptidebond from a cis to a trans configuration relative to thebackbone of the polypeptide.sheep, and bovine spongiform encephalopathy (madcow disease) in cattle. Prion diseases may manifestthemselves as infectious, genetic, or sporadic disorders.Because no viral or bacterial gene encoding the pathologicprion protein could be identified, the source andmechanism of transmission of prion disease long remainedelusive. Today it is believed that prion diseasesare protein conformation diseases transmitted by alteringthe conformation, and hence the physical properties,of proteins endogenous to the host. Human prionrelatedprotein, PrP, a glycoprotein encoded on theshort arm of chromosome 20, normally is monomericand rich in α helix. Pathologic prion proteins serve asthe templates for the conformational transformation ofnormal PrP, known as PrPc, into PrPsc. PrPsc is rich inβ sheet with many hydrophobic aminoacyl side chainsexposed to solvent. PrPsc molecules therefore associatestrongly with one other, forming insoluble protease-resistantaggregates. Since one pathologic prion or prionrelatedprotein can serve as template for the conformationaltransformation of many times its number of PrPcmolecules, prion diseases can be transmitted by the proteinalone without involvement of DNA or RNA.Alzheimer’s DiseaseRefolding or misfolding of another protein endogenousto human brain tissue, β-amyloid, is also a prominentfeature of Alzheimer’s disease. While the root cause ofAlzheimer’s disease remains elusive, the characteristicsenile plaques and neurofibrillary bundles contain aggregatesof the protein β-amyloid, a 4.3-kDa polypeptideproduced by proteolytic cleavage of a larger proteinknown as amyloid precursor protein. In Alzheimer’sdisease patients, levels of β-amyloid become elevated,and this protein undergoes a conformational transformationfrom a soluble α helix–rich state to a state richin β sheet and prone to self-aggregation. ApolipoproteinE has been implicated as a potential mediator ofthis conformational transformation.COLLAGEN ILLUSTRATES THE ROLE OFPOSTTRANSLATIONAL PROCESSING INPROTEIN MATURATIONProtein Maturation Often Involves Making& Breaking Covalent BondsThe maturation of proteins into their final structuralstate often involves the cleavage or formation (or both)of covalent bonds, a process termed posttranslationalmodification. Many polypeptides are initially synthesizedas larger precursors, called proproteins. The“extra” polypeptide segments in these proproteinsoften serve as leader sequences that target a polypeptide

38 / CHAPTER 5to a particular organelle or facilitate its passage througha membrane. Others ensure that the potentially harmfulactivity of a protein such as the proteases trypsinand chymotrypsin remains inhibited until these proteinsreach their final destination. However, once thesetransient requirements are fulfilled, the now superfluouspeptide regions are removed by selective proteolysis.Other covalent modifications may take place thatadd new chemical functionalities to a protein. The maturationof collagen illustrates both of these processes.Collagen Is a Fibrous ProteinCollagen is the most abundant of the fibrous proteinsthat constitute more than 25% of the protein mass inthe human body. Other prominent fibrous proteins includekeratin and myosin. These proteins represent aprimary source of structural strength for cells (ie, thecytoskeleton) and tissues. Skin derives its strength andflexibility from a crisscrossed mesh of collagen and keratinfibers, while bones and teeth are buttressed by anunderlying network of collagen fibers analogous to thesteel strands in reinforced concrete. Collagen also ispresent in connective tissues such as ligaments and tendons.The high degree of tensile strength required tofulfill these structural roles requires elongated proteinscharacterized by repetitive amino acid sequences and aregular secondary structure.Collagen Forms a Unique Triple HelixTropocollagen consists of three fibers, each containingabout 1000 amino acids, bundled together in a uniqueconformation, the collagen triple helix (Figure 5–10). Amature collagen fiber forms an elongated rod with anaxial ratio of about 200. Three intertwined polypeptidestrands, which twist to the left, wrap around one anotherin a right-handed fashion to form the collagentriple helix. The opposing handedness of this superhelixand its component polypeptides makes the collagentriple helix highly resistant to unwinding—the sameprinciple used in the steel cables of suspension bridges.A collagen triple helix has 3.3 residues per turn and aAmino acidsequence – Gly – X – Y – Gly – X – Y – Gly – X – Y –2º structureTriple helixFigure 5–10. Primary, secondary, and tertiary structuresof collagen.rise per residue nearly twice that of an α helix. TheR groups of each polypeptide strand of the triple helixpack so closely that in order to fit, one must be glycine.Thus, every third amino acid residue in collagen is aglycine residue. Staggering of the three strands providesappropriate positioning of the requisite glycinesthroughout the helix. Collagen is also rich in prolineand hydroxyproline, yielding a repetitive Gly-X-Y pattern(Figure 5–10) in which Y generally is proline orhydroxyproline.Collagen triple helices are stabilized by hydrogenbonds between residues in different polypeptide chains.The hydroxyl groups of hydroxyprolyl residues also participatein interchain hydrogen bonding. Additionalstability is provided by covalent cross-links formed betweenmodified lysyl residues both within and betweenpolypeptide chains.Collagen Is Synthesized as aLarger PrecursorCollagen is initially synthesized as a larger precursorpolypeptide, procollagen. Numerous prolyl and lysylresidues of procollagen are hydroxylated by prolyl hydroxylaseand lysyl hydroxylase, enzymes that requireascorbic acid (vitamin C). Hydroxyprolyl and hydroxylysylresidues provide additional hydrogen bonding capabilitythat stabilizes the mature protein. In addition,glucosyl and galactosyl transferases attach glucosyl orgalactosyl residues to the hydroxyl groups of specifichydroxylysyl residues.The central portion of the precursor polypeptidethen associates with other molecules to form the characteristictriple helix. This process is accompanied bythe removal of the globular amino terminal and carboxylterminal extensions of the precursor polypeptideby selective proteolysis. Certain lysyl residues are modifiedby lysyl oxidase, a copper-containing protein thatconverts ε-amino groups to aldehydes. The aldehydescan either undergo an aldol condensation to form aC⎯C double bond or to form a Schiff base (eneimine)with the ε-amino group of an unmodified lysyl residue,which is subsequently reduced to form a C⎯N singlebond. These covalent bonds cross-link the individualpolypeptides and imbue the fiber with exceptionalstrength and rigidity.Nutritional & Genetic Disorders Can ImpairCollagen MaturationThe complex series of events in collagen maturationprovide a model that illustrates the biologic consequencesof incomplete polypeptide maturation. Thebest-known defect in collagen biosynthesis is scurvy, aresult of a dietary deficiency of vitamin C required by

PROTEINS: HIGHER ORDERS OF STRUCTURE / 39prolyl and lysyl hydroxylases. The resulting deficit inthe number of hydroxyproline and hydroxylysineresidues undermines the conformational stability of collagenfibers, leading to bleeding gums, swelling joints,poor wound healing, and ultimately to death. Menkes’syndrome, characterized by kinky hair and growth retardation,reflects a dietary deficiency of the copper requiredby lysyl oxidase, which catalyzes a key step information of the covalent cross-links that strengthencollagen fibers.Genetic disorders of collagen biosynthesis includeseveral forms of osteogenesis imperfecta, characterizedby fragile bones. In Ehlers-Dahlos syndrome, a groupof connective tissue disorders that involve impaired integrityof supporting structures, defects in the genesthat encode α collagen-1, procollagen N-peptidase, orlysyl hydroxylase result in mobile joints and skin abnormalities.SUMMARY• Proteins may be classified on the basis of the solubility,shape, or function or of the presence of a prostheticgroup such as heme. Proteins perform complexphysical and catalytic functions by positioning specificchemical groups in a precise three-dimensionalarrangement that is both functionally efficient andphysically strong.• The gene-encoded primary structure of a polypeptideis the sequence of its amino acids. Its secondarystructure results from folding of polypeptides intohydrogen-bonded motifs such as the α helix, theβ-pleated sheet, β bends, and loops. Combinationsof these motifs can form supersecondary motifs.• Tertiary structure concerns the relationships betweensecondary structural domains. Quaternary structureof proteins with two or more polypeptides(oligomeric proteins) is a feature based on the spatialrelationships between various types of polypeptides.• Primary structures are stabilized by covalent peptidebonds. Higher orders of structure are stabilized byweak forces—multiple hydrogen bonds, salt (electrostatic)bonds, and association of hydrophobic Rgroups.• The phi (Φ) angle of a polypeptide is the angle aboutthe C α ⎯N bond; the psi (Ψ) angle is that about theC α -C o bond. Most combinations of phi-psi anglesare disallowed due to steric hindrance. The phi-psiangles that form the α helix and the β sheet fallwithin the lower and upper left-hand quadrants of aRamachandran plot, respectively.• Protein folding is a poorly understood process.Broadly speaking, short segments of newly synthesizedpolypeptide fold into secondary structuralunits. Forces that bury hydrophobic regions fromsolvent then drive the partially folded polypeptideinto a “molten globule” in which the modules of secondarystructure are rearranged to give the nativeconformation of the protein.• Proteins that assist folding include protein disulfideisomerase, proline-cis,trans,-isomerase, and the chaperonesthat participate in the folding of over half ofmammalian proteins. Chaperones shield newly synthesizedpolypeptides from solvent and provide anenvironment for elements of secondary structure toemerge and coalesce into molten globules.• Techniques for study of higher orders of proteinstructure include x-ray crystallography, NMR spectroscopy,analytical ultracentrifugation, gel filtration,and gel electrophoresis.• Silk fibroin and collagen illustrate the close linkage ofprotein structure and biologic function. Diseases ofcollagen maturation include Ehlers-Danlos syndromeand the vitamin C deficiency disease scurvy.• Prions—protein particles that lack nucleic acid—cause fatal transmissible spongiform encephalopathiessuch as Creutzfeldt-Jakob disease, scrapie, andbovine spongiform encephalopathy. Prion diseasesinvolve an altered secondary-tertiary structure of anaturally occurring protein, PrPc. When PrPc interactswith its pathologic isoform PrPSc, its conformationis transformed from a predominantly α-helicalstructure to the β-sheet structure characteristic ofPrPSc.REFERENCESBranden C, Tooze J: Introduction to Protein Structure. Garland,1991.Burkhard P, Stetefeld J, Strelkov SV: Coiled coils: A highly versatileprotein folding motif. Trends Cell Biol 2001;11:82.Collinge J: Prion diseases of humans and animals: Their causes andmolecular basis. Annu Rev Neurosci 2001;24:519.Frydman J: Folding of newly translated proteins in vivo: The roleof molecular chaperones. Annu Rev Biochem 2001;70:603.Radord S: Protein folding: Progress made and promises ahead.Trends Biochem Sci 2000;25:611.Schmid FX: Proly isomerase: Enzymatic catalysis of slow proteinfolding reactions. Ann Rev Biophys Biomol Struct 1993;22:123.Segrest MP et al: The amphipathic alpha-helix: A multifunctionalstructural motif in plasma lipoproteins. Adv Protein Chem1995;45:1.Soto C: Alzheimer’s and prion disease as disorders of protein conformation:Implications for the design of novel therapeuticapproaches. J Mol Med 1999;77:412.

Proteins: Myoglobin & Hemoglobin 6Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEThe heme proteins myoglobin and hemoglobin maintaina supply of oxygen essential for oxidative metabolism.Myoglobin, a monomeric protein of red muscle,stores oxygen as a reserve against oxygen deprivation.Hemoglobin, a tetrameric protein of erythrocytes,transports O 2 to the tissues and returns CO 2 and protonsto the lungs. Cyanide and carbon monoxide killbecause they disrupt the physiologic function of theheme proteins cytochrome oxidase and hemoglobin, respectively.The secondary-tertiary structure of the subunitsof hemoglobin resembles myoglobin. However,the tetrameric structure of hemoglobin permits cooperativeinteractions that are central to its function. For example,2,3-bisphosphoglycerate (BPG) promotes theefficient release of O 2 by stabilizing the quaternarystructure of deoxyhemoglobin. Hemoglobin and myoglobinillustrate both protein structure-function relationshipsand the molecular basis of genetic diseasessuch as sickle cell disease and the thalassemias.HEME & FERROUS IRON CONFER THEABILITY TO STORE & TO TRANSPORTOXYGENMyoglobin and hemoglobin contain heme, a cyclictetrapyrrole consisting of four molecules of pyrrolelinked by α-methylene bridges. This planar network ofconjugated double bonds absorbs visible light and colorsheme deep red. The substituents at the β-positionsof heme are methyl (M), vinyl (V), and propionate (Pr)groups arranged in the order M, V, M, V, M, Pr, Pr, M(Figure 6–1). One atom of ferrous iron (Fe + 2 ) resides atthe center of the planar tetrapyrrole. Other proteinswith metal-containing tetrapyrrole prosthetic groupsinclude the cytochromes (Fe and Cu) and chlorophyll(Mg) (see Chapter 12). Oxidation and reduction of theFe and Cu atoms of cytochromes is essential to their biologicfunction as carriers of electrons. By contrast, oxidationof the Fe + 2 of myoglobin or hemoglobin to Fe+ 3destroys their biologic activity.Myoglobin Is Rich in α HelixOxygen stored in red muscle myoglobin is released duringO 2 deprivation (eg, severe exercise) for use in musclemitochondria for aerobic synthesis of ATP (seeChapter 12). A 153-aminoacyl residue polypeptide(MW 17,000), myoglobin folds into a compact shapethat measures 4.5 × 3.5 × 2.5 nm (Figure 6–2). Unusuallyhigh proportions, about 75%, of the residues arepresent in eight right-handed, 7–20 residue α helices.Starting at the amino terminal, these are termed helicesA–H. Typical of globular proteins, the surface of myoglobinis polar, while—with only two exceptions—theinterior contains only nonpolar residues such as Leu,Val, Phe, and Met. The exceptions are His E7 and HisF8, the seventh and eighth residues in helices E and F,which lie close to the heme iron where they function inO 2 binding.Histidines F8 & E7 Perform Unique Roles inOxygen BindingThe heme of myoglobin lies in a crevice between helicesE and F oriented with its polar propionate groups facingthe surface of the globin (Figure 6–2). The remainderresides in the nonpolar interior. The fifth coordinationposition of the iron is linked to a ring nitrogen ofthe proximal histidine, His F8. The distal histidine,His E7, lies on the side of the heme ring opposite toHis F8.The Iron Moves Toward the Plane of theHeme When Oxygen Is BoundThe iron of unoxygenated myoglobin lies 0.03 nm(0.3 Å) outside the plane of the heme ring, toward HisF8. The heme therefore “puckers” slightly. When O 2occupies the sixth coordination position, the ironmoves to within 0.01 nm (0.1 Å) of the plane of theheme ring. Oxygenation of myoglobin thus is accompaniedby motion of the iron, of His F8, and of residueslinked to His F8.40

PROTEINS: MYOGLOBIN & HEMOGLOBIN / 41N– OOO –ONFe 2+Figure 6–1. Heme. The pyrrole rings and methylenebridge carbons are coplanar, and the iron atom (Fe + 2 )resides in almost the same plane. The fifth and sixth coordinationpositions of Fe + 2 are directed perpendicularto—and directly above and below—the plane of theheme ring. Observe the nature of the substituentgroups on the β carbons of the pyrrole rings, the centraliron atom, and the location of the polar side of theheme ring (at about 7 o’clock) that faces the surface ofthe myoglobin molecule.H24+ H3 NH16NA1EF3O O –C F9HC5 F6H5F1G1EF1G5E20C3F8FG2NC7C1B16NC5CD1B14G15CD2E7G19B5A16CD7D1E5B1AB1E1D7Apomyoglobin Provides a HinderedEnvironment for Heme IronWhen O 2 binds to myoglobin, the bond between the firstoxygen atom and the Fe + 2 is perpendicular to the plane ofthe heme ring. The bond linking the first and secondoxygen atoms lies at an angle of 121 degrees to the planeof the heme, orienting the second oxygen away from thedistal histidine (Figure 6–3, left). Isolated heme bindscarbon monoxide (CO) 25,000 times more strongly thanoxygen. Since CO is present in small quantities in the atmosphereand arises in cells from the catabolism of heme,why is it that CO does not completely displace O 2 fromheme iron? The accepted explanation is that the apoproteinsof myoglobin and hemoglobin create a hinderedenvironment. While CO can bind to isolated heme in itspreferred orientation, ie, with all three atoms (Fe, C, andO) perpendicular to the plane of the heme, in myoglobinand hemoglobin the distal histidine sterically precludesthis orientation. Binding at a less favored angle reducesthe strength of the heme-CO bond to about 200 timesthat of the heme-O 2 bond (Figure 6–3, right) at whichlevel the great excess of O 2 over CO normally presentdominates. Nevertheless, about 1% of myoglobin typicallyis present combined with carbon monoxide.THE OXYGEN DISSOCIATION CURVESFOR MYOGLOBIN & HEMOGLOBIN SUITTHEIR PHYSIOLOGIC ROLESWhy is myoglobin unsuitable as an O 2 transport proteinbut well suited for O 2 storage? The relationshipbetween the concentration, or partial pressure, of O 2(PO 2 ) and the quantity of O 2 bound is expressed as anO 2 saturation isotherm (Figure 6–4). The oxygen-NE7NOFeONE7NCFeOA1H1GH4NF8NF8Figure 6–2. A model of myoglobin at low resolution.Only the α-carbon atoms are shown. The α-helical regionsare named A through H. (Based on Dickerson RE in:The Proteins, 2nd ed. Vol 2. Neurath H [editor]. AcademicPress, 1964. Reproduced with permission.)NFigure 6–3. Angles for bonding of oxygen and carbonmonoxide to the heme iron of myoglobin. The distalE7 histidine hinders bonding of CO at the preferred(180 degree) angle to the plane of the heme ring.N

42 / CHAPTER 6Percent saturation10080604020MyoglobinHemoglobinReduced bloodreturning from tissuesOxygenated bloodleaving the lungsHemoglobin Is TetramericHemoglobins are tetramers comprised of pairs of twodifferent polypeptide subunits. Greek letters are used todesignate each subunit type. The subunit compositionof the principal hemoglobins are α 2 β 2 (HbA; normaladult hemoglobin), α 2 γ 2 (HbF; fetal hemoglobin), α 2 S 2(HbS; sickle cell hemoglobin), and α 2 δ 2 (HbA 2 ; aminor adult hemoglobin). The primary structures ofthe β, γ, and δ chains of human hemoglobin are highlyconserved.0 20 40 60 80 100 120Gaseous pressure of oxygen (mm Hg)140Figure 6–4. Oxygen-binding curves of both hemoglobinand myoglobin. Arterial oxygen tension is about100 mm Hg; mixed venous oxygen tension is about 40mm Hg; capillary (active muscle) oxygen tension isabout 20 mm Hg; and the minimum oxygen tension requiredfor cytochrome oxidase is about 5 mm Hg. Associationof chains into a tetrameric structure (hemoglobin)results in much greater oxygen delivery thanwould be possible with single chains. (Modified, withpermission, from Scriver CR et al [editors]: The Molecularand Metabolic Bases of Inherited Disease, 7th ed.McGraw-Hill, 1995.)binding curve for myoglobin is hyperbolic. Myoglobintherefore loads O 2 readily at the PO 2 of the lung capillarybed (100 mm Hg). However, since myoglobin releasesonly a small fraction of its bound O 2 at the PO 2values typically encountered in active muscle (20 mmHg) or other tissues (40 mm Hg), it represents an ineffectivevehicle for delivery of O 2 . However, whenstrenuous exercise lowers the PO 2 of muscle tissue toabout 5 mm Hg, myoglobin releases O 2 for mitochondrialsynthesis of ATP, permitting continued muscularactivity.THE ALLOSTERIC PROPERTIES OFHEMOGLOBINS RESULT FROM THEIRQUATERNARY STRUCTURESThe properties of individual hemoglobins are consequencesof their quaternary as well as of their secondaryand tertiary structures. The quaternary structure of hemoglobinconfers striking additional properties, absentfrom monomeric myoglobin, which adapts it to itsunique biologic roles. The allosteric (Gk allos “other,”steros “space”) properties of hemoglobin provide, in addition,a model for understanding other allosteric proteins(see Chapter 11).Myoglobin & the Subunitsof Hemoglobin Share Almost IdenticalSecondary and Tertiary StructuresDespite differences in the kind and number of aminoacids present, myoglobin and the β polypeptide of hemoglobinA have almost identical secondary and tertiarystructures. Similarities include the location of theheme and the eight helical regions and the presence ofamino acids with similar properties at comparable locations.Although it possesses seven rather than eight helicalregions, the α polypeptide of hemoglobin alsoclosely resembles myoglobin.Oxygenation of HemoglobinTriggers Conformational Changesin the ApoproteinHemoglobins bind four molecules of O 2 per tetramer,one per heme. A molecule of O 2 binds to a hemoglobintetramer more readily if other O 2 molecules are alreadybound (Figure 6–4). Termed cooperative binding,this phenomenon permits hemoglobin to maximizeboth the quantity of O 2 loaded at the PO 2 of the lungsand the quantity of O 2 released at the PO 2 of the peripheraltissues. Cooperative interactions, an exclusiveproperty of multimeric proteins, are critically importantto aerobic life.P 50 Expresses the Relative Affinitiesof Different Hemoglobins for OxygenThe quantity P 50 , a measure of O 2 concentration, is thepartial pressure of O 2 that half-saturates a given hemoglobin.Depending on the organism, P 50 can varywidely, but in all instances it will exceed the PO 2 of theperipheral tissues. For example, values of P 50 for HbAand fetal HbF are 26 and 20 mm Hg, respectively. Inthe placenta, this difference enables HbF to extract oxygenfrom the HbA in the mother’s blood. However,HbF is suboptimal postpartum since its high affinityfor O 2 dictates that it can deliver less O 2 to the tissues.The subunit composition of hemoglobin tetramersundergoes complex changes during development. The

PROTEINS: MYOGLOBIN & HEMOGLOBIN / 43human fetus initially synthesizes a ζ 2 ε 2 tetramer. By theend of the first trimester, ζ and γ subunits have been replacedby α and ε subunits, forming HbF (α 2 γ 2 ), thehemoglobin of late fetal life. While synthesis of β subunitsbegins in the third trimester, β subunits do notcompletely replace γ subunits to yield adult HbA (α 2 β 2 )until some weeks postpartum (Figure 6–5).Oxygenation of Hemoglobin IsAccompanied by LargeConformational ChangesThe binding of the first O 2 molecule to deoxyHb shiftsthe heme iron towards the plane of the heme ring froma position about 0.6 nm beyond it (Figure 6–6). Thismotion is transmitted to the proximal (F8) histidineand to the residues attached thereto, which in turncauses the rupture of salt bridges between the carboxylterminal residues of all four subunits. As a consequence,one pair of α/β subunits rotates 15 degrees with respectto the other, compacting the tetramer (Figure 6–7).Profound changes in secondary, tertiary, and quaternarystructure accompany the high-affinity O 2 -inducedtransition of hemoglobin from the low-affinity T (taut)state to the R (relaxed) state. These changes significantlyincrease the affinity of the remaining unoxygenatedhemes for O 2 , as subsequent binding events requirethe rupture of fewer salt bridges (Figure 6–8).The terms T and R also are used to refer to the lowaffinityand high-affinity conformations of allosteric enzymes,respectively.F helixStericrepulsionCHCF helixHistidine F8NNFe+O 2CCHNFeNHC CHPorphyrinplaneFigure 6–6. The iron atom moves into the plane ofthe heme on oxygenation. Histidine F8 and its associatedresidues are pulled along with the iron atom.(Slightly modified and reproduced, with permission,from Stryer L: Biochemistry, 4th ed. Freeman, 1995.)OOGlobin chain synthesis (% of total)50α chain403020100∋γ chain(fetal)and ζ chains(embryonic)δ chainβ chain (adult)3 6 Birth 3 6Gestation (months)Age (months)Figure 6–5. Developmental pattern of the quaternarystructure of fetal and newborn hemoglobins. (Reproduced,with permission, from Ganong WF: Review ofMedical Physiology, 20th ed. McGraw-Hill, 2001.)α 1 β 2α 1β 2Axisα 2α 2 β 1β 1T form15°R formFigure 6–7. During transition of the T form to the Rform of hemoglobin, one pair of subunits (α 2 /β 2 ) rotatesthrough 15 degrees relative to the other pair(α 1 /β 1 ). The axis of rotation is eccentric, and the α 2 /β 2pair also shifts toward the axis somewhat. In the diagram,the unshaded α 1 /β 1 pair is shown fixed while thecolored α 2 /β 2 pair both shifts and rotates.

44 / CHAPTER 6T structureα 1 α 2O 2β 1 β 2O 2 O 2 O 2 O 2O 2O 2 O 2 O 2 O 2O 2O 2 O 2O 2 O 2O 2R structureFigure 6–8. Transition from the T structure to the R structure. In this model, saltbridges (thin lines) linking the subunits in the T structure break progressively as oxygenis added, and even those salt bridges that have not yet ruptured are progressivelyweakened (wavy lines). The transition from T to R does not take place after a fixednumber of oxygen molecules have been bound but becomes more probable as eachsuccessive oxygen binds. The transition between the two structures is influenced byprotons, carbon dioxide, chloride, and BPG; the higher their concentration, the moreoxygen must be bound to trigger the transition. Fully oxygenated molecules in the Tstructure and fully deoxygenated molecules in the R structure are not shown becausethey are unstable. (Modified and redrawn, with permission, from Perutz MF: Hemoglobinstructure and respiratory transport. Sci Am [Dec] 1978;239:92.)After Releasing O 2 at the Tissues,Hemoglobin Transports CO 2 & Protonsto the LungsIn addition to transporting O 2 from the lungs to peripheraltissues, hemoglobin transports CO 2 , the byproductof respiration, and protons from peripheral tissuesto the lungs. Hemoglobin carries CO 2 ascarbamates formed with the amino terminal nitrogensof the polypeptide chains.OH ||CO2 + + +Hb⎯ NH 3 = 2H + Hb⎯N⎯C⎯O−Carbamates change the charge on amino terminalsfrom positive to negative, favoring salt bond formationbetween the α and β chains.Hemoglobin carbamates account for about 15% ofthe CO 2 in venous blood. Much of the remaining CO 2is carried as bicarbonate, which is formed in erythrocytesby the hydration of CO 2 to carbonic acid(H 2 CO 3 ), a process catalyzed by carbonic anhydrase. Atthe pH of venous blood, H 2 CO 3 dissociates into bicarbonateand a proton.Deoxyhemoglobin binds one proton for every twoO 2 molecules released, contributing significantly to thebuffering capacity of blood. The somewhat lower pH ofperipheral tissues, aided by carbamation, stabilizes theT state and thus enhances the delivery of O 2 . In thelungs, the process reverses. As O 2 binds to deoxyhemoglobin,protons are released and combine with bicarbonateto form carbonic acid. Dehydration of H 2 CO 3 ,catalyzed by carbonic anhydrase, forms CO 2 , which isexhaled. Binding of oxygen thus drives the exhalationof CO 2 (Figure 6–9).This reciprocal coupling of protonand O 2 binding is termed the Bohr effect. The Bohreffect is dependent upon cooperative interactions betweenthe hemes of the hemoglobin tetramer. Myoglobin,a monomer, exhibits no Bohr effect.Protons Arise From Rupture of Salt BondsWhen O 2 BindsProtons responsible for the Bohr effect arise from ruptureof salt bridges during the binding of O 2 to T state

PROTEINS: MYOGLOBIN & HEMOGLOBIN / 45Exhaled2CO 2 + 2H 2 O2H 2 CO 3CARBONICANHYDRASE2HCO 3 – + 2H +LUNGSHb • 4O 24O 22H + –+ 2HCO 34O Hb • 2H +2(buffer)2H 2 CO 3Generated bythe Krebs cyclePERIPHERALTISSUES2CO 2 + 2H 2 OCARBONICANHYDRASEFigure 6–9. The Bohr effect. Carbon dioxide generatedin peripheral tissues combines with water to formcarbonic acid, which dissociates into protons and bicarbonateions. Deoxyhemoglobin acts as a buffer bybinding protons and delivering them to the lungs. Inthe lungs, the uptake of oxygen by hemoglobin releasesprotons that combine with bicarbonate ion,forming carbonic acid, which when dehydrated by carbonicanhydrase becomes carbon dioxide, which thenis exhaled.The hemoglobin tetramer binds one molecule ofBPG in the central cavity formed by its four subunits.However, the space between the H helices of the βchains lining the cavity is sufficiently wide to accommodateBPG only when hemoglobin is in the T state.BPG forms salt bridges with the terminal amino groupsof both β chains via Val NA1 and with Lys EF6 andHis H21 (Figure 6–10). BPG therefore stabilizes deoxygenated(T state) hemoglobin by forming additionalsalt bridges that must be broken prior to conversion tothe R state.Residue H21 of the γ subunit of fetal hemoglobin(HbF) is Ser rather than His. Since Ser cannot form asalt bridge, BPG binds more weakly to HbF than toHbA. The lower stabilization afforded to the T state byBPG accounts for HbF having a higher affinity for O 2than HbA.His H21hemoglobin. Conversion to the oxygenated R statebreaks salt bridges involving β-chain residue His 146.The subsequent dissociation of protons from His 146drives the conversion of bicarbonate to carbonic acid(Figure 6–9). Upon the release of O 2 , the T structureand its salt bridges re-form. This conformationalchange increases the pK a of the β-chain His 146residues, which bind protons. By facilitating the re-formationof salt bridges, an increase in proton concentrationenhances the release of O 2 from oxygenated (Rstate) hemoglobin. Conversely, an increase in PO 2 promotesproton release.2,3-Bisphosphoglycerate (BPG) Stabilizesthe T Structure of HemoglobinA low PO 2 in peripheral tissues promotes the synthesisin erythrocytes of 2,3-bisphosphoglycerate (BPG) fromthe glycolytic intermediate 1,3-bisphosphoglycerate.Lys EF6Val NA1His H21BPGLys EF6α-NH 3+Val NA1Figure 6–10. Mode of binding of 2,3-bisphosphoglycerateto human deoxyhemoglobin. BPG interactswith three positively charged groups on each β chain.(Based on Arnone A: X-ray diffraction study of binding of2,3-diphosphoglycerate to human deoxyhemoglobin. Nature1972;237:146. Reproduced with permission.)

46 / CHAPTER 6Adaptation to High AltitudePhysiologic changes that accompany prolonged exposureto high altitude include an increase in the numberof erythrocytes and in their concentrations of hemoglobinand of BPG. Elevated BPG lowers the affinity ofHbA for O 2 (decreases P 50 ), which enhances release ofO 2 at the tissues.NUMEROUS MUTANT HUMANHEMOGLOBINS HAVE BEEN IDENTIFIEDMutations in the genes that encode the α or β subunitsof hemoglobin potentially can affect its biologic function.However, almost all of the over 800 known mutanthuman hemoglobins are both extremely rare andbenign, presenting no clinical abnormalities. When amutation does compromise biologic function, the conditionis termed a hemoglobinopathy. The URLhttp://globin.cse.psu.edu/ (Globin Gene Server) providesinformation about—and links for—normal andmutant hemoglobins.Methemoglobin & Hemoglobin MIn methemoglobinemia, the heme iron is ferric ratherthan ferrous. Methemoglobin thus can neither bind nortransport O 2 . Normally, the enzyme methemoglobinreductase reduces the Fe + 3 of methemoglobin to Fe + 2 .Methemoglobin can arise by oxidation of Fe + 2 to Fe+ 3as a side effect of agents such as sulfonamides, fromhereditary hemoglobin M, or consequent to reducedactivity of the enzyme methemoglobin reductase.In hemoglobin M, histidine F8 (His F8) has beenreplaced by tyrosine. The iron of HbM forms a tightionic complex with the phenolate anion of tyrosine thatstabilizes the Fe + 3 form. In α-chain hemoglobin M variants,the R-T equilibrium favors the T state. Oxygenaffinity is reduced, and the Bohr effect is absent.β-Chain hemoglobin M variants exhibit R-T switching,and the Bohr effect is therefore present.Mutations (eg, hemoglobin Chesapeake) that favorthe R state increase O 2 affinity. These hemoglobinstherefore fail to deliver adequate O 2 to peripheral tissues.The resulting tissue hypoxia leads to polycythemia,an increased concentration of erythrocytes.Hemoglobin SIn HbS, the nonpolar amino acid valine has replacedthe polar surface residue Glu6 of the β subunit, generatinga hydrophobic “sticky patch” on the surface ofthe β subunit of both oxyHbS and deoxyHbS. BothHbA and HbS contain a complementary sticky patchon their surfaces that is exposed only in the deoxygenated,R state. Thus, at low PO 2 , deoxyHbS can polymerizeto form long, insoluble fibers. Binding of deoxy-HbA terminates fiber polymerization, since HbA lacksthe second sticky patch necessary to bind another Hbmolecule (Figure 6–11). These twisted helical fibersdistort the erythrocyte into a characteristic sickle shape,rendering it vulnerable to lysis in the interstices of thesplenic sinusoids. They also cause multiple secondaryclinical effects. A low PO 2 such as that at high altitudesexacerbates the tendency to polymerize.Oxy ADeoxy A Oxy S Deoxy SβααβDeoxy ADeoxy SFigure 6–11. Representation of the sticky patch () on hemoglobin S and its “receptor” ()on deoxyhemoglobin A and deoxyhemoglobin S. The complementary surfaces allow deoxyhemoglobinS to polymerize into a fibrous structure, but the presence of deoxyhemoglobin A willterminate the polymerization by failing to provide sticky patches. (Modified and reproduced, withpermission, from Stryer L: Biochemistry, 4th ed. Freeman, 1995.)

PROTEINS: MYOGLOBIN & HEMOGLOBIN / 47BIOMEDICAL IMPLICATIONSMyoglobinuriaFollowing massive crush injury, myoglobin releasedfrom damaged muscle fibers colors the urine dark red.Myoglobin can be detected in plasma following a myocardialinfarction, but assay of serum enzymes (seeChapter 7) provides a more sensitive index of myocardialinjury.AnemiasAnemias, reductions in the number of red blood cells orof hemoglobin in the blood, can reflect impaired synthesisof hemoglobin (eg, in iron deficiency; Chapter51) or impaired production of erythrocytes (eg, in folicacid or vitamin B 12 deficiency; Chapter 45). Diagnosisof anemias begins with spectroscopic measurement ofblood hemoglobin levels.ThalassemiasThe genetic defects known as thalassemias result fromthe partial or total absence of one or more α or β chainsof hemoglobin. Over 750 different mutations havebeen identified, but only three are common. Either theα chain (alpha thalassemias) or β chain (beta thalassemias)can be affected. A superscript indicateswhether a subunit is completely absent (α 0 or β 0 ) orwhether its synthesis is reduced (α + or β + ). Apart frommarrow transplantation, treatment is symptomatic.Certain mutant hemoglobins are common in manypopulations, and a patient may inherit more than onetype. Hemoglobin disorders thus present a complexpattern of clinical phenotypes. The use of DNA probesfor their diagnosis is considered in Chapter 40.Glycosylated Hemoglobin (HbA 1c )When blood glucose enters the erythrocytes it glycosylatesthe ε-amino group of lysine residues and theamino terminals of hemoglobin. The fraction of hemoglobinglycosylated, normally about 5%, is proportionateto blood glucose concentration. Since the half-life ofan erythrocyte is typically 60 days, the level of glycosylatedhemoglobin (HbA 1c ) reflects the mean blood glucoseconcentration over the preceding 6–8 weeks.Measurement of HbA 1c therefore provides valuable informationfor management of diabetes mellitus.SUMMARY• Myoglobin is monomeric; hemoglobin is a tetramerof two subunit types (α 2 β 2 in HbA). Despite havingdifferent primary structures, myoglobin and the subunitsof hemoglobin have nearly identical secondaryand tertiary structures.• Heme, an essentially planar, slightly puckered, cyclictetrapyrrole, has a central Fe + 2 linked to all four nitrogenatoms of the heme, to histidine F8, and, inoxyMb and oxyHb, also to O 2 .• The O 2 -binding curve for myoglobin is hyperbolic,but for hemoglobin it is sigmoidal, a consequence ofcooperative interactions in the tetramer. Cooperativitymaximizes the ability of hemoglobin both to loadO 2 at the PO 2 of the lungs and to deliver O 2 at thePO 2 of the tissues.• Relative affinities of different hemoglobins for oxygenare expressed as P 50 , the PO 2 that half-saturatesthem with O 2 . Hemoglobins saturate at the partialpressures of their respective respiratory organ, eg, thelung or placenta.• On oxygenation of hemoglobin, the iron, histidineF8, and linked residues move toward the heme ring.Conformational changes that accompany oxygenationinclude rupture of salt bonds and loosening ofquaternary structure, facilitating binding of additionalO 2 .• 2,3-Bisphosphoglycerate (BPG) in the central cavityof deoxyHb forms salt bonds with the β subunitsthat stabilize deoxyHb. On oxygenation, the centralcavity contracts, BPG is extruded, and the quaternarystructure loosens.• Hemoglobin also functions in CO 2 and protontransport from tissues to lungs. Release of O 2 fromoxyHb at the tissues is accompanied by uptake ofprotons due to lowering of the pK a of histidineresidues.• In sickle cell hemoglobin (HbS), Val replaces the β6Glu of HbA, creating a “sticky patch” that has acomplement on deoxyHb (but not on oxyHb). DeoxyHbSpolymerizes at low O 2 concentrations,forming fibers that distort erythrocytes into sickleshapes.• Alpha and beta thalassemias are anemias that resultfrom reduced production of α and β subunits ofHbA, respectively.REFERENCESBettati S et al: Allosteric mechanism of haemoglobin: Rupture ofsalt-bridges raises the oxygen affinity of the T-structure. JMol Biol 1998;281:581.Bunn HF: Pathogenesis and treatment of sickle cell disease. N EnglJ Med 1997;337:762.Faustino P et al: Dominantly transmitted beta-thalassemia arisingfrom the production of several aberrant mRNA species andone abnormal peptide. Blood 1998;91:685.

48 / CHAPTER 6Manning JM et al: Normal and abnormal protein subunit interactionsin hemoglobins. J Biol Chem 1998;273:19359.Mario N, Baudin B, Giboudeau J: Qualitative and quantitativeanalysis of hemoglobin variants by capillary isoelectric focusing.J Chromatogr B Biomed Sci Appl 1998;706:123.Reed W, Vichinsky EP: New considerations in the treatment ofsickle cell disease. Annu Rev Med 1998;49:461.Unzai S et al: Rate constants for O 2 and CO binding to the alphaand beta subunits within the R and T states of human hemoglobin.J Biol Chem 1998;273:23150.Weatherall DJ et al: The hemoglobinopathies. Chapter 181 in TheMetabolic and Molecular Bases of Inherited Disease, 8th ed.Scriver CR et al (editors). McGraw-Hill, 2000.

Enzymes: Mechanism of Action 7Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEEnzymes are biologic polymers that catalyze the chemicalreactions which make life as we know it possible.The presence and maintenance of a complete and balancedset of enzymes is essential for the breakdown ofnutrients to supply energy and chemical buildingblocks; the assembly of those building blocks into proteins,DNA, membranes, cells, and tissues; and the harnessingof energy to power cell motility and musclecontraction. With the exception of a few catalytic RNAmolecules, or ribozymes, the vast majority of enzymesare proteins. Deficiencies in the quantity or catalytic activityof key enzymes can result from genetic defects,nutritional deficits, or toxins. Defective enzymes can resultfrom genetic mutations or infection by viral or bacterialpathogens (eg, Vibrio cholerae). Medical scientistsaddress imbalances in enzyme activity by using pharmacologicagents to inhibit specific enzymes and are investigatinggene therapy as a means to remedy deficits inenzyme level or function.ENZYMES ARE EFFECTIVE & HIGHLYSPECIFIC CATALYSTSThe enzymes that catalyze the conversion of one ormore compounds (substrates) into one or more differentcompounds (products) enhance the rates of thecorresponding noncatalyzed reaction by factors of atleast 10 6 . Like all catalysts, enzymes are neither consumednor permanently altered as a consequence oftheir participation in a reaction.In addition to being highly efficient, enzymes arealso extremely selective catalysts. Unlike most catalystsused in synthetic chemistry, enzymes are specific bothfor the type of reaction catalyzed and for a single substrateor a small set of closely related substrates. Enzymesare also stereospecific catalysts and typically catalyzereactions only of specific stereoisomers of a givencompound—for example, D- but not L-sugars, L- butnot D-amino acids. Since they bind substrates throughat least “three points of attachment,” enzymes can evenconvert nonchiral substrates to chiral products. Figure7–1 illustrates why the enzyme-catalyzed reduction ofthe nonchiral substrate pyruvate produces L-lactaterather a racemic mixture of D- and L-lactate. The exquisitespecificity of enzyme catalysts imbues living cells49with the ability to simultaneously conduct and independentlycontrol a broad spectrum of chemicalprocesses.ENZYMES ARE CLASSIFIED BY REACTIONTYPE & MECHANISMA system of enzyme nomenclature that is comprehensive,consistent, and at the same time easy to use hasproved elusive. The common names for most enzymesderive from their most distinctive characteristic: theirability to catalyze a specific chemical reaction. In general,an enzyme’s name consists of a term that identifiesthe type of reaction catalyzed followed by the suffix-ase. For example, dehydrogenases remove hydrogenatoms, proteases hydrolyze proteins, and isomerases catalyzerearrangements in configuration. One or moremodifiers usually precede this name. Unfortunately,while many modifiers name the specific substrate involved(xanthine oxidase), others identify the source ofthe enzyme (pancreatic ribonuclease), specify its modeof regulation (hormone-sensitive lipase), or name a distinguishingcharacteristic of its mechanism (a cysteineprotease). When it was discovered that multiple formsof some enzymes existed, alphanumeric designatorswere added to distinguish between them (eg, RNApolymerase III; protein kinase Cβ). To address the ambiguityand confusion arising from these inconsistenciesin nomenclature and the continuing discovery of newenzymes, the International Union of Biochemists (IUB)developed a complex but unambiguous system of enzymenomenclature. In the IUB system, each enzymehas a unique name and code number that reflect thetype of reaction catalyzed and the substrates involved.Enzymes are grouped into six classes, each with severalsubclasses. For example, the enzyme commonly called“hexokinase” is designated “ATP:D-hexose-6-phosphotransferaseE.C. 2.7.1.1.” This identifies hexokinase as amember of class 2 (transferases), subclass 7 (transfer of aphosphoryl group), sub-subclass 1 (alcohol is the phosphorylacceptor). Finally, the term “hexose-6” indicatesthat the alcohol phosphorylated is that of carbon six ofa hexose. Listed below are the six IUB classes of enzymesand the reactions they catalyze.1. Oxidoreductases catalyze oxidations and reductions.

50 / CHAPTER 7132 2Enzyme site14SubstrateFigure 7–1. Planar representation of the “threepointattachment” of a substrate to the active site of anenzyme. Although atoms 1 and 4 are identical, onceatoms 2 and 3 are bound to their complementary siteson the enzyme, only atom 1 can bind. Once bound toan enzyme, apparently identical atoms thus may be distinguishable,permitting a stereospecific chemicalchange.2. Transferases catalyze transfer of groups such asmethyl or glycosyl groups from a donor moleculeto an acceptor molecule.3. Hydrolases catalyze the hydrolytic cleavage ofC⎯ C, C⎯O, C⎯N, P⎯ O, and certain otherbonds, including acid anhydride bonds.4. Lyases catalyze cleavage of C⎯ C, C⎯ O, C⎯N,and other bonds by elimination, leaving doublebonds, and also add groups to double bonds.5. Isomerases catalyze geometric or structuralchanges within a single molecule.6. Ligases catalyze the joining together of two molecules,coupled to the hydrolysis of a pyrophosphorylgroup in ATP or a similar nucleoside triphosphate.Despite the many advantages of the IUB system,texts tend to refer to most enzymes by their older andshorter, albeit sometimes ambiguous names.PROSTHETIC GROUPS, COFACTORS,& COENZYMES PLAY IMPORTANTROLES IN CATALYSISMany enzymes contain small nonprotein molecules andmetal ions that participate directly in substrate bindingor catalysis. Termed prosthetic groups, cofactors, andcoenzymes, these extend the repertoire of catalytic capabilitiesbeyond those afforded by the limited numberof functional groups present on the aminoacyl sidechains of peptides.3Prosthetic Groups Are Tightly IntegratedInto an Enzyme’s StructureProsthetic groups are distinguished by their tight, stableincorporation into a protein’s structure by covalent ornoncovalent forces. Examples include pyridoxal phosphate,flavin mononucleotide (FMN), flavin dinucleotide(FAD), thiamin pyrophosphate, biotin, andthe metal ions of Co, Cu, Mg, Mn, Se, and Zn. Metalsare the most common prosthetic groups. The roughlyone-third of all enzymes that contain tightly boundmetal ions are termed metalloenzymes. Metal ions thatparticipate in redox reactions generally are complexedto prosthetic groups such as heme (Chapter 6) or ironsulfurclusters (Chapter 12). Metals also may facilitatethe binding and orientation of substrates, the formationof covalent bonds with reaction intermediates (Co 2+ incoenzyme B 12 ), or interaction with substrates to renderthem more electrophilic (electron-poor) or nucleophilic(electron-rich).Cofactors Associate Reversibly WithEnzymes or SubstratesCofactors serve functions similar to those of prostheticgroups but bind in a transient, dissociable manner eitherto the enzyme or to a substrate such as ATP. Unlikethe stably associated prosthetic groups, cofactorstherefore must be present in the medium surroundingthe enzyme for catalysis to occur. The most commoncofactors also are metal ions. Enzymes that require ametal ion cofactor are termed metal-activated enzymesto distinguish them from the metalloenzymes forwhich metal ions serve as prosthetic groups.Coenzymes Serve as Substrate ShuttlesCoenzymes serve as recyclable shuttles—or grouptransfer reagents—that transport many substrates fromtheir point of generation to their point of utilization.Association with the coenzyme also stabilizes substratessuch as hydrogen atoms or hydride ions that are unstablein the aqueous environment of the cell. Otherchemical moieties transported by coenzymes includemethyl groups (folates), acyl groups (coenzyme A), andoligosaccharides (dolichol).Many Coenzymes, Cofactors, & ProstheticGroups Are Derivatives of B VitaminsThe water-soluble B vitamins supply important componentsof numerous coenzymes. Many coenzymes contain,in addition, the adenine, ribose, and phosphorylmoieties of AMP or ADP (Figure 7–2). Nicotinamideand riboflavin are components of the redox coenzymes

ENZYMES: MECHANISM OF ACTION / 51OArg 145OOOPOPO –O –ONNH 2+NCH 2OH HHO OHNH 2NN NCH 2OH HHO ORHis 196OCGlu 72HNNOHOHCHZn 2+His 69CONH 2NNHOCNCNHCOHCH2NH 2HNH2OTyr 248Figure 7–3. Two-dimensional representation of adipeptide substrate, glycyl-tyrosine, bound within theactive site of carboxypeptidase A.Figure 7–2. Structure of NAD + and NADP + . ForNAD + , R = H. For NADP + , R = PO 2− 3 .NAD and NADP and FMN and FAD, respectively.Pantothenic acid is a component of the acyl group carriercoenzyme A. As its pyrophosphate, thiamin participatesin decarboxylation of α-keto acids and folic acidand cobamide coenzymes function in one-carbon metabolism.CATALYSIS OCCURS AT THE ACTIVE SITEThe extreme substrate specificity and high catalytic efficiencyof enzymes reflect the existence of an environmentthat is exquisitely tailored to a single reaction.Termed the active site, this environment generallytakes the form of a cleft or pocket. The active sites ofmultimeric enzymes often are located at the interfacebetween subunits and recruit residues from more thanone monomer. The three-dimensional active site bothshields substrates from solvent and facilitates catalysis.Substrates bind to the active site at a region complementaryto a portion of the substrate that will not undergochemical change during the course of the reaction.This simultaneously aligns portions of thesubstrate that will undergo change with the chemicalfunctional groups of peptidyl aminoacyl residues. Theactive site also binds and orients cofactors or prostheticgroups. Many amino acyl residues drawn from diverseportions of the polypeptide chain (Figure 7–3) contributeto the extensive size and three-dimensional characterof the active site.ENZYMES EMPLOY MULTIPLEMECHANISMS TO FACILITATECATALYSISFour general mechanisms account for the ability of enzymesto achieve dramatic catalytic enhancement of therates of chemical reactions.Catalysis by ProximityFor molecules to react, they must come within bondformingdistance of one another. The higher their concentration,the more frequently they will encounter oneanother and the greater will be the rate of their reaction.When an enzyme binds substrate molecules in its activesite, it creates a region of high local substrate concentration.This environment also orients the substrate moleculesspatially in a position ideal for them to interact, resultingin rate enhancements of at least a thousandfold.Acid-Base CatalysisThe ionizable functional groups of aminoacyl sidechains and (where present) of prosthetic groups contributeto catalysis by acting as acids or bases. Acid-basecatalysis can be either specific or general. By “specific”we mean only protons (H 3 O + ) or OH – ions. In specificacid or specific base catalysis, the rate of reaction issensitive to changes in the concentration of protons but

52 / CHAPTER 7independent of the concentrations of other acids (protondonors) or bases (proton acceptors) present in solutionor at the active site. Reactions whose rates are responsiveto all the acids or bases present are said to besubject to general acid or general base catalysis.Catalysis by StrainEnzymes that catalyze lytic reactions which involvebreaking a covalent bond typically bind their substratesin a conformation slightly unfavorable for the bondthat will undergo cleavage. The resulting strainstretches or distorts the targeted bond, weakening itand making it more vulnerable to cleavage.Covalent CatalysisThe process of covalent catalysis involves the formationof a covalent bond between the enzyme and one or moresubstrates. The modified enzyme then becomes a reactant.Covalent catalysis introduces a new reaction pathwaythat is energetically more favorable—and thereforefaster—than the reaction pathway in homogeneous solution.The chemical modification of the enzyme is,however, transient. On completion of the reaction, theenzyme returns to its original unmodified state. Its rolethus remains catalytic. Covalent catalysis is particularlycommon among enzymes that catalyze group transferreactions. Residues on the enzyme that participate in covalentcatalysis generally are cysteine or serine and occasionallyhistidine. Covalent catalysis often follows a“ping-pong” mechanism—one in which the first substrateis bound and its product released prior to thebinding of the second substrate (Figure 7–4).SUBSTRATES INDUCECONFORMATIONAL CHANGESIN ENZYMESEarly in the last century, Emil Fischer compared thehighly specific fit between enzymes and their substratesto that of a lock and its key. While the “lock and keymodel” accounted for the exquisite specificity of enzyme-substrateinteractions, the implied rigidity of theenzyme’s active site failed to account for the dynamicchanges that accompany catalysis. This drawback wasaddressed by Daniel Koshland’s induced fit model,which states that when substrates approach and bind toan enzyme they induce a conformational change, achange analogous to placing a hand (substrate) into aglove (enzyme) (Figure 7–5). A corollary is that the enzymeinduces reciprocal changes in its substrates, harnessingthe energy of binding to facilitate the transformationof substrates into products. The induced fitmodel has been amply confirmed by biophysical studiesof enzyme motion during substrate binding.HIV PROTEASE ILLUSTRATESACID-BASE CATALYSISEnzymes of the aspartic protease family, which includesthe digestive enzyme pepsin, the lysosomalcathepsins, and the protease produced by the human immunodeficiencyvirus (HIV), share a common catalyticmechanism. Catalysis involves two conserved aspartylresidues which act as acid-base catalysts. In the first stageof the reaction, an aspartate functioning as a general base(Asp X, Figure 7–6) extracts a proton from a water molecule,making it more nucleophilic. This resulting nucleophilethen attacks the electrophilic carbonyl carbon ofthe peptide bond targeted for hydrolysis, forming atetrahedral transition state intermediate. A second aspartate(Asp Y, Figure 7–6) then facilitates the decompositionof this tetrahedral intermediate by donating a protonto the amino group produced by rupture of thepeptide bond. Two different active site aspartates thuscan act simultaneously as a general base or as a generalacid. This is possible because their immediate environmentfavors ionization of one but not the other.CHYMOTRYPSIN & FRUCTOSE-2,6-BISPHOSPHATASE ILLUSTRATECOVALENT CATALYSISChymotrypsinWhile catalysis by aspartic proteases involves the directhydrolytic attack of water on a peptide bond, catalysisAlaE CHO ECHOAlaECH 2 NH 2PyrPyrCHOGluKG CH 2 NH 2E CH 2 NH 2 EEKGGluECHOFigure 7–4. Ping-pong mechanism for transamination. E⎯CHO and E⎯CH 2 NH 2 represent the enzymepyridoxalphosphate and enzyme-pyridoxamine complexes, respectively. (Ala, alanine; Pyr, pyruvate; KG,α-ketoglutarate; Glu, glutamate).

ENZYMES: MECHANISM OF ACTION / 53R′OAB1HN..C....ORHHOCOHOCOABCH 2Asp YCH 2Asp XR′OAB2N..HHCOHRHOCOOCOCH 2CH 2Figure 7–5. Two-dimensional representation ofKoshland’s induced fit model of the active site of alyase. Binding of the substrate A⎯B induces conformationalchanges in the enzyme that aligns catalyticresidues which participate in catalysis and strains thebond between A and B, facilitating its cleavage.by the serine protease chymotrypsin involves prior formationof a covalent acyl enzyme intermediate. Ahighly reactive seryl residue, serine 195, participates ina charge-relay network with histidine 57 and aspartate102. Far apart in primary structure, in the active sitethese residues are within bond-forming distance of oneanother. Aligned in the order Asp 102-His 57-Ser 195,they constitute a “charge-relay network” that functionsas a “proton shuttle.”Binding of substrate initiates proton shifts that in effecttransfer the hydroxyl proton of Ser 195 to Asp 102(Figure 7–7). The enhanced nucleophilicity of the seryloxygen facilitates its attack on the carbonyl carbon ofthe peptide bond of the substrate, forming a covalentacyl-enzyme intermediate. The hydrogen on Asp 102then shuttles through His 57 to the amino group liberatedwhen the peptide bond is cleaved. The portion ofthe original peptide with a free amino group then leavesthe active site and is replaced by a water molecule. Thecharge-relay network now activates the water moleculeby withdrawing a proton through His 57 to Asp 102.The resulting hydroxide ion attacks the acyl-enzyme in-3Asp YOR′CHCH 2Asp YONHO+ CHOHAsp XORCOCH 2Asp XFigure 7–6. Mechanism for catalysis by an asparticprotease such as HIV protease. Curved arrows indicatedirections of electron movement. 1 Aspartate X actsas a base to activate a water molecule by abstracting aproton. 2 The activated water molecule attacks thepeptide bond, forming a transient tetrahedral intermediate.3 Aspartate Y acts as an acid to facilitate breakdownof the tetrahedral intermediate and release of thesplit products by donating a proton to the newlyformed amino group. Subsequent shuttling of the protonon Asp X to Asp Y restores the protease to its initialstate.

54 / CHAPTER 712OCAsp 102OAsp 102HO H N N HOR 1 N C R 2His 57HO H N N HOOR 1 N C R 2His 57OOSer 195Ser 195R 1 NH 2 C R 2termediate and a reverse proton shuttle returns a protonto Ser 195, restoring its original state. While modifiedduring the process of catalysis, chymotrypsin emergesunchanged on completion of the reaction. Trypsin andelastase employ a similar catalytic mechanism, but thenumbers of the residues in their Ser-His-Asp protonshuttles differ.Fructose-2,6-BisphosphataseFructose-2,6-bisphosphatase, a regulatory enzyme ofgluconeogenesis (Chapter 19), catalyzes the hydrolyticrelease of the phosphate on carbon 2 of fructose 2,6-bisphosphate. Figure 7–8 illustrates the roles of sevenactive site residues. Catalysis involves a “catalytic triad”of one Glu and two His residues and a covalent phosphohistidylintermediate.3456OAsp 102OAsp 102OAsp 102OO H N N OHis 57His 57Ser 195OHOC R 2OO H N N HSer 195HO H N N HOAsp 102His 57H N N HHis 57OO C R 2OHOOCOSer 195R 2Ser 195Figure 7–7. Catalysis by chymotrypsin. 1 Thecharge-relay system removes a proton from Ser 195,making it a stronger nucleophile. 2 Activated Ser 195attacks the peptide bond, forming a transient tetrahedralintermediate. 3 Release of the amino terminal peptideis facilitated by donation of a proton to the newlyformed amino group by His 57 of the charge-relay system,yielding an acyl-Ser 195 intermediate. 4 His 57 andAsp 102 collaborate to activate a water molecule, whichattacks the acyl-Ser 195, forming a second tetrahedral intermediate.5 The charge-relay system donates a protonto Ser 195, facilitating breakdown of tetrahedral intermediateto release the carboxyl terminal peptide 6 .CATALYTIC RESIDUES AREHIGHLY CONSERVEDMembers of an enzyme family such as the aspartic orserine proteases employ a similar mechanism to catalyzea common reaction type but act on different substrates.Enzyme families appear to arise through gene duplicationevents that create a second copy of the gene whichencodes a particular enzyme. The proteins encoded bythe two genes can then evolve independently to recognizedifferent substrates—resulting, for example, inchymotrypsin, which cleaves peptide bonds on the carboxylterminal side of large hydrophobic amino acids;and trypsin, which cleaves peptide bonds on the carboxylterminal side of basic amino acids. The commonancestry of enzymes can be inferred from the presenceof specific amino acids in the same position in eachfamily member. These residues are said to be conservedresidues. Proteins that share a large number of conservedresidues are said to be homologous to one another.Table 7–1 illustrates the primary structural conservationof two components of the charge-relaynetwork for several serine proteases. Among the mosthighly conserved residues are those that participate directlyin catalysis.ISOZYMES ARE DISTINCT ENZYMEFORMS THAT CATALYZE THESAME REACTIONHigher organisms often elaborate several physically distinctversions of a given enzyme, each of which catalyzesthe same reaction. Like the members of otherprotein families, these protein catalysts or isozymesarise through gene duplication. Isozymes may exhibitsubtle differences in properties such as sensitivity to

ENZYMES: MECHANISM OF ACTION / 55Glu327His392Arg 257+6–P +2–– O++ H P+E • Fru-2,6-P 2H HOGlu327His392Arg 257E-P • H 2 O+–++ H P+His 258+His 258Lys 356Arg352Arg 3071Lys 356Arg352Arg 3073Glu327His392Arg 2572–E-P • Fru-6-P–Glu+327His392Arg 257E • P i+6–P +– O H+P++His 258+Lys 356Arg352Arg 3072Lys 356Arg352Arg 307P + i+4His 258Figure 7–8. Catalysis by fructose-2,6-bisphosphatase.(1) Lys 356 and Arg 257, 307, and 352 stabilizethe quadruple negative charge of the substrate bycharge-charge interactions. Glu 327 stabilizes the positivecharge on His 392. (2) The nucleophile His 392 attacksthe C-2 phosphoryl group and transfers it to His258, forming a phosphoryl-enzyme intermediate. Fructose6-phosphate leaves the enzyme. (3) Nucleophilicattack by a water molecule, possibly assisted by Glu 327acting as a base, forms inorganic phosphate. (4) Inorganicorthophosphate is released from Arg 257 and Arg307. (Reproduced, with permission, from Pilkis SJ et al: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: Ametabolic signaling enzyme. Annu Rev Biochem1995;64:799.)particular regulatory factors (Chapter 9) or substrateaffinity (eg, hexokinase and glucokinase) that adaptthem to specific tissues or circumstances. Some isozymesmay also enhance survival by providing a “backup”copy of an essential enzyme.THE CATALYTIC ACTIVITY OF ENZYMESFACILITATES THEIR DETECTIONThe minute quantities of enzymes present in cells complicatedetermination of their presence and concentration.However, the ability to rapidly transform thousandsof molecules of a specific substrate into productsimbues each enzyme with the ability to reveal its presence.Assays of the catalytic activity of enzymes are frequentlyused in research and clinical laboratories.Under appropriate conditions (see Chapter 8), the rateof the catalytic reaction being monitored is proportionateto the amount of enzyme present, which allows itsconcentration to be inferred.Enzyme-Linked ImmunoassaysThe sensitivity of enzyme assays can also be exploited todetect proteins that lack catalytic activity. Enzymelinkedimmunoassays (ELISAs) use antibodies covalentlylinked to a “reporter enzyme” such as alkalinephosphatase or horseradish peroxidase, enzymes whoseproducts are readily detected. When serum or othersamples to be tested are placed in a plastic microtiterplate, the proteins adhere to the plastic surface and areimmobilized. Any remaining absorbing areas of the wellare then “blocked” by adding a nonantigenic proteinsuch as bovine serum albumin. A solution of antibodycovalently linked to a reporter enzyme is then added.The antibodies adhere to the immobilized antigen andthese are themselves immobilized. Excess free antibodymolecules are then removed by washing. The presenceand quantity of bound antibody are then determinedby adding the substrate for the reporter enzyme.Table 7–1. Amino acid sequences in the neighborhood of the catalytic sites of severalbovine proteases. Regions shown are those on either side of the catalytic site seryl (S) andhistidyl (H) residues.Enzyme Sequence Around Serine S Sequence Around Histidine HTrypsin D S C Q D G S G G P V V C S G K V V S A A H C Y K S GChymotrypsin A S S C M G D S G G P L V C K K N V V T A A H G G V T TChymotrypsin B S S C M G D S G G P L V C Q K N V V T A A H C G V T TThrombin D A C E G D S G G P F V M K S P V L T A A H C L L Y P

56 / CHAPTER 7NAD(P) + -Dependent Dehydrogenases AreAssayed SpectrophotometricallyThe physicochemical properties of the reactants in anenzyme-catalyzed reaction dictate the options for theassay of enzyme activity. Spectrophotometric assays exploitthe ability of a substrate or product to absorblight. The reduced coenzymes NADH and NADPH,written as NAD(P)H, absorb light at a wavelength of340 nm, whereas their oxidized forms NAD(P) + do not(Figure 7–9). When NAD(P) + is reduced, the absorbanceat 340 nm therefore increases in proportionto—and at a rate determined by—the quantity ofNAD(P)H produced. Conversely, for a dehydrogenasethat catalyzes the oxidation of NAD(P)H, a decrease inabsorbance at 340 nm will be observed. In each case,the rate of change in optical density at 340 nm will beproportionate to the quantity of enzyme present.Many Enzymes Are Assayed by Couplingto a DehydrogenaseThe assay of enzymes whose reactions are not accompaniedby a change in absorbance or fluorescence is generallymore difficult. In some instances, the product or remainingsubstrate can be transformed into a morereadily detected compound. In other instances, the reactionproduct may have to be separated from unreactedsubstrate prior to measurement—a process facilitatedby the use of radioactive substrates. An alternativestrategy is to devise a synthetic substrate whose productabsorbs light. For example, p-nitrophenyl phosphate isan artificial substrate for certain phosphatases and forchymotrypsin that does not absorb visible light. However,following hydrolysis, the resulting p-nitrophenylateanion absorbs light at 419 nm.Another quite general approach is to employ a “coupled”assay (Figure 7–10). Typically, a dehydrogenasewhose substrate is the product of the enzyme of interestis added in catalytic excess. The rate of appearance ordisappearance of NAD(P)H then depends on the rateof the enzyme reaction to which the dehydrogenase hasbeen coupled.THE ANALYSIS OF CERTAIN ENZYMESAIDS DIAGNOSISOf the thousands of different enzymes present in thehuman body, those that fulfill functions indispensableto cell vitality are present throughout the body tissues.Other enzymes or isozymes are expressed only in specificcell types, during certain periods of development,or in response to specific physiologic or pathophysiologicchanges. Analysis of the presence and distributionof enzymes and isozymes—whose expression is normallytissue-, time-, or circumstance-specific—oftenaids diagnosis.1.0Glucose0.8HEXOKINASEATP, Mg 2+Optical density0.60.4NADHADP, Mg 2+Glucose 6-phosphateNADP +GLUCOSE-6-PHOSPHATEDEHYDROGENASENADPH + H +0.26-Phosphogluconolactone0NAD +200 250 300 350 400Wavelength (nm)Figure 7–9. Absorption spectra of NAD + and NADH.Densities are for a 44 mg/L solution in a cell with a 1 cmlight path. NADP + and NADPH have spectrums analogousto NAD + and NADH, respectively.Figure 7–10. Coupled enzyme assay for hexokinaseactivity. The production of glucose 6-phosphate byhexokinase is coupled to the oxidation of this productby glucose-6-phosphate dehydrogenase in the presenceof added enzyme and NADP + . When an excess ofglucose-6-phosphate dehydrogenase is present, therate of formation of NADPH, which can be measured at340 nm, is governed by the rate of formation of glucose6-phosphate by hexokinase.

ENZYMES: MECHANISM OF ACTION / 57Nonfunctional Plasma Enzymes AidDiagnosis & PrognosisCertain enzymes, proenzymes, and their substrates arepresent at all times in the circulation of normal individualsand perform a physiologic function in the blood.Examples of these functional plasma enzymes includelipoprotein lipase, pseudocholinesterase, and the proenzymesof blood coagulation and blood clot dissolution(Chapters 9 and 51). The majority of these enzymes aresynthesized in and secreted by the liver.Plasma also contains numerous other enzymes thatperform no known physiologic function in blood.These apparently nonfunctional plasma enzymes arisefrom the routine normal destruction of erythrocytes,leukocytes, and other cells. Tissue damage or necrosisresulting from injury or disease is generally accompaniedby increases in the levels of several nonfunctionalplasma enzymes. Table 7–2 lists several enzymes usedin diagnostic enzymology.Isozymes of Lactate Dehydrogenase AreUsed to Detect Myocardial InfarctionsL-Lactate dehydrogenase is a tetrameric enzyme whosefour subunits occur in two isoforms, designated H (forTable 7–2. Principal serum enzymes used inclinical diagnosis. Many of the enzymes are notspecific for the disease listed.Serum EnzymeAminotransferasesAspartate aminotransferase(AST, or SGOT)Alanine aminotransferase(ALT, or SGPT)AmylaseCeruloplasminCreatine kinaseγ-Glutamyl transpeptidaseLactate dehydrogenase(isozymes)LipasePhosphatase, acidPhosphatase, alkaline(isozymes)Major Diagnostic UseMyocardial infarctionViral hepatitisAcute pancreatitisHepatolenticular degeneration(Wilson’s disease)Muscle disorders and myocardialinfarctionVarious liver diseasesMyocardial infarctionAcute pancreatitisMetastatic carcinoma of theprostateVarious bone disorders, obstructiveliver diseasesheart) and M (for muscle). The subunits can combineas shown below to yield catalytically active isozymes ofL-lactate dehydrogenase:LactateDehydrogenaseIsozymeI 1I 2I 3I 4I 5SubunitsHHHHHHHMHHMMHMMMMMMMDistinct genes whose expression is differentially regulatedin various tissues encode the H and M subunits.Since heart expresses the H subunit almost exclusively,isozyme I 1 predominates in this tissue. By contrast,isozyme I 5 predominates in liver. Small quantities oflactate dehydrogenase are normally present in plasma.Following a myocardial infarction or in liver disease,the damaged tissues release characteristic lactate dehydrogenaseisoforms into the blood. The resulting elevationin the levels of the I 1 or I 5 isozymes is detected byseparating the different oligomers of lactate dehydrogenaseby electrophoresis and assaying their catalytic activity(Figure 7–11).ENZYMES FACILITATE DIAGNOSISOF GENETIC DISEASESWhile many diseases have long been known to resultfrom alterations in an individual’s DNA, tools for thedetection of genetic mutations have only recently becomewidely available. These techniques rely upon thecatalytic efficiency and specificity of enzyme catalysts.For example, the polymerase chain reaction (PCR) reliesupon the ability of enzymes to serve as catalytic amplifiersto analyze the DNA present in biologic andforensic samples. In the PCR technique, a thermostableDNA polymerase, directed by appropriate oligonucleotideprimers, produces thousands of copies of asample of DNA that was present initially at levels toolow for direct detection.The detection of restriction fragment length polymorphisms(RFLPs) facilitates prenatal detection ofhereditary disorders such as sickle cell trait, betathalassemia,infant phenylketonuria, and Huntington’sdisease. Detection of RFLPs involves cleavage of double-strandedDNA by restriction endonucleases, whichcan detect subtle alterations in DNA that affect theirrecognized sites. Chapter 40 provides further detailsconcerning the use of PCR and restriction enzymes fordiagnosis.

58 / CHAPTER 7+ –(Lactate)SH 2LACTATEDEHYDROGENASES(Pyruvate)HeartANAD +NADH + H +NormalBReduced PMSOxidized PMSOxidized NBT(colorless)Reduced NBT(blue formazan)LiverC54 3 2 1Figure 7–11. Normal and pathologic patterns of lactate dehydrogenase (LDH) isozymes in humanserum. LDH isozymes of serum were separated by electrophoresis and visualized using the coupled reactionscheme shown on the left. (NBT, nitroblue tetrazolium; PMS, phenazine methylsulfate). At right isshown the stained electropherogram. Pattern A is serum from a patient with a myocardial infarct; B is normalserum; and C is serum from a patient with liver disease. Arabic numerals denote specific LDH isozymes.RECOMBINANT DNA PROVIDES ANIMPORTANT TOOL FOR STUDYINGENZYMESRecombinant DNA technology has emerged as an importantasset in the study of enzymes. Highly purifiedsamples of enzymes are necessary for the study of theirstructure and function. The isolation of an individualenzyme, particularly one present in low concentration,from among the thousands of proteins present in a cellcan be extremely difficult. If the gene for the enzyme ofinterest has been cloned, it generally is possible to producelarge quantities of its encoded protein in Escherichiacoli or yeast. However, not all animal proteinscan be expressed in active form in microbial cells, nordo microbes perform certain posttranslational processingtasks. For these reasons, a gene may be expressed incultured animal cell systems employing the baculovirusexpression vector to transform cultured insect cells. Formore details concerning recombinant DNA techniques,see Chapter 40.Recombinant Fusion Proteins Are Purifiedby Affinity ChromatographyRecombinant DNA technology can also be used to createmodified proteins that are readily purified by affinitychromatography. The gene of interest is linked to anoligonucleotide sequence that encodes a carboxyl oramino terminal extension to the encoded protein. Theresulting modified protein, termed a fusion protein,contains a domain tailored to interact with a specificaffinity support. One popular approach is to attach anoligonucleotide that encodes six consecutive histidineresidues. The expressed “His tag” protein binds to chromatographicsupports that contain an immobilized divalentmetal ion such as Ni 2+ . Alternatively, the substratebindingdomain of glutathione S-transferase (GST) canserve as a “GST tag.” Figure 7–12 illustrates the purificationof a GST-fusion protein using an affinity supportcontaining bound glutathione. Fusion proteins alsooften encode a cleavage site for a highly specific proteasesuch as thrombin in the region that links the two portionsof the protein. This permits removal of the addedfusion domain following affinity purification.Site-Directed Mutagenesis ProvidesMechanistic InsightsOnce the ability to express a protein from its clonedgene has been established, it is possible to employ sitedirectedmutagenesis to change specific aminoacylresidues by altering their codons. Used in combinationwith kinetic analyses and x-ray crystallography, this approachfacilitates identification of the specific roles ofgiven aminoacyl residues in substrate binding and catalysis.For example, the inference that a particularaminoacyl residue functions as a general acid can betested by replacing it with an aminoacyl residue incapableof donating a proton.

ENZYMES: MECHANISM OF ACTION / 59GSTPlasmid encoding GSTwith thrombin site (T)SepharosebeadGSHTGSTGSTGSHTTGSTCloned DNAencoding enzymeLigate togetherEnzymeEnzymeEnzymeTransfect cells, addinducing agent, thenbreak cellsApply to glutathione (GSH)affinity columnTElute with GSH,treat with thrombinEnzymeFigure 7–12. Use of glutathione S-transferase (GST)fusion proteins to purify recombinant proteins. (GSH,glutathione.)SUMMARY• Enzymes are highly effective and extremely specificcatalysts.• Organic and inorganic prosthetic groups, cofactors,and coenzymes play important roles in catalysis.Coenzymes, many of which are derivatives of B vitamins,serve as “shuttles.”• Catalytic mechanisms employed by enzymes includethe introduction of strain, approximation of reactants,acid-base catalysis, and covalent catalysis.• Aminoacyl residues that participate in catalysis arehighly conserved among all classes of a given enzymeactivity.• Substrates and enzymes induce mutual conformationalchanges in one another that facilitate substraterecognition and catalysis.• The catalytic activity of enzymes reveals their presence,facilitates their detection, and provides the basisfor enzyme-linked immunoassays.• Many enzymes can be assayed spectrophotometricallyby coupling them to an NAD(P) + -dependentdehydrogenase.• Assay of plasma enzymes aids diagnosis and prognosis.For example, a myocardial infarction elevatesserum levels of lactate dehydrogenase isozyme I 1 .• Restriction endonucleases facilitate diagnosis of geneticdiseases by revealing restriction fragment lengthpolymorphisms.• Site-directed mutagenesis, used to change residuessuspected of being important in catalysis or substratebinding, provides insights into the mechanisms ofenzyme action.• Recombinant fusion proteins such as His-tagged orGST fusion enzymes are readily purified by affinitychromatography.REFERENCESConyers GB et al: Metal requirements of a diadenosine pyrophosphatasefrom Bartonella bacilliformis. Magnetic resonance andkinetic studies of the role of Mn 2+ . Biochemistry 2000;39:2347.Fersht A: Structure and Mechanism in Protein Science: A Guide toEnzyme Catalysis and Protein Folding. Freeman, 1999.Suckling CJ: Enzyme Chemistry. Chapman & Hall, 1990.Walsh CT: Enzymatic Reaction Mechanisms. Freeman, 1979.

Enzymes: Kinetics 8Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEEnzyme kinetics is the field of biochemistry concernedwith the quantitative measurement of the rates of enzyme-catalyzedreactions and the systematic study of factorsthat affect these rates. Kinetic analyses permit scientiststo reconstruct the number and order of theindividual steps by which enzymes transform substratesinto products. The study of enzyme kinetics also representsthe principal way to identify potential therapeuticagents that selectively enhance or inhibit the rates of specificenzyme-catalyzed processes. Together with sitedirectedmutagenesis and other techniques that probeprotein structure, kinetic analysis can also reveal detailsof the catalytic mechanism. A complete, balanced set ofenzyme activities is of fundamental importance for maintaininghomeostasis. An understanding of enzyme kineticsthus is important for understanding how physiologicstresses such as anoxia, metabolic acidosis or alkalosis,toxins, and pharmacologic agents affect that balance.CHEMICAL REACTIONS ARE DESCRIBEDUSING BALANCED EQUATIONSA balanced chemical equation lists the initial chemicalspecies (substrates) present and the new chemicalspecies (products) formed for a particular chemical reaction,all in their correct proportions or stoichiometry.For example, balanced equation (1) below describesthe reaction of one molecule each of substrates A and Bto form one molecule each of products P and Q.A+ B ← → P+QThe double arrows indicate reversibility, an intrinsicproperty of all chemical reactions. Thus, for reaction(1), if A and B can form P and Q, then P and Q canalso form A and B. Designation of a particular reactantas a “substrate” or “product” is therefore somewhat arbitrarysince the products for a reaction written in onedirection are the substrates for the reverse reaction. Theterm “products” is, however, often used to designatethe reactants whose formation is thermodynamically favored.Reactions for which thermodynamic factorsstrongly favor formation of the products to which thearrow points often are represented with a single arrowas if they were “irreversible”:(1)60A+ B → P+QUnidirectional arrows are also used to describe reactionsin living cells where the products of reaction (2)are immediately consumed by a subsequent enzymecatalyzedreaction. The rapid removal of product P orQ therefore precludes occurrence of the reverse reaction,rendering equation (2) functionally irreversibleunder physiologic conditions.CHANGES IN FREE ENERGY DETERMINETHE DIRECTION & EQUILIBRIUM STATEOF CHEMICAL REACTIONSThe Gibbs free energy change ∆G (also called either thefree energy or Gibbs energy) describes both the directionin which a chemical reaction will tend to proceedand the concentrations of reactants and products thatwill be present at equilibrium. ∆G for a chemical reactionequals the sum of the free energies of formation ofthe reaction products ∆G P minus the sum of the freeenergies of formation of the substrates ∆G S . ∆G 0 denotesthe change in free energy that accompanies transitionfrom the standard state, one-molar concentrationsof substrates and products, to equilibrium. A more usefulbiochemical term is ∆G 0′ , which defines ∆G 0 at astandard state of 10 −7 M protons, pH 7.0 (Chapter 10).If the free energy of the products is lower than that ofthe substrates, the signs of ∆G 0 and ∆G 0′ will be negative,indicating that the reaction as written is favored inthe direction left to right. Such reactions are referred toas spontaneous. The sign and the magnitude of thefree energy change determine how far the reaction willproceed. Equation (3)—∆G 0 =− RTln Keq—illustrates the relationship between the equilibriumconstant K eq and ∆G 0 , where R is the gas constant (1.98cal/mol/°K or 8.31 J/mol/°K) and T is the absolutetemperature in degrees Kelvin. K eq is equal to the productof the concentrations of the reaction products, eachraised to the power of their stoichiometry, divided bythe product of the substrates, each raised to the powerof their stoichiometry.(2)(3)

ENZYMES: KINETICS / 61For the reaction A + B → P+Q—and for reaction (5)P QK eq = [ ][ ][ A][ B]A+ A → ← PPK eq = [ ][ A] 2—∆G 0 may be calculated from equation (3) if the concentrationsof substrates and products present at equilibriumare known. If ∆G 0 is a negative number, K eqwill be greater than unity and the concentration ofproducts at equilibrium will exceed that of substrates. If∆G 0 is positive, K eq will be less than unity and the formationof substrates will be favored.Notice that, since ∆G 0 is a function exclusively ofthe initial and final states of the reacting species, it canprovide information only about the direction and equilibriumstate of the reaction. ∆G 0 is independent of themechanism of the reaction and therefore provides noinformation concerning rates of reactions. Consequently—andas explained below—although a reactionmay have a large negative ∆G 0 or ∆G 0′ , it may neverthelesstake place at a negligible rate.THE RATES OF REACTIONSARE DETERMINED BY THEIRACTIVATION ENERGYReactions Proceed via Transition StatesThe concept of the transition state is fundamental tounderstanding the chemical and thermodynamic basisof catalysis. Equation (7) depicts a displacement reactionin which an entering group E displaces a leavinggroup L, attached initially to R.E+ R−L → ← E −R+L(7)Midway through the displacement, the bond betweenR and L has weakened but has not yet been completelysevered, and the new bond between E and R is as yetincompletely formed. This transient intermediate—inwhich neither free substrate nor product exists—istermed the transition state, ERL. Dotted linesrepresent the “partial” bonds that are undergoing formationand rupture.Reaction (7) can be thought of as consisting of two“partial reactions,” the first corresponding to the formation(F) and the second to the subsequent decay (D) ofthe transition state intermediate. As for all reactions,(4)(5)(6)characteristic changes in free energy, ∆G F, and ∆G Dareassociated with each partial reaction.E+ R− L → ← ELLR L ∆GFELL R L → ← E −R+L ∆G DE+ R − L ←→ E − R + L ∆ G = ∆ GF+ ∆ GD(8)(9)(8-10)For the overall reaction (10), ∆G is the sum of ∆G Fand∆G D. As for any equation of two terms, it is not possibleto infer from ∆G either the sign or the magnitudeof ∆G For ∆G D.Many reactions involve multiple transition states,each with an associated change in free energy. For thesereactions, the overall ∆G represents the sum of all ofthe free energy changes associated with the formationand decay of all of the transition states. Therefore, it isnot possible to infer from the overall G the numberor type of transition states through which the reactionproceeds. Stated another way: overall thermodynamicstells us nothing about kinetics.∆G F Defines the Activation EnergyRegardless of the sign or magnitude of ∆G, ∆G Ffor theoverwhelming majority of chemical reactions has a positivesign. The formation of transition state intermediatestherefore requires surmounting of energy barriers.For this reason, ∆G Fis often termed the activation energy,E act , the energy required to surmount a given energybarrier. The ease—and hence the frequency—withwhich this barrier is overcome is inversely related toE act . The thermodynamic parameters that determinehow fast a reaction proceeds thus are the ∆G Fvalues forformation of the transition states through which the reactionproceeds. For a simple reaction, where means“proportionate to,”−EactRate ∝ e RT(11)The activation energy for the reaction proceeding in theopposite direction to that drawn is equal to −∆G D.NUMEROUS FACTORS AFFECTTHE REACTION RATEThe kinetic theory—also called the collision theory—of chemical kinetics states that for two molecules toreact they must (1) approach within bond-forming distanceof one another, or “collide”; and (2) must possesssufficient kinetic energy to overcome the energy barrierfor reaching the transition state. It therefore follows

62 / CHAPTER 8that anything which increases the frequency or energy ofcollision between substrates will increase the rate of thereaction in which they participate.TemperatureRaising the temperature increases the kinetic energy ofmolecules. As illustrated in Figure 8–1, the total numberof molecules whose kinetic energy exceeds the energybarrier E act (vertical bar) for formation of productsincreases from low (A), through intermediate (B), tohigh (C) temperatures. Increasing the kinetic energy ofmolecules also increases their motion and therefore thefrequency with which they collide. This combination ofmore frequent and more highly energetic and productivecollisions increases the reaction rate.Reactant ConcentrationThe frequency with which molecules collide is directlyproportionate to their concentrations. For two differentmolecules A and B, the frequency with which they collidewill double if the concentration of either A or B isdoubled. If the concentrations of both A and B are doubled,the probability of collision will increase fourfold.For a chemical reaction proceeding at constant temperaturethat involves one molecule each of A and B,Number ofmolecules∞0Figure 8–1.reactions.AA+ B→PEnergy barrierBKinetic energyThe energy barrier for chemicalC∞(12)the number of molecules that possess kinetic energysufficient to overcome the activation energy barrier willbe a constant. The number of collisions with sufficientenergy to produce product P therefore will be directlyproportionate to the number of collisions between Aand B and thus to their molar concentrations, denotedby square brackets.Rate ∝ [ A][ B]Similarly, for the reaction represented byA+ 2B→P(13)(14)which can also be written asthe corresponding rate expression isor(15)(16)(17)For the general case when n molecules of A react withm molecules of B,the rate expression is(18)(19)Replacing the proportionality constant with an equalsign by introducing a proportionality or rate constantk characteristic of the reaction under study gives equations(20) and (21), in which the subscripts 1 and −1refer to the rate constants for the forward and reversereactions, respectively.(20)(21)K eq Is a Ratio of Rate ConstantsWhile all chemical reactions are to some extent reversible,at equilibrium the overall concentrations of reactantsand products remain constant. At equilibrium,the rate of conversion of substrates to products thereforeequals the rate at which products are converted tosubstrates.Therefore,andA+ B+ B→PRate ∝ [ A][ B][ B]Rate ∝ [ A][ B] 2nA + mB →Pn mRate ∝ [ A] [ B]n mRate1= k 1[ A] [ B]Rate−1= k −1[ P]Rate1= Rate−1n mk1[ A] [ B] = k − 1[ P]k 1 [ P]=kn m−1 [ A] [ B](22)(23)(24)The ratio of k 1 to k −1 is termed the equilibrium constant,K eq . The following important properties of a systemat equilibrium must be kept in mind:(1) The equilibrium constant is a ratio of the reactionrate constants (not the reaction rates).

ENZYMES: KINETICS / 63(2) At equilibrium, the reaction rates (not the rateconstants) of the forward and back reactions areequal.(3) Equilibrium is a dynamic state. Although there isno net change in the concentration of substratesor products, individual substrate and productmolecules are continually being interconverted.(4) The numeric value of the equilibrium constantK eq can be calculated either from the concentrationsof substrates and products at equilibrium orfrom the ratio k 1 /k −1 .THE KINETICS OFENZYMATIC CATALYSISEnzymes Lower the Activation EnergyBarrier for a ReactionAll enzymes accelerate reaction rates by providing transitionstates with a lowered ∆G Ffor formation of thetransition states. However, they may differ in the waythis is achieved. Where the mechanism or the sequenceof chemical steps at the active site is essentially the sameas those for the same reaction proceeding in the absenceof a catalyst, the environment of the active site lowersG Fby stabilizing the transition state intermediates. Asdiscussed in Chapter 7, stabilization can involve (1)acid-base groups suitably positioned to transfer protonsto or from the developing transition state intermediate,(2) suitably positioned charged groups or metal ionsthat stabilize developing charges, or (3) the impositionof steric strain on substrates so that their geometry approachesthat of the transition state. HIV protease (Figure7–6) illustrates catalysis by an enzyme that lowersthe activation barrier by stabilizing a transition state intermediate.Catalysis by enzymes that proceeds via a unique reactionmechanism typically occurs when the transitionstate intermediate forms a covalent bond with the enzyme(covalent catalysis). The catalytic mechanism ofthe serine protease chymotrypsin (Figure 7–7) illustrateshow an enzyme utilizes covalent catalysis to providea unique reaction pathway.ENZYMES DO NOT AFFECT K eqEnzymes accelerate reaction rates by lowering the activationbarrier ∆G F. While they may undergo transientmodification during the process of catalysis, enzymesemerge unchanged at the completion of the reaction.The presence of an enzyme therefore has no effect on∆G 0 for the overall reaction, which is a function solelyof the initial and final states of the reactants. Equation(25) shows the relationship between the equilibriumconstant for a reaction and the standard free energychange for that reaction:o∆G =− RTln K eq(25)If we include the presence of the enzyme (E) in the calculationof the equilibrium constant for a reaction,A + B + Enz → ← P+ Q +Enzthe expression for the equilibrium constant,(26)[ P][ Q][ Enz]K eq = (27)[ A][ B][ Enz]reduces to one identical to that for the reaction in theabsence of the enzyme:P QK eq = [ ][ ][ A][ B]Enzymes therefore have no effect on K eq .(28)MULTIPLE FACTORS AFFECT THE RATESOF ENZYME-CATALYZED REACTIONSTemperatureRaising the temperature increases the rate of both uncatalyzedand enzyme-catalyzed reactions by increasing thekinetic energy and the collision frequency of the reactingmolecules. However, heat energy can also increasethe kinetic energy of the enzyme to a point that exceedsthe energy barrier for disrupting the noncovalent interactionsthat maintain the enzyme’s three-dimensionalstructure. The polypeptide chain then begins to unfold,or denature, with an accompanying rapid loss of catalyticactivity. The temperature range over which anenzyme maintains a stable, catalytically competent conformationdepends upon—and typically moderatelyexceeds—the normal temperature of the cells in whichit resides. Enzymes from humans generally exhibit stabilityat temperatures up to 45–55 °C. By contrast,enzymes from the thermophilic microorganisms that residein volcanic hot springs or undersea hydrothermalvents may be stable up to or above 100 °C.The Q 10 , or temperature coefficient, is the factorby which the rate of a biologic process increases for a10 °C increase in temperature. For the temperaturesover which enzymes are stable, the rates of most biologicprocesses typically double for a 10 °C rise in temperature(Q 10 = 2). Changes in the rates of enzymecatalyzedreactions that accompany a rise or fall in bodytemperature constitute a prominent survival feature for“cold-blooded” life forms such as lizards or fish, whosebody temperatures are dictated by the external environment.However, for mammals and other homeothermicorganisms, changes in enzyme reaction rates with temperatureassume physiologic importance only in circumstancessuch as fever or hypothermia.

64 / CHAPTER 8Hydrogen Ion ConcentrationThe rate of almost all enzyme-catalyzed reactions exhibitsa significant dependence on hydrogen ion concentration.Most intracellular enzymes exhibit optimalactivity at pH values between 5 and 9. The relationshipof activity to hydrogen ion concentration (Figure 8–2)reflects the balance between enzyme denaturation athigh or low pH and effects on the charged state of theenzyme, the substrates, or both. For enzymes whosemechanism involves acid-base catalysis, the residues involvedmust be in the appropriate state of protonationfor the reaction to proceed. The binding and recognitionof substrate molecules with dissociable groups alsotypically involves the formation of salt bridges with theenzyme. The most common charged groups are thenegative carboxylate groups and the positively chargedgroups of protonated amines. Gain or loss of criticalcharged groups thus will adversely affect substrate bindingand thus will retard or abolish catalysis.ASSAYS OF ENZYME-CATALYZEDREACTIONS TYPICALLY MEASURETHE INITIAL VELOCITYMost measurements of the rates of enzyme-catalyzed reactionsemploy relatively short time periods, conditionsthat approximate initial rate conditions. Under theseconditions, only traces of product accumulate, hencethe rate of the reverse reaction is negligible. The initialvelocity (v i ) of the reaction thus is essentially that of100%XSH + E –the rate of the forward reaction. Assays of enzyme activityalmost always employ a large (10 3 –10 7 ) molar excessof substrate over enzyme. Under these conditions, v i isproportionate to the concentration of enzyme. Measuringthe initial velocity therefore permits one to estimatethe quantity of enzyme present in a biologic sample.SUBSTRATE CONCENTRATION AFFECTSREACTION RATEIn what follows, enzyme reactions are treated as if theyhad only a single substrate and a single product. Whilemost enzymes have more than one substrate, the principlesdiscussed below apply with equal validity to enzymeswith multiple substrates.For a typical enzyme, as substrate concentration isincreased, v i increases until it reaches a maximum valueV max (Figure 8–3). When further increases in substrateconcentration do not further increase v i , the enzyme issaid to be “saturated” with substrate. Note that theshape of the curve that relates activity to substrate concentration(Figure 8–3) is hyperbolic. At any given instant,only substrate molecules that are combined withthe enzyme as an ES complex can be transformed intoproduct. Second, the equilibrium constant for the formationof the enzyme-substrate complex is not infinitelylarge. Therefore, even when the substrate is presentin excess (points A and B of Figure 8–4), only afraction of the enzyme may be present as an ES complex.At points A or B, increasing or decreasing [S]therefore will increase or decrease the number of EScomplexes with a corresponding change in v i . At pointC (Figure 8–4), essentially all the enzyme is present asthe ES complex. Since no free enzyme remains availablefor forming ES, further increases in [S] cannot increasethe rate of the reaction. Under these saturating conditions,v i depends solely on—and thus is limited by—the rapidity with which free enzyme is released to combinewith more substrate.0Low HighFigure 8–2. Effect of pH on enzyme activity. Consider,for example, a negatively charged enzyme (EH − )that binds a positively charged substrate (SH + ). Shownis the proportion (%) of SH + [\\\] and of EH − [///] as afunction of pH. Only in the cross-hatched area do boththe enzyme and the substrate bear an appropriatecharge.pHV maxCV max /2v iBAV max /2K m[S]Figure 8–3. Effect of substrate concentration on theinitial velocity of an enzyme-catalyzed reaction.

ENZYMES: KINETICS / 65= S= EA B CFigure 8–4. Representation of an enzyme at low (A), at high (C), and at a substrate concentrationequal to K m (B). Points A, B, and C correspond to those points in Figure 8–3.THE MICHAELIS-MENTEN & HILLEQUATIONS MODEL THE EFFECTSOF SUBSTRATE CONCENTRATIONThe Michaelis-Menten EquationThe Michaelis-Menten equation (29) illustrates inmathematical terms the relationship between initial reactionvelocity v i and substrate concentration [S],shown graphically in Figure 8–3.V Svi = max [ ]Km+ [ S](29)The Michaelis constant K m is the substrate concentrationat which v i is half the maximal velocity(V max /2) attainable at a particular concentration ofenzyme. K m thus has the dimensions of substrate concentration.The dependence of initial reaction velocityon [S] and K m may be illustrated by evaluating theMichaelis-Menten equation under three conditions.(1) When [S] is much less than K m (point A in Figures8–3 and 8–4), the term K m + [S] is essentially equalto K m . Replacing K m + [S] with K m reduces equation(29) toVVVv1=v1≈ ≈ ⎛ ⎞max[ S]max[ S]K +K ⎝ ⎜ max⎟ [ S]m [ S]m Km⎠(30)where ≈ means “approximately equal to.” Since V maxand K m are both constants, their ratio is a constant. Inother words, when [S] is considerably below K m , v i ∝k[S]. The initial reaction velocity therefore is directlyproportionate to [S].(2) When [S] is much greater than K m (point C inFigures 8–3 and 8–4), the term K m + [S] is essentiallyequal to [S]. Replacing K m + [S] with [S] reduces equation(29) toV Svi= max[ ]Km+ [ S]V Sv ≈ max[ ]i ≈Vmax[S](31)Thus, when [S] greatly exceeds K m , the reaction velocityis maximal (V max ) and unaffected by further increases insubstrate concentration.(3) When [S] = K m (point B in Figures 8–3 and8–4).V S Svi= max[ ] V= max[ ] V= maxKm+ [ S]2[ S]2(32)Equation (32) states that when [S] equals K m , the initialvelocity is half-maximal. Equation (32) also reveals thatK m is—and may be determined experimentally from—the substrate concentration at which the initial velocityis half-maximal.A Linear Form of the Michaelis-MentenEquation Is Used to Determine K m & V maxThe direct measurement of the numeric value of V maxand therefore the calculation of K m often requires impracticallyhigh concentrations of substrate to achievesaturating conditions. A linear form of the Michaelis-Menten equation circumvents this difficulty and permitsV max and K m to be extrapolated from initial velocitydata obtained at less than saturating concentrationsof substrate. Starting with equation (29),Svi = Vmax [ ]Km+ [ S](29)

66 / CHAPTER 8invertfactorand simplify–1vi1 1 1= ⎛ mvi⎝ ⎜ K ⎞⎟ +V max ⎠ [ S]V max1K mK= m +V max [ S]1v i1=v10Km+ [ S]Vmax [ S ]Slope =1V max1[S]K mV maxFigure 8–5. Double reciprocal or Lineweaver-Burkplot of 1/v i versus 1/[S] used to evaluate K m and V max .(33)(34)(35)Equation (35) is the equation for a straight line, y = ax+ b, where y = 1/v i and x = 1/[S]. A plot of 1/v i as y as afunction of 1/[S] as x therefore gives a straight linewhose y intercept is 1/V max and whose slope is K m /V max .Such a plot is called a double reciprocal orLineweaver-Burk plot (Figure 8–5). Setting the y termof equation (36) equal to zero and solving for x revealsthat the x intercept is −1/K m .−b−10 = ax + b; therefore, x = =a KmK m is thus most easily calculated from the x intercept.K m May Approximate a Binding Constant(36)The affinity of an enzyme for its substrate is the inverseof the dissociation constant K d for dissociation of theenzyme substrate complex ES.kE+ S → 1← ESk −1kKd = −1k 1[ S]V max [ S](37)(38)Stated another way, the smaller the tendency of the enzymeand its substrate to dissociate, the greater the affinityof the enzyme for its substrate. While the Michaelisconstant K m often approximates the dissociation constantK d , this is by no means always the case. For a typicalenzyme-catalyzed reaction,the value of [S] that gives v i = V max /2 isWhen k −1 » k 2 , thenandk kE+ S → 1 2← ES → E + Pk −1[ S]k k= −1+2 = Kmk1k−1+ k2 ≈k−1[ S]k 1 ≈ ≈ K dk −1(39)(40)(41)(42)Hence, 1/K m only approximates 1/K d under conditionswhere the association and dissociation of the ES complexis rapid relative to the rate-limiting step in catalysis.For the many enzyme-catalyzed reactions for whichk −1 + k 2 is not approximately equal to k −1 , 1/K m willunderestimate 1/K d .The Hill Equation Describes the Behaviorof Enzymes That Exhibit CooperativeBinding of SubstrateWhile most enzymes display the simple saturation kineticsdepicted in Figure 8–3 and are adequately describedby the Michaelis-Menten expression, some enzymesbind their substrates in a cooperative fashionanalogous to the binding of oxygen by hemoglobin(Chapter 6). Cooperative behavior may be encounteredfor multimeric enzymes that bind substrate at multiplesites. For enzymes that display positive cooperativity inbinding substrate, the shape of the curve that relateschanges in v i to changes in [S] is sigmoidal (Figure8–6). Neither the Michaelis-Menten expression nor itsderived double-reciprocal plots can be used to evaluatecooperative saturation kinetics. Enzymologists thereforeemploy a graphic representation of the Hill equationoriginally derived to describe the cooperative binding ofO 2 by hemoglobin. Equation (43) represents the Hillequation arranged in a form that predicts a straight line,where k′ is a complex constant.

ENZYMES: KINETICS / 67∞first substrate molecule then enhances the affinity of theenzyme for binding additional substrate. The greaterthe value for n, the higher the degree of cooperativityand the more sigmoidal will be the plot of v i versus [S].A perpendicular dropped from the point where the yterm log v i /(V max − v i ) is zero intersects the x axis at asubstrate concentration termed S 50 , the substrate concentrationthat results in half-maximal velocity. S 50 thusis analogous to the P 50 for oxygen binding to hemoglobin(Chapter 6).v i0Figure 8–6. Representation of sigmoid substratesaturation kinetics.log v1= n log[S] − log k′(43)Vmax− v1Equation (43) states that when [S] is low relative to k′,the initial reaction velocity increases as the nth powerof [S].A graph of log v i /(V max − v i ) versus log[S] gives astraight line (Figure 8–7), where the slope of the line nis the Hill coefficient, an empirical parameter whosevalue is a function of the number, kind, and strength ofthe interactions of the multiple substrate-binding siteson the enzyme. When n = 1, all binding sites behave independently,and simple Michaelis-Menten kinetic behavioris observed. If n is greater than 1, the enzyme issaid to exhibit positive cooperativity. Binding of thev iV max – v iLog10– 1[S]∞Slope = nKINETIC ANALYSIS DISTINGUISHESCOMPETITIVE FROMNONCOMPETITIVE INHIBITIONInhibitors of the catalytic activities of enzymes provideboth pharmacologic agents and research tools for studyof the mechanism of enzyme action. Inhibitors can beclassified based upon their site of action on the enzyme,on whether or not they chemically modify the enzyme,or on the kinetic parameters they influence. Kinetically,we distinguish two classes of inhibitors based uponwhether raising the substrate concentration does ordoes not overcome the inhibition.Competitive Inhibitors TypicallyResemble SubstratesThe effects of competitive inhibitors can be overcomeby raising the concentration of the substrate. Most frequently,in competitive inhibition the inhibitor, I,binds to the substrate-binding portion of the active siteand blocks access by the substrate. The structures ofmost classic competitive inhibitors therefore tend to resemblethe structures of a substrate and thus are termedsubstrate analogs. Inhibition of the enzyme succinatedehydrogenase by malonate illustrates competitive inhibitionby a substrate analog. Succinate dehydrogenasecatalyzes the removal of one hydrogen atom from eachof the two methylene carbons of succinate (Figure 8–8).Both succinate and its structural analog malonate( − OOC ⎯ CH 2 ⎯ COO − ) can bind to the active site ofsuccinate dehydrogenase, forming an ES or an EI complex,respectively. However, since malonate contains– 4 S 50 – 3Log [S]Figure 8–7. A graphic representation of a linearform of the Hill equation is used to evaluate S 50 , thesubstrate concentration that produces half-maximalvelocity, and the degree of cooperativity n.HHC COO ––2HC COO –– OOC C H– OOC C HSUCCINATEDEHYDROGENASEHSuccinateFigure 8–8.HFumarateThe succinate dehydrogenase reaction.

68 / CHAPTER 8only one methylene carbon, it cannot undergo dehydrogenation.The formation and dissociation of the EIcomplex is a dynamic process described byk 1EnzI → ← Enz + Ik −1for which the equilibrium constant K i is[ Enz][ I]k 1K1= =[ EnzI] k −1(44)(45)In effect, a competitive inhibitor acts by decreasingthe number of free enzyme molecules available tobind substrate, ie, to form ES, and thus eventuallyto form product, as described below:E± I± S(46)A competitive inhibitor and substrate exert reciprocaleffects on the concentration of the EI and ES complexes.Since binding substrate removes free enzymeavailable to combine with inhibitor, increasing the [S]decreases the concentration of the EI complex andraises the reaction velocity. The extent to which [S]must be increased to completely overcome the inhibitiondepends upon the concentration of inhibitor present,its affinity for the enzyme K i , and the K m of theenzyme for its substrate.Double Reciprocal Plots Facilitate theEvaluation of InhibitorsDouble reciprocal plots distinguish between competitiveand noncompetitive inhibitors and simplify evaluationof inhibition constants K i . v i is determined at severalsubstrate concentrations both in the presence andin the absence of inhibitor. For classic competitive inhibition,the lines that connect the experimental datapoints meet at the y axis (Figure 8–9). Since the y interceptis equal to 1/V max , this pattern indicates that when1/[S] approaches 0, v i is independent of the presenceof inhibitor. Note, however, that the intercept on thex axis does vary with inhibitor concentration—and thatsince −1/K m ′ is smaller than 1/K m , K m ′ (the “apparentK m ”) becomes larger in the presence of increasing concentrationsof inhibitor. Thus, a competitive inhibitorhas no effect on V max but raises K ′ m , the apparentK m for the substrate.E-IE-SE + P–1K m–1v i1K′ m0+ InhibitorNo inhibitor1V maxFigure 8–9. Lineweaver-Burk plot of competitive inhibition.Note the complete relief of inhibition at high[S] (ie, low 1/[S]).For simple competitive inhibition, the intercept onthe x axis is1[S]−1 ⎛ [] I ⎞x = ⎜ 1 +K⎟m⎝Ki⎠(47)Once K m has been determined in the absence of inhibitor,K i can be calculated from equation (47). K i valuesare used to compare different inhibitors of the sameenzyme. The lower the value for K i , the more effectivethe inhibitor. For example, the statin drugs that act ascompetitive inhibitors of HMG-CoA reductase (Chapter26) have K i values several orders of magnitude lowerthan the K m for the substrate HMG-CoA.Simple Noncompetitive Inhibitors LowerV max but Do Not Affect K mIn noncompetitive inhibition, binding of the inhibitordoes not affect binding of substrate. Formation of bothEI and EIS complexes is therefore possible. However,while the enzyme-inhibitor complex can still bind substrate,its efficiency at transforming substrate to product,reflected by V max , is decreased. Noncompetitiveinhibitors bind enzymes at sites distinct from the substrate-bindingsite and generally bear little or no structuralresemblance to the substrate.For simple noncompetitive inhibition, E and EIpossess identical affinity for substrate, and the EIS complexgenerates product at a negligible rate (Figure 8–10).More complex noncompetitive inhibition occurs whenbinding of the inhibitor does affect the apparent affinityof the enzyme for substrate, causing the lines to interceptin either the third or fourth quadrants of a doublereciprocal plot (not shown).

ENZYMES: KINETICS / 69A B P Q1v i–1V ′ max–1K m+ InhibitorNo inhibitorEEAEAB-EPQEQA BP QEAEQE01V max1[S]EEAB-EPQEBEPEFigure 8–10. Lineweaver-Burk plot for simple noncompetitiveinhibition.B AQ PA P B QIrreversible Inhibitors “Poison” EnzymesIn the above examples, the inhibitors form a dissociable,dynamic complex with the enzyme. Fully active enzymecan therefore be recovered simply by removingthe inhibitor from the surrounding medium. However,a variety of other inhibitors act irreversibly by chemicallymodifying the enzyme. These modifications generallyinvolve making or breaking covalent bonds withaminoacyl residues essential for substrate binding, catalysis,or maintenance of the enzyme’s functional conformation.Since these covalent changes are relatively stable,an enzyme that has been “poisoned” by anirreversible inhibitor remains inhibited even after removalof the remaining inhibitor from the surroundingmedium.MOST ENZYME-CATALYZED REACTIONSINVOLVE TWO OR MORE SUBSTRATESWhile many enzymes have a single substrate, many othershave two—and sometimes more than two—substratesand products. The fundamental principles discussedabove, while illustrated for single-substrateenzymes, apply also to multisubstrate enzymes. Themathematical expressions used to evaluate multisubstratereactions are, however, complex. While detailedkinetic analysis of multisubstrate reactions exceeds thescope of this chapter, two-substrate, two-product reactions(termed “Bi-Bi” reactions) are considered below.Sequential or SingleDisplacement ReactionsIn sequential reactions, both substrates must combinewith the enzyme to form a ternary complex beforecatalysis can proceed (Figure 8–11, top). Sequential reactionsare sometimes referred to as single displacementEEA-FPF FB-EQ EFigure 8–11. Representations of three classes of Bi-Bi reaction mechanisms. Horizontal lines represent theenzyme. Arrows indicate the addition of substrates anddeparture of products. Top: An ordered Bi-Bi reaction,characteristic of many NAD(P)H-dependent oxidoreductases.Center: A random Bi-Bi reaction, characteristicof many kinases and some dehydrogenases. Bottom:A ping-pong reaction, characteristic ofaminotransferases and serine proteases.reactions because the group undergoing transfer is usuallypassed directly, in a single step, from one substrateto the other. Sequential Bi-Bi reactions can be furtherdistinguished based on whether the two substrates addin a random or in a compulsory order. For randomorderreactions, either substrate A or substrate B maycombine first with the enzyme to form an EA or an EBcomplex (Figure 8–11, center). For compulsory-orderreactions, A must first combine with E before B cancombine with the EA complex. One explanation for acompulsory-order mechanism is that the addition of Ainduces a conformational change in the enzyme thataligns residues which recognize and bind B.Ping-Pong ReactionsThe term “ping-pong” applies to mechanisms inwhich one or more products are released from the enzymebefore all the substrates have been added. Pingpongreactions involve covalent catalysis and a transient,modified form of the enzyme (Figure 7–4).Ping-pong Bi-Bi reactions are double displacement reactions.The group undergoing transfer is first displacedfrom substrate A by the enzyme to form product

70 / CHAPTER 8Increasing[S 2 ]S 11and y intercepts, but not the slope.Figure 8–12. Lineweaver-Burk plot for a two-substratev i1ping-pong reaction. An increase in concentra-tion of one substrate (S 1 ) while that of the other substrate(S 2 ) is maintained constant changes both the xP and a modified form of the enzyme (F). The subsequentgroup transfer from F to the second substrate B,forming product Q and regenerating E, constitutes thesecond displacement (Figure 8–11, bottom).Most Bi-Bi Reactions Conform toMichaelis-Menten KineticsMost Bi-Bi reactions conform to a somewhat morecomplex form of Michaelis-Menten kinetics in whichV max refers to the reaction rate attained when both substratesare present at saturating levels. Each substratehas its own characteristic K m value which correspondsto the concentration that yields half-maximal velocitywhen the second substrate is present at saturating levels.As for single-substrate reactions, double-reciprocal plotscan be used to determine V max and K m . v i is measured asa function of the concentration of one substrate (thevariable substrate) while the concentration of the othersubstrate (the fixed substrate) is maintained constant. Ifthe lines obtained for several fixed-substrate concentrationsare plotted on the same graph, it is possible to distinguishbetween a ping-pong enzyme, which yieldsparallel lines, and a sequential mechanism, which yieldsa pattern of intersecting lines (Figure 8–12).Product inhibition studies are used to complementkinetic analyses and to distinguish between ordered andrandom Bi-Bi reactions. For example, in a randomorderBi-Bi reaction, each product will be a competitiveinhibitor regardless of which substrate is designated thevariable substrate. However, for a sequential mechanism(Figure 8–11, bottom), only product Q will givethe pattern indicative of competitive inhibition when Ais the variable substrate, while only product P will producethis pattern with B as the variable substrate. Theother combinations of product inhibitor and variablesubstrate will produce forms of complex noncompetitiveinhibition.SUMMARY• The study of enzyme kinetics—the factors that affectthe rates of enzyme-catalyzed reactions—reveals theindividual steps by which enzymes transform substratesinto products.• ∆G, the overall change in free energy for a reaction,is independent of reaction mechanism and providesno information concerning rates of reactions.• Enzymes do not affect K eq. K eq , a ratio of reactionrate constants, may be calculated from the concentrationsof substrates and products at equilibrium orfrom the ratio k 1 /k −1 .• Reactions proceed via transition states in which ∆G Fis the activation energy. Temperature, hydrogen ionconcentration, enzyme concentration, substrate concentration,and inhibitors all affect the rates of enzyme-catalyzedreactions.• A measurement of the rate of an enzyme-catalyzedreaction generally employs initial rate conditions, forwhich the essential absence of product precludes thereverse reaction.• A linear form of the Michaelis-Menten equation simplifiesdetermination of K m and V max .• A linear form of the Hill equation is used to evaluatethe cooperative substrate-binding kinetics exhibitedby some multimeric enzymes. The slope n, the Hillcoefficient, reflects the number, nature, and strengthof the interactions of the substrate-binding sites. A

ENZYMES: KINETICS / 71value of n greater than 1 indicates positive cooperativity.• The effects of competitive inhibitors, which typicallyresemble substrates, are overcome by raising the concentrationof the substrate. Noncompetitive inhibitorslower V max but do not affect K m .• Substrates may add in a random order (either substratemay combine first with the enzyme) or in acompulsory order (substrate A must bind before substrateB).• In ping-pong reactions, one or more products are releasedfrom the enzyme before all the substrates haveadded.REFERENCESFersht A: Structure and Mechanism in Protein Science: A Guide toEnzyme Catalysis and Protein Folding. Freeman, 1999.Schultz AR: Enzyme Kinetics: From Diastase to Multi-enzyme Systems.Cambridge Univ Press, 1994.Segel IH: Enzyme Kinetics. Wiley Interscience, 1975.

Enzymes: Regulation of Activities 9Victor W. Rodwell, PhD, & Peter J. Kennelly, PhDBIOMEDICAL IMPORTANCEThe 19th-century physiologist Claude Bernard enunciatedthe conceptual basis for metabolic regulation. Heobserved that living organisms respond in ways that areboth quantitatively and temporally appropriate to permitthem to survive the multiple challenges posed bychanges in their external and internal environments.Walter Cannon subsequently coined the term “homeostasis”to describe the ability of animals to maintain aconstant intracellular environment despite changes intheir external environment. We now know that organismsrespond to changes in their external and internalenvironment by balanced, coordinated changes in therates of specific metabolic reactions. Many human diseases,including cancer, diabetes, cystic fibrosis, andAlzheimer’s disease, are characterized by regulatory dysfunctionstriggered by pathogenic agents or genetic mutations.For example, many oncogenic viruses elaborateprotein-tyrosine kinases that modify the regulatoryevents which control patterns of gene expression, contributingto the initiation and progression of cancer. Thetoxin from Vibrio cholerae, the causative agent of cholera,disables sensor-response pathways in intestinal epithelialcells by ADP-ribosylating the GTP-binding proteins(G-proteins) that link cell surface receptors to adenylylcyclase. The consequent activation of the cyclase triggersthe flow of water into the intestines, resulting in massivediarrhea and dehydration. Yersinia pestis, the causativeagent of plague, elaborates a protein-tyrosine phosphatasethat hydrolyzes phosphoryl groups on key cytoskeletalproteins. Knowledge of factors that control therates of enzyme-catalyzed reactions thus is essential to anunderstanding of the molecular basis of disease. Thischapter outlines the patterns by which metabolicprocesses are controlled and provides illustrative examples.Subsequent chapters provide additional examples.REGULATION OF METABOLITE FLOWCAN BE ACTIVE OR PASSIVEEnzymes that operate at their maximal rate cannot respondto an increase in substrate concentration, andcan respond only to a precipitous decrease in substrateconcentration. For most enzymes, therefore, the averageintracellular concentration of their substrate tendsto be close to the K m value, so that changes in substrate72concentration generate corresponding changes in metaboliteflux (Figure 9–1). Responses to changes in substratelevel represent an important but passive means forcoordinating metabolite flow and maintaining homeostasisin quiescent cells. However, they offer limitedscope for responding to changes in environmental variables.The mechanisms that regulate enzyme activity inan active manner in response to internal and externalsignals are discussed below.Metabolite Flow Tendsto Be UnidirectionalDespite the existence of short-term oscillations inmetabolite concentrations and enzyme levels, livingcells exist in a dynamic steady state in which the meanconcentrations of metabolic intermediates remain relativelyconstant over time (Figure 9–2). While all chemicalreactions are to some extent reversible, in living cellsthe reaction products serve as substrates for—and areremoved by—other enzyme-catalyzed reactions. Manynominally reversible reactions thus occur unidirectionally.This succession of coupled metabolic reactions isaccompanied by an overall change in free energy thatfavors unidirectional metabolite flow (Chapter 10). Theunidirectional flow of metabolites through a pathwaywith a large overall negative change in free energy isanalogous to the flow of water through a pipe in whichone end is lower than the other. Bends or kinks in thepipe simulate individual enzyme-catalyzed steps with asmall negative or positive change in free energy. Flow ofwater through the pipe nevertheless remains unidirectionaldue to the overall change in height, which correspondsto the overall change in free energy in a pathway(Figure 9–3).COMPARTMENTATION ENSURESMETABOLIC EFFICIENCY& SIMPLIFIES REGULATIONIn eukaryotes, anabolic and catabolic pathways that interconvertcommon products may take place in specificsubcellular compartments. For example, many of theenzymes that degrade proteins and polysaccharides resideinside organelles called lysosomes. Similarly, fattyacid biosynthesis occurs in the cytosol, whereas fatty

ENZYMES: REGULATION OF ACTIVITIES / 73∆V B[ S ]V ∆V AK mA∆S∆SBFigure 9–1. Differential response of the rate of anenzyme-catalyzed reaction, ∆V, to the same incrementalchange in substrate concentration at a substrateconcentration of K m (∆V A ) or far above K m (∆V B ).Figure 9–3. Hydrostatic analogy for a pathway witha rate-limiting step (A) and a step with a ∆G value nearzero (B).acid oxidation takes place within mitochondria (Chapters21 and 22). Segregation of certain metabolic pathwayswithin specialized cell types can provide furtherphysical compartmentation. Alternatively, possession ofone or more unique intermediates can permit apparentlyopposing pathways to coexist even in the absence ofphysical barriers. For example, despite many shared intermediatesand enzymes, both glycolysis and gluconeogenesisare favored energetically. This cannot be true ifall the reactions were the same. If one pathway was favoredenergetically, the other would be accompanied bya change in free energy G equal in magnitude but oppositein sign. Simultaneous spontaneity of both pathwaysresults from substitution of one or more reactionsby different reactions favored thermodynamically in theopposite direction. The glycolytic enzyme phosphofructokinase(Chapter 17) is replaced by the gluconeogenicenzyme fructose-1,6-bisphosphatase (Chapter19). The ability of enzymes to discriminate between thestructurally similar coenzymes NAD + and NADP + alsoresults in a form of compartmentation, since it segregatesthe electrons of NADH that are destined for ATPNutrientsSmallmoleculesLargemolecules~P ~PSmallmoleculesSmallmoleculesWastesFigure 9–2. An idealized cell in steady state. Notethat metabolite flow is unidirectional.generation from those of NADPH that participate inthe reductive steps in many biosynthetic pathways.Controlling an Enzyme That Catalyzesa Rate-Limiting Reaction Regulatesan Entire Metabolic PathwayWhile the flux of metabolites through metabolic pathwaysinvolves catalysis by numerous enzymes, activecontrol of homeostasis is achieved by regulation of onlya small number of enzymes. The ideal enzyme for regulatoryintervention is one whose quantity or catalytic efficiencydictates that the reaction it catalyzes is slow relativeto all others in the pathway. Decreasing thecatalytic efficiency or the quantity of the catalyst for the“bottleneck” or rate-limiting reaction immediately reducesmetabolite flux through the entire pathway. Conversely,an increase in either its quantity or catalytic efficiencyenhances flux through the pathway as a whole.For example, acetyl-CoA carboxylase catalyzes the synthesisof malonyl-CoA, the first committed reaction offatty acid biosynthesis (Chapter 21). When synthesis ofmalonyl-CoA is inhibited, subsequent reactions of fattyacid synthesis cease due to lack of substrates. Enzymesthat catalyze rate-limiting steps serve as natural “governors”of metabolic flux. Thus, they constitute efficienttargets for regulatory intervention by drugs. For example,inhibition by “statin” drugs of HMG-CoA reductase,which catalyzes the rate-limiting reaction of cholesterogenesis,curtails synthesis of cholesterol.REGULATION OF ENZYME QUANTITYThe catalytic capacity of the rate-limiting reaction in ametabolic pathway is the product of the concentrationof enzyme molecules and their intrinsic catalytic efficiency.It therefore follows that catalytic capacity can be

74 / CHAPTER 9influenced both by changing the quantity of enzyme Enzyme levels in mammalian tissues respond to apresent and by altering its intrinsic catalytic efficiency. wide range of physiologic, hormonal, or dietary factors.For example, glucocorticoids increase the concentrationof tyrosine aminotransferase by stimulating k s , andControl of Enzyme Synthesisglucagon—despite its antagonistic physiologic effects—increases k s fourfold to fivefold. Regulation of liverEnzymes whose concentrations remain essentially constantover time are termed constitutive enzymes. By a protein-rich meal, liver arginase levels rise and argi-arginase can involve changes either in k s or in k deg . Aftercontrast, the concentrations of many other enzymes dependupon the presence of inducers, typically sub-also rise in starvation, but here arginase degradation deninesynthesis decreases (Chapter 29). Arginase levelsstrates or structurally related compounds, that initiate creases, whereas k s remains unchanged. Similarly, injectionof glucocorticoids and ingestion of tryptophantheir synthesis. Escherichia coli grown on glucose will,for example, only catabolize lactose after addition of a both elevate levels of tryptophan oxygenase. While theβ-galactoside, an inducer that initiates synthesis of a hormone raises k s for oxygenase synthesis, tryptophanβ-galactosidase and a galactoside permease (Figure 39–3). specifically lowers k deg by stabilizing the oxygenaseInducible enzymes of humans include tryptophan pyrrolase,threonine dehydrase, tyrosine-α-ketoglutarateagainst proteolytic digestion.aminotransferase, enzymes of the urea cycle, HMG-CoAreductase, and cytochrome P450. Conversely, an excessof a metabolite may curtail synthesis of its cognate MULTIPLE OPTIONS ARE AVAILABLE FORenzyme via repression. Both induction and repression REGULATING CATALYTIC ACTIVITYinvolve cis elements, specific DNA sequences located upstreamof regulated genes, and trans-acting regulatory plex multistep process that typically requires hours toIn humans, the induction of protein synthesis is a com-proteins. The molecular mechanisms of induction and produce significant changes in overall enzyme level. Byrepression are discussed in Chapter 39.contrast, changes in intrinsic catalytic efficiency effectedby binding of dissociable ligands (allosteric regulation)or by covalent modification achieve regulationof enzymic activity within seconds. Changes inControl of Enzyme DegradationThe absolute quantity of an enzyme reflects the net balancebetween enzyme synthesis and enzyme degrada-protein level serve long-term adaptive requirements,whereas changes in catalytic efficiency are best suitedtion, where k s and k deg represent the rate constants forfor rapid and transient alterations in metabolite flux.the overall processes of synthesis and degradation, respectively.Changes in both the k s and k deg of specificenzymes occur in human subjects.ALLOSTERIC EFFECTORS REGULATECERTAIN ENZYMESEnzymeFeedback inhibition refers to inhibition of an enzymek s k degin a biosynthetic pathway by an end product of thatpathway. For example, for the biosynthesis of D from Acatalyzed by enzymes Enz 1 through Enz 3 ,Amino acidsProtein turnover represents the net result of enzymesynthesis and degradation. By measuring the ratesof incorporation of 15 N-labeled amino acids into proteinand the rates of loss of 15 N from protein, Schoenheimerdeduced that body proteins are in a state of “dynamicequilibrium” in which they are continuouslysynthesized and degraded. Mammalian proteins are degradedboth by ATP and ubiquitin-dependent pathwaysand by ATP-independent pathways (Chapter 29).Susceptibility to proteolytic degradation can be influencedby the presence of ligands such as substrates,coenzymes, or metal ions that alter protein conformation.Intracellular ligands thus can influence the rates atwhich specific enzymes are degraded.Enz1Enz 2 Enz3A → B → C → Dhigh concentrations of D inhibit conversion of A to B.Inhibition results not from the “backing up” of intermediatesbut from the ability of D to bind to and inhibitEnz 1 . Typically, D binds at an allosteric site spatiallydistinct from the catalytic site of the targetenzyme. Feedback inhibitors thus are allosteric effectorsand typically bear little or no structural similarity to thesubstrates of the enzymes they inhibit. In this example,the feedback inhibitor D acts as a negative allostericeffector of Enz 1 .

ENZYMES: REGULATION OF ACTIVITIES / 75In a branched biosynthetic pathway, the initial reactionsparticipate in the synthesis of several products.Figure 9–4 shows a hypothetical branched biosyntheticpathway in which curved arrows lead from feedback inhibitorsto the enzymes whose activity they inhibit. Thesequences S 3 → A, S 4 → B, S 4 → C, and S 3 →→Deach represent linear reaction sequences that are feedback-inhibitedby their end products. The pathways ofnucleotide biosynthesis (Chapter 34) provide specificexamples.The kinetics of feedback inhibition may be competitive,noncompetitive, partially competitive, or mixed.Feedback inhibitors, which frequently are the smallmolecule building blocks of macromolecules (eg, aminoacids for proteins, nucleotides for nucleic acids), typicallyinhibit the first committed step in a particularbiosynthetic sequence. A much-studied example is inhibitionof bacterial aspartate transcarbamoylase by CTP(see below and Chapter 34).Multiple feedback loops can provide additional finecontrol. For example, as shown in Figure 9–5, the presenceof excess product B decreases the requirement forsubstrate S 2 . However, S 2 is also required for synthesisof A, C, and D. Excess B should therefore also curtailsynthesis of all four end products. To circumvent thispotential difficulty, each end product typically onlypartially inhibits catalytic activity. The effect of an excessof two or more end products may be strictly additiveor, alternatively, may be greater than their individualeffect (cooperative feedback inhibition).S 1 S 2 S 3 S 4Figure 9–4. Sites of feedback inhibition in abranched biosynthetic pathway. S 1 –S 5 are intermediatesin the biosynthesis of end products A–D. Straightarrows represent enzymes catalyzing the indicated conversions.Curved arrows represent feedback loops andindicate sites of feedback inhibition by specific endproducts.AS 5DCBS 1 S 2 S 3 S 4Figure 9–5. Multiple feedback inhibition in abranched biosynthetic pathway. Superimposed on simplefeedback loops (dashed, curved arrows) are multiplefeedback loops (solid, curved arrows) that regulateenzymes common to biosynthesis of several end products.Aspartate Transcarbamoylase Is a ModelAllosteric EnzymeAspartate transcarbamoylase (ATCase), the catalyst forthe first reaction unique to pyrimidine biosynthesis(Figure 34–7), is feedback-inhibited by cytidine triphosphate(CTP). Following treatment with mercurials,ATCase loses its sensitivity to inhibition by CTPbut retains its full activity for synthesis of carbamoyl aspartate.This suggests that CTP is bound at a different(allosteric) site from either substrate. ATCase consistsof multiple catalytic and regulatory subunits. Each catalyticsubunit contains four aspartate (substrate) sitesand each regulatory subunit at least two CTP (regulatory)sites (Chapter 34).Allosteric & Catalytic Sites AreSpatially DistinctThe lack of structural similarity between a feedback inhibitorand the substrate for the enzyme whose activityit regulates suggests that these effectors are not isostericwith a substrate but allosteric (“occupy anotherspace”). Jacques Monod therefore proposed the existenceof allosteric sites that are physically distinct fromthe catalytic site. Allosteric enzymes thus are thosewhose activity at the active site may be modulated bythe presence of effectors at an allosteric site. This hypothesishas been confirmed by many lines of evidence,including x-ray crystallography and site-directed mutagenesis,demonstrating the existence of spatially distinctactive and allosteric sites on a variety of enzymes.Allosteric Effects May Be on K m or on V maxTo refer to the kinetics of allosteric inhibition as “competitive”or “noncompetitive” with substrate carriesmisleading mechanistic implications. We refer insteadto two classes of regulated enzymes: K-series and V-seriesenzymes. For K-series allosteric enzymes, the substratesaturation kinetics are competitive in the sensethat K m is raised without an effect on V max . For V-seriesallosteric enzymes, the allosteric inhibitor lowers V maxAS 5DCB

76 / CHAPTER 9without affecting the K m . Alterations in K m or V maxprobably result from conformational changes at the catalyticsite induced by binding of the allosteric effectorat the allosteric site. For a K-series allosteric enzyme,this conformational change may weaken the bonds betweensubstrate and substrate-binding residues. For aV-series allosteric enzyme, the primary effect may be toalter the orientation or charge of catalytic residues, loweringV max . Intermediate effects on K m and V max , however,may be observed consequent to these conformationalchanges.FEEDBACK REGULATIONIS NOT SYNONYMOUS WITHFEEDBACK INHIBITIONIn both mammalian and bacterial cells, end products“feed back” and control their own synthesis, in manyinstances by feedback inhibition of an early biosyntheticenzyme. We must, however, distinguish betweenfeedback regulation, a phenomenologic term devoidof mechanistic implications, and feedback inhibition,a mechanism for regulation of enzyme activity. For example,while dietary cholesterol decreases hepatic synthesisof cholesterol, this feedback regulation does notinvolve feedback inhibition. HMG-CoA reductase, therate-limiting enzyme of cholesterologenesis, is affected,but cholesterol does not feedback-inhibit its activity.Regulation in response to dietary cholesterol involvescurtailment by cholesterol or a cholesterol metabolite ofthe expression of the gene that encodes HMG-CoA reductase(enzyme repression) (Chapter 26).MANY HORMONES ACT THROUGHALLOSTERIC SECOND MESSENGERSNerve impulses—and binding of hormones to cell surfacereceptors—elicit changes in the rate of enzymecatalyzedreactions within target cells by inducing the releaseor synthesis of specialized allosteric effectors calledsecond messengers. The primary or “first” messenger isthe hormone molecule or nerve impulse. Second messengersinclude 3′,5′-cAMP, synthesized from ATP bythe enzyme adenylyl cyclase in response to the hormoneepinephrine, and Ca 2+ , which is stored inside the endoplasmicreticulum of most cells. Membrane depolarizationresulting from a nerve impulse opens a membranechannel that releases calcium ion into the cytoplasm,where it binds to and activates enzymes involved in theregulation of contraction and the mobilization of storedglucose from glycogen. Glucose then supplies the increasedenergy demands of muscle contraction. Othersecond messengers include 3′,5′-cGMP and polyphosphoinositols,produced by the hydrolysis of inositolphospholipids by hormone-regulated phospholipases.REGULATORY COVALENTMODIFICATIONS CAN BEREVERSIBLE OR IRREVERSIBLEIn mammalian cells, the two most common forms ofcovalent modification are partial proteolysis andphosphorylation. Because cells lack the ability to reunitethe two portions of a protein produced by hydrolysisof a peptide bond, proteolysis constitutes an irreversiblemodification. By contrast, phosphorylation is areversible modification process. The phosphorylation ofproteins on seryl, threonyl, or tyrosyl residues, catalyzedby protein kinases, is thermodynamically spontaneous.Equally spontaneous is the hydrolytic removal of thesephosphoryl groups by enzymes called protein phosphatases.PROTEASES MAY BE SECRETED ASCATALYTICALLY INACTIVE PROENZYMESCertain proteins are synthesized and secreted as inactiveprecursor proteins known as proproteins. The proproteinsof enzymes are termed proenzymes or zymogens.Selective proteolysis converts a proprotein by one ormore successive proteolytic “clips” to a form that exhibitsthe characteristic activity of the mature protein,eg, its enzymatic activity. Proteins synthesized as proproteinsinclude the hormone insulin (proprotein =proinsulin), the digestive enzymes pepsin, trypsin, andchymotrypsin (proproteins = pepsinogen, trypsinogen,and chymotrypsinogen, respectively), several factors ofthe blood clotting and blood clot dissolution cascades(see Chapter 51), and the connective tissue protein collagen(proprotein = procollagen).Proenzymes Facilitate RapidMobilization of an Activity in Responseto Physiologic DemandThe synthesis and secretion of proteases as catalyticallyinactive proenzymes protects the tissue of origin (eg,the pancreas) from autodigestion, such as can occur inpancreatitis. Certain physiologic processes such as digestionare intermittent but fairly regular and predictable.Others such as blood clot formation, clot dissolution,and tissue repair are brought “on line” only inresponse to pressing physiologic or pathophysiologicneed. The processes of blood clot formation and dissolutionclearly must be temporally coordinated toachieve homeostasis. Enzymes needed intermittentlybut rapidly often are secreted in an initially inactiveform since the secretion process or new synthesis of therequired proteins might be insufficiently rapid for responseto a pressing pathophysiologic demand such asthe loss of blood.

ENZYMES: REGULATION OF ACTIVITIES / 771 13 14 15 16 146 149245Pro-CT1 13 14 15 16 146 149245π-CT14-15 147-1481 13 16 146 149245α-CTSSSFigure 9–6. Selective proteolysis and associated conformational changes form theactive site of chymotrypsin, which includes the Asp102-His57-Ser195 catalytic triad.Successive proteolysis forms prochymotrypsin (pro-CT), π-chymotrypsin (π-CT), and ultimatelyα-chymotrypsin (α-CT), an active protease whose three peptides remain associatedby covalent inter-chain disulfide bonds.SActivation of ProchymotrypsinRequires Selective ProteolysisSelective proteolysis involves one or more highly specificproteolytic clips that may or may not be accompaniedby separation of the resulting peptides. Most importantly,selective proteolysis often results inconformational changes that “create” the catalytic siteof an enzyme. Note that while His 57 and Asp 102 resideon the B peptide of α-chymotrypsin, Ser 195 resideson the C peptide (Figure 9–6). The conformationalchanges that accompany selective proteolysis ofprochymotrypsin (chymotrypsinogen) align the threeresidues of the charge-relay network, creating the catalyticsite. Note also that contact and catalytic residuescan be located on different peptide chains but still bewithin bond-forming distance of bound substrate.REVERSIBLE COVALENT MODIFICATIONREGULATES KEY MAMMALIAN ENZYMESMammalian proteins are the targets of a wide range ofcovalent modification processes. Modifications such asglycosylation, hydroxylation, and fatty acid acylationintroduce new structural features into newly synthesizedproteins that tend to persist for the lifetime of theprotein. Among the covalent modifications that regulateprotein function (eg, methylation, adenylylation),the most common by far is phosphorylation-dephosphorylation.Protein kinases phosphorylate proteins bycatalyzing transfer of the terminal phosphoryl group ofATP to the hydroxyl groups of seryl, threonyl, or tyrosylresidues, forming O-phosphoseryl, O-phosphothreonyl,or O-phosphotyrosyl residues, respectively (Figure9–7). Some protein kinases target the side chains of histidyl,lysyl, arginyl, and aspartyl residues. The unmodifiedform of the protein can be regenerated by hydrolyticremoval of phosphoryl groups, catalyzed byprotein phosphatases.A typical mammalian cell possesses over 1000 phosphorylatedproteins and several hundred protein kinasesand protein phosphatases that catalyze their interconversion.The ease of interconversion of enzymes betweentheir phospho- and dephospho- forms in partATPOH2–Enz Ser Enz Ser O PO 3P iMg 2+KINASEPHOSPHATASEMg 2+ADPH 2 OFigure 9–7. Covalent modification of a regulated enzymeby phosphorylation-dephosphorylation of a serylresidue.

78 / CHAPTER 9accounts for the frequency of phosphorylation-dephosphorylationas a mechanism for regulatory control.Phosphorylation-dephosphorylation permits the functionalproperties of the affected enzyme to be alteredonly for as long as it serves a specific need. Once theneed has passed, the enzyme can be converted back toits original form, poised to respond to the next stimulatoryevent. A second factor underlying the widespreaduse of protein phosphorylation-dephosphorylation liesin the chemical properties of the phosphoryl group itself.In order to alter an enzyme’s functional properties,any modification of its chemical structure must influencethe protein’s three-dimensional configuration.The high charge density of protein-bound phosphorylgroups—generally −2 at physiologic pH—and theirpropensity to form salt bridges with arginyl residuesmake them potent agents for modifying protein structureand function. Phosphorylation generally targetsamino acids distant from the catalytic site itself. Consequentconformational changes then influence an enzyme’sintrinsic catalytic efficiency or other properties.In this sense, the sites of phosphorylation and other covalentmodifications can be considered another form ofallosteric site. However, in this case the “allosteric ligand”binds covalently to the protein.PROTEIN PHOSPHORYLATIONIS EXTREMELY VERSATILEProtein phosphorylation-dephosphorylation is a highlyversatile and selective process. Not all proteins are subjectto phosphorylation, and of the many hydroxylgroups on a protein’s surface, only one or a small subsetare targeted. While the most common enzyme functionaffected is the protein’s catalytic efficiency, phosphorylationcan also alter the affinity for substrates, locationwithin the cell, or responsiveness to regulation by allostericligands. Phosphorylation can increase an enzyme’scatalytic efficiency, converting it to its activeform in one protein, while phosphorylation of anotherconverts it into an intrinsically inefficient, or inactive,form (Table 9–1).Many proteins can be phosphorylated at multiplesites or are subject to regulation both by phosphorylation-dephosphorylationand by the binding of allostericligands. Phosphorylation-dephosphorylation at any onesite can be catalyzed by multiple protein kinases or proteinphosphatases. Many protein kinases and most proteinphosphatases act on more than one protein and arethemselves interconverted between active and inactiveforms by the binding of second messengers or by covalentmodification by phosphorylation-dephosphorylation.The interplay between protein kinases and proteinphosphatases, between the functional consequences ofTable 9–1. Examples of mammalian enzymeswhose catalytic activity is altered by covalentphosphorylation-dephosphorylation.Activity State 1Enzyme Low HighAcetyl-CoA carboxylase EP EGlycogen synthase EP EPyruvate dehydrogenase EP EHMG-CoA reductase EP EGlycogen phosphorylase E EPCitrate lyase E EPPhosphorylase b kinase E EPHMG-CoA reductase kinase E EP1 E, dephosphoenzyme; EP, phosphoenzyme.phosphorylation at different sites, or between phosphorylationsites and allosteric sites provides the basis forregulatory networks that integrate multiple environmentalinput signals to evoke an appropriate coordinatedcellular response. In these sophisticated regulatorynetworks, individual enzymes respond to differentenvironmental signals. For example, if an enzyme canbe phosphorylated at a single site by more than oneprotein kinase, it can be converted from a catalyticallyefficient to an inefficient (inactive) form, or vice versa,in response to any one of several signals. If the proteinkinase is activated in response to a signal different fromthe signal that activates the protein phosphatase, thephosphoprotein becomes a decision node. The functionaloutput, generally catalytic activity, reflects thephosphorylation state. This state or degree of phosphorylationis determined by the relative activities ofthe protein kinase and protein phosphatase, a reflectionof the presence and relative strength of the environmentalsignals that act through each. The ability ofmany protein kinases and protein phosphatases to targetmore than one protein provides a means for an environmentalsignal to coordinately regulate multiplemetabolic processes. For example, the enzymes 3-hydroxy-3-methylglutaryl-CoAreductase and acetyl-CoAcarboxylase—the rate-controlling enzymes for cholesteroland fatty acid biosynthesis, respectively—arephosphorylated and inactivated by the AMP-activatedprotein kinase. When this protein kinase is activated eitherthrough phosphorylation by yet another proteinkinase or in response to the binding of its allosteric activator5′-AMP, the two major pathways responsible forthe synthesis of lipids from acetyl-CoA both are inhibited.Interconvertible enzymes and the enzymes responsiblefor their interconversion do not act as mere onand off switches working independently of one another.

ENZYMES: REGULATION OF ACTIVITIES / 79They form the building blocks of biomolecular computersthat maintain homeostasis in cells that carry outa complex array of metabolic processes that must beregulated in response to a broad spectrum of environmentalfactors.Covalent Modification RegulatesMetabolite FlowRegulation of enzyme activity by phosphorylationdephosphorylationhas analogies to regulation by feedbackinhibition. Both provide for short-term, readilyreversible regulation of metabolite flow in response tospecific physiologic signals. Both act without alteringgene expression. Both act on early enzymes of a protracted,often biosynthetic metabolic sequence, andboth act at allosteric rather than catalytic sites. Feedbackinhibition, however, involves a single protein andlacks hormonal and neural features. By contrast, regulationof mammalian enzymes by phosphorylationdephosphorylationinvolves several proteins and ATPand is under direct neural and hormonal control.SUMMARY• Homeostasis involves maintaining a relatively constantintracellular and intra-organ environment despitewide fluctuations in the external environmentvia appropriate changes in the rates of biochemicalreactions in response to physiologic need.• The substrates for most enzymes are usually presentat a concentration close to K m . This facilitates passivecontrol of the rates of product formation response tochanges in levels of metabolic intermediates.• Active control of metabolite flux involves changes inthe concentration, catalytic activity, or both of an enzymethat catalyzes a committed, rate-limiting reaction.• Selective proteolysis of catalytically inactive proenzymesinitiates conformational changes that form theactive site. Secretion as an inactive proenzyme facilitatesrapid mobilization of activity in response to injuryor physiologic need and may protect the tissueof origin (eg, autodigestion by proteases).• Binding of metabolites and second messengers tosites distinct from the catalytic site of enzymes triggersconformational changes that alter V max or theK m .• Phosphorylation by protein kinases of specific seryl,threonyl, or tyrosyl residues—and subsequent dephosphorylationby protein phosphatases—regulatesthe activity of many human enzymes. The protein kinasesand phosphatases that participate in regulatorycascades which respond to hormonal or second messengersignals constitute a “bio-organic computer”that can process and integrate complex environmentalinformation to produce an appropriate and comprehensivecellular response.REFERENCESBray D: Protein molecules as computational elements in livingcells. Nature 1995;376:307.Graves DJ, Martin BL, Wang JH: Co- and Post-translational Modificationof Proteins: Chemical Principles and Biological Effects.Oxford Univ Press, 1994.Johnson LN, Barford D: The effect of phosphorylation on thestructure and function of proteins. Annu Rev Biophys BiomolStruct 1993;22:199.Marks F (editor): Protein Phosphorylation. VCH Publishers, 1996.Pilkis SJ et al: 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase:A metabolic signaling enzyme. Annu Rev Biochem1995;64:799.Scriver CR et al (editors): The Metabolic and Molecular Bases ofInherited Disease, 8th ed. McGraw-Hill, 2000.Sitaramayya A (editor): Introduction to Cellular Signal Transduction.Birkhauser, 1999.Stadtman ER, Chock PB (editors): Current Topics in Cellular Regulation.Academic Press, 1969 to the present.Weber G (editor): Advances in Enzyme Regulation. Pergamon Press,1963 to the present.

SECTION IIBioenergetics & the Metabolismof Carbohydrates & LipidsBioenergetics:The Role of ATP 10Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEBioenergetics, or biochemical thermodynamics, is thestudy of the energy changes accompanying biochemicalreactions. Biologic systems are essentially isothermicand use chemical energy to power living processes.How an animal obtains suitable fuel from its food toprovide this energy is basic to the understanding of normalnutrition and metabolism. Death from starvationoccurs when available energy reserves are depleted, andcertain forms of malnutrition are associated with energyimbalance (marasmus). Thyroid hormones control therate of energy release (metabolic rate), and disease resultswhen they malfunction. Excess storage of surplusenergy causes obesity, one of the most common diseasesof Western society.the system to another or may be transformed into anotherform of energy. In living systems, chemical energymay be transformed into heat or into electrical, radiant,or mechanical energy.The second law of thermodynamics states that thetotal entropy of a system must increase if a processis to occur spontaneously. Entropy is the extent ofdisorder or randomness of the system and becomesmaximum as equilibrium is approached. Under conditionsof constant temperature and pressure, the relationshipbetween the free energy change (∆G) of a reactingsystem and the change in entropy (∆S) isexpressed by the following equation, which combinesthe two laws of thermodynamics:∆G =∆H − T∆SFREE ENERGY IS THE USEFUL ENERGYIN A SYSTEMGibbs change in free energy (∆G) is that portion of thetotal energy change in a system that is available fordoing work—ie, the useful energy, also known as thechemical potential.Biologic Systems Conform to the GeneralLaws of ThermodynamicsThe first law of thermodynamics states that the totalenergy of a system, including its surroundings, remainsconstant. It implies that within the total system,energy is neither lost nor gained during any change.However, energy may be transferred from one part of80where ∆H is the change in enthalpy (heat) and T is theabsolute temperature.In biochemical reactions, because ∆H is approximatelyequal to ∆E, the total change in internal energyof the reaction, the above relationship may be expressedin the following way:∆G =∆E − T∆SIf ∆G is negative, the reaction proceeds spontaneouslywith loss of free energy; ie, it is exergonic. If,in addition, ∆G is of great magnitude, the reaction goesvirtually to completion and is essentially irreversible.On the other hand, if ∆G is positive, the reaction proceedsonly if free energy can be gained; ie, it is endergonic.If, in addition, the magnitude of ∆G is great, the

BIOENERGETICS: THE ROLE OF ATP / 81system is stable, with little or no tendency for a reactionto occur. If ∆G is zero, the system is at equilibrium andno net change takes place.When the reactants are present in concentrations of1.0 mol/L, ∆G 0 is the standard free energy change. Forbiochemical reactions, a standard state is defined ashaving a pH of 7.0. The standard free energy change atthis standard state is denoted by ∆G 0′ .The standard free energy change can be calculatedfrom the equilibrium constant K eq .∆G 0′ = −RT ln K ′ eqwhere R is the gas constant and T is the absolute temperature(Chapter 8). It is important to note that theactual ∆G may be larger or smaller than ∆G 0′ dependingon the concentrations of the various reactants, includingthe solvent, various ions, and proteins.In a biochemical system, an enzyme only speeds upthe attainment of equilibrium; it never alters the finalconcentrations of the reactants at equilibrium.ENDERGONIC PROCESSES PROCEED BYCOUPLING TO EXERGONIC PROCESSESThe vital processes—eg, synthetic reactions, muscularcontraction, nerve impulse conduction, and activetransport—obtain energy by chemical linkage, or coupling,to oxidative reactions. In its simplest form, thistype of coupling may be represented as shown in Figure10–1. The conversion of metabolite A to metabolite Boccurs with release of free energy. It is coupled to anotherreaction, in which free energy is required to convertmetabolite C to metabolite D. The terms exergonicand endergonic rather than the normal chemicalterms “exothermic” and “endothermic” are used to indicatethat a process is accompanied by loss or gain, respectively,of free energy in any form, not necessarily asheat. In practice, an endergonic process cannot exist independentlybut must be a component of a coupled exergonic-endergonicsystem where the overall net changeis exergonic. The exergonic reactions are termed catabolism(generally, the breakdown or oxidation of fuelmolecules), whereas the synthetic reactions that buildup substances are termed anabolism. The combinedcatabolic and anabolic processes constitute metabolism.If the reaction shown in Figure 10–1 is to go fromleft to right, then the overall process must be accompaniedby loss of free energy as heat. One possible mechanismof coupling could be envisaged if a common obligatoryintermediate (I) took part in both reactions, ie,A + C → I → B+DSome exergonic and endergonic reactions in biologicsystems are coupled in this way. This type of system hasa built-in mechanism for biologic control of the rate ofoxidative processes since the common obligatory intermediateallows the rate of utilization of the product ofthe synthetic path (D) to determine by mass action therate at which A is oxidized. Indeed, these relationshipssupply a basis for the concept of respiratory control,the process that prevents an organism from burning outof control. An extension of the coupling concept is providedby dehydrogenation reactions, which are coupledto hydrogenations by an intermediate carrier (Figure10–2).An alternative method of coupling an exergonic toan endergonic process is to synthesize a compound ofhigh-energy potential in the exergonic reaction and toincorporate this new compound into the endergonic reaction,thus effecting a transference of free energy fromthe exergonic to the endergonic pathway (Figure 10–3).The biologic advantage of this mechanism is that thecompound of high potential energy, ∼E , unlike I∆G =∆H − T∆SCoupling of an exergonic to an ender-Figure 10–1.gonic reaction.Figure 10–2. Coupling of dehydrogenation and hydrogenationreactions by an intermediate carrier.

82 / CHAPTER 10Figure 10–4. Adenosine triphosphate (ATP) shownas the magnesium complex. ADP forms a similar complexwith Mg 2+ .Figure 10–3. Transfer of free energy from an exergonicto an endergonic reaction via a high-energy intermediatecompound (∼E ).in the previous system, need not be structurally relatedto A, B, C, or D, allowing E to serve as a transducer ofenergy from a wide range of exergonic reactions to anequally wide range of endergonic reactions or processes,such as biosyntheses, muscular contraction, nervous excitation,and active transport. In the living cell, theprincipal high-energy intermediate or carrier compound(designated ∼E in Figure 10–3) is adenosinetriphosphate (ATP).HIGH-ENERGY PHOSPHATES PLAY ACENTRAL ROLE IN ENERGY CAPTUREAND TRANSFERIn order to maintain living processes, all organismsmust obtain supplies of free energy from their environment.Autotrophic organisms utilize simple exergonicprocesses; eg, the energy of sunlight (green plants), thereaction Fe 2+ → Fe 3+ (some bacteria). On the otherhand, heterotrophic organisms obtain free energy bycoupling their metabolism to the breakdown of complexorganic molecules in their environment. In allthese organisms, ATP plays a central role in the transferenceof free energy from the exergonic to the endergonicprocesses (Figure 10–3). ATP is a nucleosidetriphosphate containing adenine, ribose, and threephosphate groups. In its reactions in the cell, it functionsas the Mg 2+ complex (Figure 10–4).The importance of phosphates in intermediary metabolismbecame evident with the discovery of the roleof ATP, adenosine diphosphate (ADP), and inorganicphosphate (P i ) in glycolysis (Chapter 17).The Intermediate Value for the FreeEnergy of Hydrolysis of ATP Has ImportantBioenergetic SignificanceThe standard free energy of hydrolysis of a number ofbiochemically important phosphates is shown in Table10–1. An estimate of the comparative tendency of eachof the phosphate groups to transfer to a suitable acceptormay be obtained from the ∆G 0′ of hydrolysis at37 °C. The value for the hydrolysis of the terminalTable 10–1. Standard free energy of hydrolysisof some organophosphates of biochemicalimportance. 1,2G 0 Compound kJ/mol kcal/molPhosphoenolpyruvate −61.9 −14.8Carbamoyl phosphate −51.4 −12.31,3-Bisphosphoglycerate −49.3 −11.8(to 3-phosphoglycerate)Creatine phosphate −43.1 −10.3ATP → ADP + P i −30.5 −7.3ADP → AMP + P i −27.6 −6.6Pyrophosphate −27.6 −6.6Glucose 1-phosphate −20.9 −5.0Fructose 6-phosphate −15.9 −3.8AMP −14.2 −3.4Glucose 6-phosphate −13.8 −3.3Glycerol 3-phosphate −9.2 −2.21 P i , inorganic orthophosphate.2 Values for ATP and most others taken from Krebs and Kornberg(1957). They differ between investigators depending on the preciseconditions under which the measurements are made.

BIOENERGETICS: THE ROLE OF ATP / 83phosphate of ATP divides the list into two groups.Low-energy phosphates, exemplified by the esterphosphates found in the intermediates of glycolysis,have ∆G 0′ values smaller than that of ATP, while inhigh-energy phosphates the value is higher than thatof ATP. The components of this latter group, includingATP, are usually anhydrides (eg, the 1-phosphate of1,3-bisphosphoglycerate), enolphosphates (eg, phosphoenolpyruvate),and phosphoguanidines (eg, creatinephosphate, arginine phosphate). The intermediate positionof ATP allows it to play an important role in energytransfer. The high free energy change on hydrolysisof ATP is due to relief of charge repulsion of adjacentnegatively charged oxygen atoms and to stabilization ofthe reaction products, especially phosphate, as resonancehybrids. Other “high-energy compounds” arethiol esters involving coenzyme A (eg, acetyl-CoA), acylcarrier protein, amino acid esters involved in proteinsynthesis, S-adenosylmethionine (active methionine),UDPGlc (uridine diphosphate glucose), and PRPP(5-phosphoribosyl-1-pyrophosphate).High-Energy Phosphates AreDesignated by ~ PThe symbol ∼P indicates that the group attached tothe bond, on transfer to an appropriate acceptor, resultsin transfer of the larger quantity of free energy. For thisreason, the term group transfer potential is preferredby some to “high-energy bond.” Thus, ATP containstwo high-energy phosphate groups and ADP containsone, whereas the phosphate in AMP (adenosine monophosphate)is of the low-energy type, since it is a normalester link (Figure 10–5).HIGH-ENERGY PHOSPHATES ACT AS THE“ENERGY CURRENCY” OF THE CELLATP is able to act as a donor of high-energy phosphateto form those compounds below it in Table 10–1. Likewise,with the necessary enzymes, ADP can accepthigh-energy phosphate to form ATP from those compoundsabove ATP in the table. In effect, an ATP/ADP cycle connects those processes that generate ∼Pto those processes that utilize ∼P (Figure 10–6), continuouslyconsuming and regenerating ATP. This occursat a very rapid rate, since the total ATP/ADP poolis extremely small and sufficient to maintain an activetissue for only a few seconds.There are three major sources of ∼P taking part inenergy conservation or energy capture:(1) Oxidative phosphorylation: The greatest quantitativesource of ∼P in aerobic organisms. Free energyFigure 10–5. Structure of ATP, ADP, and AMP showingthe position and the number of high-energy phosphates(∼P ).Figure 10–6. Role of ATP/ADP cycle in transfer ofhigh-energy phosphate.

84 / CHAPTER 10(1) Glucose+P i → Glucose 6- phosphate+ H 2 O( ∆G 0′ = +13.8 kJ/ mol)To take place, the reaction must be coupled with another—moreexergonic—reaction such as the hydrolysisof the terminal phosphate of ATP.Figure 10–7. Transfer of high-energy phosphate betweenATP and creatine.comes from respiratory chain oxidation using molecularO 2 within mitochondria (Chapter 11).(2) Glycolysis: A net formation of two ∼P resultsfrom the formation of lactate from one molecule of glucose,generated in two reactions catalyzed by phosphoglyceratekinase and pyruvate kinase, respectively (Figure17–2).(3) The citric acid cycle: One ∼P is generated directlyin the cycle at the succinyl thiokinase step (Figure16–3).Phosphagens act as storage forms of high-energyphosphate and include creatine phosphate, occurring invertebrate skeletal muscle, heart, spermatozoa, andbrain; and arginine phosphate, occurring in invertebratemuscle. When ATP is rapidly being utilized as asource of energy for muscular contraction, phosphagenspermit its concentrations to be maintained, but whenthe ATP/ADP ratio is high, their concentration can increaseto act as a store of high-energy phosphate (Figure10–7).When ATP acts as a phosphate donor to form thosecompounds of lower free energy of hydrolysis (Table10–1), the phosphate group is invariably converted toone of low energy, eg,When (1) and (2) are coupled in a reaction catalyzed byhexokinase, phosphorylation of glucose readily proceedsin a highly exergonic reaction that under physiologicconditions is irreversible. Many “activation” reactionsfollow this pattern.Adenylyl Kinase (Myokinase)Interconverts Adenine NucleotidesThis enzyme is present in most cells. It catalyzes the followingreaction:This allows:(2) ATP→ ADP+P i (∆G 0′ =−30.5 kJ/mol)(1) High-energy phosphate in ADP to be used inthe synthesis of ATP.(2) AMP, formed as a consequence of several activatingreactions involving ATP, to be recovered byrephosphorylation to ADP.(3) AMP to increase in concentration when ATPbecomes depleted and act as a metabolic (allosteric) signalto increase the rate of catabolic reactions, which inturn lead to the generation of more ATP (Chapter 19).When ATP Forms AMP, InorganicPyrophosphate (PP i ) Is ProducedThis occurs, for example, in the activation of longchainfatty acids (Chapter 22):ATP Allows the Coupling ofThermodynamically UnfavorableReactions to Favorable OnesThe phosphorylation of glucose to glucose 6-phosphate,the first reaction of glycolysis (Figure 17–2), ishighly endergonic and cannot proceed under physiologicconditions.This reaction is accompanied by loss of free energyas heat, which ensures that the activation reaction willgo to the right; and is further aided by the hydrolyticsplitting of PP i , catalyzed by inorganic pyrophosphatase,a reaction that itself has a large ∆G 0′ of −27.6 kJ/

BIOENERGETICS: THE ROLE OF ATP / 85Thus, adenylyl kinase is a specialized monophosphatekinase.Figure 10–8. Phosphate cycles and interchange ofadenine nucleotides.mol. Note that activations via the pyrophosphate pathwayresult in the loss of two ∼P rather than one ∼P asoccurs when ADP and P i are formed.A combination of the above reactions makes it possiblefor phosphate to be recycled and the adenine nucleotidesto interchange (Figure 10–8).SUMMARY• Biologic systems use chemical energy to power theliving processes.• Exergonic reactions take place spontaneously withloss of free energy (∆G is negative). Endergonic reactionsrequire the gain of free energy (∆G is positive)and only occur when coupled to exergonic reactions.• ATP acts as the “energy currency” of the cell, transferringfree energy derived from substances of higherenergy potential to those of lower energy potential.REFERENCESde Meis L: The concept of energy-rich phosphate compounds:Water, transport ATPases, and entropy energy. Arch BiochemBiophys 1993;306:287.Ernster L (editor): Bioenergetics. Elsevier, 1984.Harold FM: The Vital Force: A Study of Bioenergetics. Freeman,1986.Klotz IM: Introduction to Biomolecular Energetics. Academic Press,1986.Krebs HA, Kornberg HL: Energy Transformations in Living Matter.Springer, 1957.Other Nucleoside TriphosphatesParticipate in the Transfer ofHigh-Energy PhosphateBy means of the enzyme nucleoside diphosphate kinase,UTP, GTP, and CTP can be synthesized fromtheir diphosphates, eg,All of these triphosphates take part in phosphorylationsin the cell. Similarly, specific nucleoside monophosphatekinases catalyze the formation of nucleosidediphosphates from the corresponding monophosphates.

Biologic Oxidation 11Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEChemically, oxidation is defined as the removal of electronsand reduction as the gain of electrons. Thus, oxidationis always accompanied by reduction of an electronacceptor. This principle of oxidation-reductionapplies equally to biochemical systems and is an importantconcept underlying understanding of the nature ofbiologic oxidation. Note that many biologic oxidationscan take place without the participation of molecularoxygen, eg, dehydrogenations. The life of higher animalsis absolutely dependent upon a supply of oxygenfor respiration, the process by which cells derive energyin the form of ATP from the controlled reaction of hydrogenwith oxygen to form water. In addition, molecularoxygen is incorporated into a variety of substratesby enzymes designated as oxygenases; many drugs, pollutants,and chemical carcinogens (xenobiotics) are metabolizedby enzymes of this class, known as the cytochromeP450 system. Administration of oxygen canbe lifesaving in the treatment of patients with respiratoryor circulatory failure.FREE ENERGY CHANGES CANBE EXPRESSED IN TERMSOF REDOX POTENTIALIn reactions involving oxidation and reduction, the freeenergy change is proportionate to the tendency of reactantsto donate or accept electrons. Thus, in addition toexpressing free energy change in terms of ∆G 0′ (Chapter10), it is possible, in an analogous manner, to express itnumerically as an oxidation-reduction or redox potential(E′ 0 ). The redox potential of a system (E 0 ) isusually compared with the potential of the hydrogenelectrode (0.0 volts at pH 0.0). However, for biologicsystems, the redox potential (E′ 0 )is normally expressedat pH 7.0, at which pH the electrode potential of thehydrogen electrode is −0.42 volts. The redox potentialsof some redox systems of special interest in mammalianbiochemistry are shown in Table 11–1. The relative positionsof redox systems in the table allows prediction ofthe direction of flow of electrons from one redox coupleto another.Enzymes involved in oxidation and reduction arecalled oxidoreductases and are classified into four86groups: oxidases, dehydrogenases, hydroperoxidases,and oxygenases.OXIDASES USE OXYGEN AS AHYDROGEN ACCEPTOROxidases catalyze the removal of hydrogen from a substrateusing oxygen as a hydrogen acceptor.* They formwater or hydrogen peroxide as a reaction product (Figure11–1).Some Oxidases Contain CopperCytochrome oxidase is a hemoprotein widely distributedin many tissues, having the typical heme prostheticgroup present in myoglobin, hemoglobin, andother cytochromes (Chapter 6). It is the terminal componentof the chain of respiratory carriers found in mitochondriaand transfers electrons resulting from theoxidation of substrate molecules by dehydrogenases totheir final acceptor, oxygen. The enzyme is poisoned bycarbon monoxide, cyanide, and hydrogen sulfide. It hasalso been termed cytochrome a 3 . It is now known thatcytochromes a and a 3 are combined in a single protein,and the complex is known as cytochrome aa 3 . It containstwo molecules of heme, each having one Fe atomthat oscillates between Fe 3+ and Fe 2+ during oxidationand reduction. Furthermore, two atoms of Cu are present,each associated with a heme unit.Other Oxidases Are FlavoproteinsFlavoprotein enzymes contain flavin mononucleotide(FMN) or flavin adenine dinucleotide (FAD) as prostheticgroups. FMN and FAD are formed in the bodyfrom the vitamin riboflavin (Chapter 45). FMN andFAD are usually tightly—but not covalently—bound totheir respective apoenzyme proteins. Metalloflavoproteinscontain one or more metals as essential cofactors.Examples of flavoprotein enzymes include L-aminoacid oxidase, an FMN-linked enzyme found in kidneywith general specificity for the oxidative deamination of* The term “oxidase” is sometimes used collectively to denote allenzymes that catalyze reactions involving molecular oxygen.

BIOLOGIC OXIDATION / 87Table 11–1. Some redox potentials of specialinterest in mammalian oxidation systems.SystemE 0 VoltsH + /H 2 −0.42NAD + /NADH −0.32Lipoate; ox/red −0.29Acetoacetate/3-hydroxybutyrate −0.27Pyruvate/lactate −0.19Oxaloacetate/malate −0.17Fumarate/succinate +0.03Cytochrome b; Fe 3+ /Fe 2+ +0.08Ubiquinone; ox/red +0.10Cytochrome c 1 ; Fe 3+ /Fe 2+ +0.22Cytochrome a; Fe 3+ /Fe 2+ +0.29Oxygen/water +0.82the naturally occurring L-amino acids; xanthine oxidase,which contains molybdenum and plays an importantrole in the conversion of purine bases to uric acid(Chapter 34), and is of particular significance in uricotelicanimals (Chapter 29); and aldehyde dehydrogenase,an FAD-linked enzyme present in mammalianlivers, which contains molybdenum and nonheme ironand acts upon aldehydes and N-heterocyclic substrates.The mechanisms of oxidation and reduction of theseenzymes are complex. Evidence suggests a two-step reactionas shown in Figure 11–2.DEHYDROGENASES CANNOT USEOXYGEN AS A HYDROGEN ACCEPTORThere are a large number of enzymes in this class. Theyperform two main functions:AH 1 2/2O 2 AH 2 O 2(Red)OXIDASEOXIDASEAH 2 OAH 2 O 2(Ox)ABFigure 11–1. Oxidation of a metabolite catalyzed byan oxidase (A) forming H 2 O, (B) forming H 2 O 2 .(1) Transfer of hydrogen from one substrate to anotherin a coupled oxidation-reduction reaction (Figure11–3). These dehydrogenases are specific for their substratesbut often utilize common coenzymes or hydrogencarriers, eg, NAD + . Since the reactions are reversible,these properties enable reducing equivalents tobe freely transferred within the cell. This type of reaction,which enables one substrate to be oxidized at theexpense of another, is particularly useful in enabling oxidativeprocesses to occur in the absence of oxygen,such as during the anaerobic phase of glycolysis (Figure17–2).(2) As components in the respiratory chain of electrontransport from substrate to oxygen (Figure 12–3).Many Dehydrogenases Dependon Nicotinamide CoenzymesThese dehydrogenases use nicotinamide adenine dinucleotide(NAD + ) or nicotinamide adenine dinucleotidephosphate (NADP + )—or both—and areformed in the body from the vitamin niacin (Chapter45). The coenzymes are reduced by the specific substrateof the dehydrogenase and reoxidized by a suitableelectron acceptor (Figure 11–4).They may freely andreversibly dissociate from their respective apoenzymes.Generally, NAD-linked dehydrogenases catalyze oxidoreductionreactions in the oxidative pathways of metabolism,particularly in glycolysis, in the citric acidcycle, and in the respiratory chain of mitochondria.NADP-linked dehydrogenases are found characteristicallyin reductive syntheses, as in the extramitochondrialpathway of fatty acid synthesis and steroid synthesis—andalso in the pentose phosphate pathway.Other Dehydrogenases Dependon RiboflavinThe flavin groups associated with these dehydrogenasesare similar to FMN and FAD occurring in oxidases.They are generally more tightly bound to their apoenzymesthan are the nicotinamide coenzymes. Most ofthe riboflavin-linked dehydrogenases are concernedwith electron transport in (or to) the respiratory chain(Chapter 12). NADH dehydrogenase acts as a carrierof electrons between NADH and the components ofhigher redox potential (Figure 12–3). Other dehydrogenasessuch as succinate dehydrogenase, acyl-CoAdehydrogenase, and mitochondrial glycerol-3-phosphatedehydrogenase transfer reducing equivalents directlyfrom the substrate to the respiratory chain (Figure12–4). Another role of the flavin-dependentdehydrogenases is in the dehydrogenation (by dihydrolipoyldehydrogenase) of reduced lipoate, an intermediatein the oxidative decarboxylation of pyruvateand α-ketoglutarate (Figures 12–4 and 17–5). Theelectron-transferring flavoprotein is an intermediarycarrier of electrons between acyl-CoA dehydrogenaseand the respiratory chain (Figure 12–4).

88 / CHAPTER 11H 3 CRNNOH 3 CRNHNOH 3 CRNHNOH 3 CNONHH 3 CNNHHOH(H + + e – ) (H + + e – )Figure 11–2. Oxidoreduction of isoalloxazine ring in flavin nucleotides via a semiquinone(free radical) intermediate (center).H 3 CNHONHCytochromes May Also Be Regardedas DehydrogenasesThe cytochromes are iron-containing hemoproteins inwhich the iron atom oscillates between Fe 3+ and Fe 2+during oxidation and reduction. Except for cytochromeoxidase (previously described), they are classified as dehydrogenases.In the respiratory chain, they are involvedas carriers of electrons from flavoproteins on theone hand to cytochrome oxidase on the other (Figure12–4). Several identifiable cytochromes occur in therespiratory chain, ie, cytochromes b, c 1 , c, a, and a 3 (cytochromeoxidase). Cytochromes are also found inother locations, eg, the endoplasmic reticulum (cytochromesP450 and b 5 ), and in plant cells, bacteria,and yeasts.HYDROPEROXIDASES USE HYDROGENPEROXIDE OR AN ORGANIC PEROXIDEAS SUBSTRATETwo type of enzymes found both in animals and plantsfall into this category: peroxidases and catalase.Hydroperoxidases protect the body against harmfulperoxides. Accumulation of peroxides can lead to generationof free radicals, which in turn can disrupt membranesand perhaps cause cancer and atherosclerosis.(See Chapters 14 and 45.)AH 2(Red)A(Ox)DEHYDROGENASESPECIFIC FOR ACarrier(Ox)Carrier–H 2(Red)DEHYDROGENASESPECIFIC FOR BBH 2(Red)B(Ox)Figure 11–3. Oxidation of a metabolite catalyzed bycoupled dehydrogenases.Peroxidases Reduce Peroxides UsingVarious Electron AcceptorsPeroxidases are found in milk and in leukocytes,platelets, and other tissues involved in eicosanoid metabolism(Chapter 23). The prosthetic group is protoheme.In the reaction catalyzed by peroxidase, hydrogenperoxide is reduced at the expense of severalsubstances that will act as electron acceptors, such asascorbate, quinones, and cytochrome c. The reactioncatalyzed by peroxidase is complex, but the overall reactionis as follows:H 2 O 2 + AH 2PEROXIDASE2H 2 O + AIn erythrocytes and other tissues, the enzyme glutathioneperoxidase, containing selenium as a prostheticgroup, catalyzes the destruction of H 2 O 2 andlipid hydroperoxides by reduced glutathione, protectingmembrane lipids and hemoglobin against oxidation byperoxides (Chapter 20).Catalase Uses Hydrogen Peroxide asElectron Donor & Electron AcceptorCatalase is a hemoprotein containing four heme groups.In addition to possessing peroxidase activity, it is ableto use one molecule of H 2 O 2 as a substrate electrondonor and another molecule of H 2 O 2 as an oxidant orelectron acceptor.CATALASE2H 2 O 2 2H 2 O + O 2Under most conditions in vivo, the peroxidase activityof catalase seems to be favored. Catalase is found inblood, bone marrow, mucous membranes, kidney, andliver. Its function is assumed to be the destruction ofhydrogen peroxide formed by the action of oxidases.

HHBIOLOGIC OXIDATION / 89HFigure 11–4. Mechanism of oxidationand reduction of nicotinamide coenzymes.There is stereospecificity aboutposition 4 of nicotinamide when it is reducedby a substrate AH 2 . One of the hydrogenatoms is removed from the substrateas a hydrogen nucleus with twoelectrons (hydride ion, H − ) and is transferredto the 4 position, where it may beattached in either the A or the B positionaccording to the specificity determinedby the particular dehydrogenase catalyzingthe reaction. The remaining hydrogenof the hydrogen pair removed fromthe substrate remains free as a hydrogenion.H4+NRCONH 2AH 2NAD + + AH 2DEHYDROGENASESPECIFIC FOR AAH 2A + H +DEHYDROGENASESPECIFIC FOR BNADH + H + + A4NR4NRHCONH 2A FormCONH 2B FormPeroxisomes are found in many tissues, including liver.They are rich in oxidases and in catalase, Thus, the enzymesthat produce H 2 O 2 are grouped with the enzymethat destroys it. However, mitochondrial and microsomalelectron transport systems as well as xanthine oxidasemust be considered as additional sources of H 2 O 2 .OXYGENASES CATALYZE THE DIRECTTRANSFER & INCORPORATIONOF OXYGEN INTO A SUBSTRATEMOLECULEOxygenases are concerned with the synthesis or degradationof many different types of metabolites. They catalyzethe incorporation of oxygen into a substrate moleculein two steps: (1) oxygen is bound to the enzyme atthe active site, and (2) the bound oxygen is reduced ortransferred to the substrate. Oxygenases may be dividedinto two subgroups, as follows.Dioxygenases Incorporate Both Atomsof Molecular Oxygen Into the SubstrateThe basic reaction is shown below:A+ O →AOExamples include the liver enzymes, homogentisatedioxygenase (oxidase) and 3-hydroxyanthranilatedioxygenase (oxidase), that contain iron; and L-tryptophandioxygenase (tryptophan pyrrolase) (Chapter30), that utilizes heme.Monooxygenases (Mixed-FunctionOxidases, Hydroxylases) IncorporateOnly One Atom of Molecular OxygenInto the SubstrateThe other oxygen atom is reduced to water, an additionalelectron donor or cosubstrate (Z) being necessaryfor this purpose.A— H+ O2+ ZH2→ A—OH+ H2O+ ZCytochromes P450 Are MonooxygenasesImportant for the Detoxification of ManyDrugs & for the Hydroxylation of SteroidsCytochromes P450 are an important superfamily ofheme-containing monooxgenases, and more than 1000such enzymes are known. Both NADH and NADPHdonate reducing equivalents for the reduction of thesecytochromes (Figure 11–5), which in turn are oxidizedby substrates in a series of enzymatic reactions collectivelyknown as the hydroxylase cycle (Figure 11–6). In livermicrosomes, cytochromes P450 are found together withcytochrome b 5 and have an important role in detoxifica-2 2tion. Benzpyrene, aminopyrine, aniline, morphine, andbenzphetamine are hydroxylated, increasing their solubilityand aiding their excretion. Many drugs such as phenobarbitalhave the ability to induce the formation of microsomalenzymes and of cytochromes P450.Mitochondrial cytochrome P450 systems are foundin steroidogenic tissues such as adrenal cortex, testis,ovary, and placenta and are concerned with the biosyn-

90 / CHAPTER 11Amine oxidase, etcNADHNADPHCN –Flavoprotein 2 Cyt b 5–Flavoprotein 3Flavoprotein 1 Cyt P450Stearyl-CoA desaturaseHydroxylationLipid peroxidationHeme oxygenaseFigure 11–5.indicated step.Electron transport chain in microsomes. Cyanide (CN − ) inhibits thethesis of steroid hormones from cholesterol (hydroxylationat C 22 and C 20 in side-chain cleavage and at the11β and 18 positions). In addition, renal systems catalyzing1α- and 24-hydroxylations of 25-hydroxycholecalciferolin vitamin D metabolism—and cholesterol7α-hydroxylase and sterol 27-hydroxylase involved inbile acid biosynthesis in the liver (Chapter 26)—areP450 enzymes.SUPEROXIDE DISMUTASE PROTECTSAEROBIC ORGANISMS AGAINSTOXYGEN TOXICITYTransfer of a single electron to O 2 generates the potentiallydamaging superoxide anion free radical (O 2− ⋅ ),the destructive effects of which are amplified by its givingrise to free radical chain reactions (Chapter 14).The ease with which superoxide can be formed fromoxygen in tissues and the occurrence of superoxide dismutase,the enzyme responsible for its removal in allaerobic organisms (although not in obligate anaerobes)indicate that the potential toxicity of oxygen is due toits conversion to superoxide.Superoxide is formed when reduced flavins—present,for example, in xanthine oxidase—are reoxidizedunivalently by molecular oxygen.−Enz −Flavin−H2+ O2→ Enz −Flavin−H+ O ⋅ +2 + HSuperoxide can reduce oxidized cytochrome c−O ⋅2 +32Cyt c ( Fe + ) → O2+ Cyt c ( Fe+ )Substrate A-HP450-A-HFe 3+P450Fe 3+NADPH-CYT P450 REDUCTASENADP + 2Fe 2 S 22+FADH 22Fe 2 S 23+e –P450-A-HFe 2+O 2e ––NADPH + H + FADCO2H + P450-A-HFe 2+ O 2A-OHH 2 OP450-A-HFe 2+ –O 2Figure 11–6. Cytochrome P450 hydroxylase cycle in microsomes. The system shown is typicalof steroid hydroxylases of the adrenal cortex. Liver microsomal cytochrome P450 hydroxylase doesnot require the iron-sulfur protein Fe 2 S 2 . Carbon monoxide (CO) inhibits the indicated step.

BIOLOGIC OXIDATION / 91or be removed by superoxide dismutase.SUPEROXIDEDISMUTASEO −2. + O −2. + 2H + H 2 O 2 + O 2In this reaction, superoxide acts as both oxidant andreductant. Thus, superoxide dismutase protects aerobicorganisms against the potential deleterious effects of superoxide.The enzyme occurs in all major aerobic tissuesin the mitochondria and the cytosol. Although exposureof animals to an atmosphere of 100% oxygencauses an adaptive increase in superoxide dismutase,particularly in the lungs, prolonged exposure leads tolung damage and death. Antioxidants, eg, α-tocopherol(vitamin E), act as scavengers of free radicals and reducethe toxicity of oxygen (Chapter 45).SUMMARY• In biologic systems, as in chemical systems, oxidation(loss of electrons) is always accompanied by reductionof an electron acceptor.• Oxidoreductases have a variety of functions in metabolism;oxidases and dehydrogenases play majorroles in respiration; hydroperoxidases protect thebody against damage by free radicals; and oxygenasesmediate the hydroxylation of drugs and steroids.• Tissues are protected from oxygen toxicity caused bythe superoxide free radical by the specific enzyme superoxidedismutase.REFERENCESBabcock GT, Wikstrom M: Oxygen activation and the conservationof energy in cell respiration. Nature 1992;356:301.Coon MJ et al: Cytochrome P450: Progress and predictions.FASEB J 1992;6:669.Ernster L (editor): Bioenergetics. Elsevier, 1984.Mammaerts GP, Van Veldhoven PP: Role of peroxisomes in mammalianmetabolism. Cell Biochem Funct 1992;10:141.Nicholls DG: Cytochromes and Cell Respiration. Carolina BiologicalSupply Company, 1984.Raha S, Robinson BH: Mitochondria, oxygen free radicals, diseaseand aging. Trends Biochem Sci 2000;25:502.Tyler DD: The Mitochondrion in Health and Disease. VCH Publishers,1992.Tyler DD, Sutton CM: Respiratory enzyme systems in mitochondrialmembranes. In: Membrane Structure and Function, vol5. Bittar EE (editor). Wiley, 1984.Yang CS, Brady JF, Hong JY: Dietary effects on cytochromesP450, xenobiotic metabolism, and toxicity. FASEB J 1992;6:737.

The Respiratory Chain &Oxidative Phosphorylation 12Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEAerobic organisms are able to capture a far greater proportionof the available free energy of respiratory substratesthan anaerobic organisms. Most of this takesplace inside mitochondria, which have been termed the“powerhouses” of the cell. Respiration is coupled to thegeneration of the high-energy intermediate, ATP, byoxidative phosphorylation, and the chemiosmotictheory offers insight into how this is accomplished. Anumber of drugs (eg, amobarbital) and poisons (eg,cyanide, carbon monoxide) inhibit oxidative phosphorylation,usually with fatal consequences. Several inheriteddefects of mitochondria involving componentsof the respiratory chain and oxidative phosphorylationhave been reported. Patients present with myopathyand encephalopathy and often have lactic acidosis.SPECIFIC ENZYMES ACT AS MARKERSOF COMPARTMENTS SEPARATED BYTHE MITOCHONDRIAL MEMBRANESMitochondria have an outer membrane that is permeableto most metabolites, an inner membrane that isselectively permeable, and a matrix within (Figure12–1). The outer membrane is characterized by thepresence of various enzymes, including acyl-CoA synthetaseand glycerolphosphate acyltransferase. Adenylylkinase and creatine kinase are found in the intermembranespace. The phospholipid cardiolipin is concentratedin the inner membrane together with the enzymesof the respiratory chain.THE RESPIRATORY CHAIN COLLECTS& OXIDIZES REDUCING EQUIVALENTSMost of the energy liberated during the oxidation ofcarbohydrate, fatty acids, and amino acids is madeavailable within mitochondria as reducing equivalents(⎯H or electrons) (Figure 12–2). Mitochondria containthe respiratory chain, which collects and transportsreducing equivalents directing them to their finalreaction with oxygen to form water, the machinery for92trapping the liberated free energy as high-energy phosphate,and the enzymes of β-oxidation and of the citricacid cycle (Chapters 22 and 16) that produce most ofthe reducing equivalents.Components of the Respiratory ChainAre Arranged in Order of IncreasingRedox PotentialHydrogen and electrons flow through the respiratorychain (Figure 12–3) through a redox span of 1.1 Vfrom NAD + /NADH to O 2 /2H 2 O (Table 11–1). Therespiratory chain consists of a number of redox carriersthat proceed from the NAD-linked dehydrogenase systems,through flavoproteins and cytochromes, to molecularoxygen. Not all substrates are linked to the respiratorychain through NAD-specific dehydrogenases;some, because their redox potentials are more positive(eg, fumarate/succinate; Table 11–1), are linked directlyto flavoprotein dehydrogenases, which in turn arelinked to the cytochromes of the respiratory chain (Figure12–4).Ubiquinone or Q (coenzyme Q) (Figure 12–5)links the flavoproteins to cytochrome b, the member ofthe cytochrome chain of lowest redox potential. Q existsin the oxidized quinone or reduced quinol formunder aerobic or anaerobic conditions, respectively.The structure of Q is very similar to that of vitamin Kand vitamin E (Chapter 45) and of plastoquinone,found in chloroplasts. Q acts as a mobile component ofthe respiratory chain that collects reducing equivalentsfrom the more fixed flavoprotein complexes and passesthem on to the cytochromes.An additional component is the iron-sulfur protein(FeS; nonheme iron) (Figure 12–6). It is associatedwith the flavoproteins (metalloflavoproteins) and withcytochrome b. The sulfur and iron are thought to takepart in the oxidoreduction mechanism between flavinand Q, which involves only a single e − change, the ironatom undergoing oxidoreduction between Fe 2+ andFe 3+ .Pyruvate and α-ketoglutarate dehydrogenase havecomplex systems involving lipoate and FAD prior tothe passage of electrons to NAD, while electron trans-

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION / 93MATRIXCristaeINNERMEMBRANEPhosphorylatingcomplexesOUTERMEMBRANEFigure 12–1. Structure of the mitochondrial membranes.Note that the inner membrane contains manyfolds, or cristae.fers from other dehydrogenases, eg, L(+)-3-hydroxyacyl-CoA dehydrogenase, couple directly with NAD.The reduced NADH of the respiratory chain is inturn oxidized by a metalloflavoprotein enzyme—NADHdehydrogenase. This enzyme contains FeS and FMN,is tightly bound to the respiratory chain, and passes reducingequivalents on to Q.Electrons flow from Q through the series of cytochromesin order of increasing redox potential to molecularoxygen (Figure 12–4). The terminal cytochromeaa 3 (cytochrome oxidase), responsible for the final combinationof reducing equivalents with molecular oxygen,has a very high affinity for oxygen, allowing therespiratory chain to function at maximum rate until thetissue has become depleted of O 2 . Since this is an irreversiblereaction (the only one in the chain), it gives directionto the movement of reducing equivalents and tothe production of ATP, to which it is coupled.Functionally and structurally, the components ofthe respiratory chain are present in the inner mitochondrialmembrane as four protein-lipid respiratory chaincomplexes that span the membrane. Cytochrome c isthe only soluble cytochrome and, together with Q,seems to be a more mobile component of the respiratorychain connecting the fixed complexes (Figures12–7 and 12–8).THE RESPIRATORY CHAIN PROVIDESMOST OF THE ENERGY CAPTUREDDURING CATABOLISMADP captures, in the form of high-energy phosphate, asignificant proportion of the free energy released bycatabolic processes. The resulting ATP has been calledthe energy “currency” of the cell because it passes onthis free energy to drive those processes requiring energy(Figure 10–6).There is a net direct capture of two high-energyphosphate groups in the glycolytic reactions (Table17–1), equivalent to approximately 103.2 kJ/mol ofglucose. (In vivo, ∆G for the synthesis of ATP fromADP has been calculated as approximately 51.6 kJ/mol.(It is greater than ∆G 0′ for the hydrolysis of ATP asgiven in Table 10–1, which is obtained under standardFOODFatCarbohydrateProteinDigestion and absorptionFatty acids+GlycerolGlucose, etcAmino acidsAcetyl – CoAβ-OxidationMITOCHONDRIONCitricacidcycle2HADPExtramitochondrial sources ofreducing equivalentsATPRespiratory chainFigure 12–2. Role of the respiratory chain of mitochondria in the conversion of food energy to ATP. Oxidationof the major foodstuffs leads to the generation of reducing equivalents (2H) that are collected by the respiratorychain for oxidation and coupled generation of ATP.O 2H 2 O

94 / CHAPTER 12AH 2 NAD + FpH 2A Fp2Fe 2+1 /2O 2H + 2H +2Fe 3+ 2H + Figure 12–3. Transport of reducingSubstrate Flavoprotein CytochromesH 2 OH +NADHequivalents through the respiratorychain.concentrations of 1.0 mol/L.) Since 1 mol of glucoseyields approximately 2870 kJ on complete combustion,the energy captured by phosphorylation in glycolysis issmall. Two more high-energy phosphates per mole ofglucose are captured in the citric acid cycle during theconversion of succinyl CoA to succinate. All of thesephosphorylations occur at the substrate level. Whensubstrates are oxidized via an NAD-linked dehydrogenaseand the respiratory chain, approximately 3 mol ofinorganic phosphate are incorporated into 3 mol ofADP to form 3 mol of ATP per half mol of O 2 consumed;ie, the P:O ratio = 3 (Figure 12–7). On theother hand, when a substrate is oxidized via a flavoprotein-linkeddehydrogenase, only 2 mol of ATP areformed; ie, P:O = 2. These reactions are known as oxidativephosphorylation at the respiratory chainlevel. Such dehydrogenations plus phosphorylations atthe substrate level can now account for 68% of the freeenergy resulting from the combustion of glucose, capturedin the form of high-energy phosphate. It is evidentthat the respiratory chain is responsible for a largeproportion of total ATP formation.Respiratory Control Ensuresa Constant Supply of ATPThe rate of respiration of mitochondria can be controlledby the availability of ADP. This is because oxidationand phosphorylation are tightly coupled; ie, oxidationcannot proceed via the respiratory chain withoutconcomitant phosphorylation of ADP. Table 12–1shows the five conditions controlling the rate of respirationin mitochondria. Most cells in the resting state arein state 4, and respiration is controlled by the availabilityof ADP. When work is performed, ATP is convertedto ADP, allowing more respiration to occur,which in turn replenishes the store of ATP. Under certainconditions, the concentration of inorganic phosphatecan also affect the rate of functioning of the respiratorychain. As respiration increases (as in exercise),PyruvateProline3-Hydroxyacyl-CoA3-HydroxybutyrateGlutamateMalateIsocitrateSuccinateCholineFp(FAD)FeSLipoateFp(FAD)NADFp(FMN)FeSQCyt bFeSCyt c 1 Cyt c Cyt aa 3CuO 2α-KetoglutarateFp(FAD)FeSFeSETF(FAD)Glycerol 3-phosphateFp(FAD)Acyl-CoASarcosineDimethylglycineFeS: Iron-sulfur proteinETF: Electron-transferring flavoproteinFp: FlavoproteinQ: UbiquinoneCyt: CytochromeFigure 12–4. Components of the respiratory chain in mitochondria, showing the collecting points for reducingequivalents from important substrates. FeS occurs in the sequences on the O 2 side of Fp or Cyt b.

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION / 95OH(H + + e – )OHH(H + + e – )CH 3 OCH 3 OOHCH 3 CH 3[CH 2 CHCCH 2 ] n HOFully oxidized orquinone form• OSemiquinone form(free radical)OHReduced or quinol form(hydroquinone)Figure 12–5. Structure of ubiquinone (Q). n = Number of isoprenoid units, which is10 in higher animals, ie, Q 10 .the cell approaches state 3 or state 5 when either the capacityof the respiratory chain becomes saturated or thePO 2 decreases below the K m for cytochrome a 3 . There isalso the possibility that the ADP/ATP transporter (Figure12–9), which facilitates entry of cytosolic ADP intoand ATP out of the mitochondrion, becomes ratelimiting.Thus, the manner in which biologic oxidativeprocesses allow the free energy resulting from the oxidationof foodstuffs to become available and to be capturedis stepwise, efficient (approximately 68%), andcontrolled—rather than explosive, inefficient, and uncontrolled,as in many nonbiologic processes. The remainingfree energy that is not captured as high-energyphosphate is liberated as heat. This need not be considered“wasted,” since it ensures that the respiratory systemas a whole is sufficiently exergonic to be removedfrom equilibrium, allowing continuous unidirectionalflow and constant provision of ATP. It also contributesto maintenance of body temperature.PrPrCysCysSSSFeFeSSCysSSPrCysFigure 12–6. Iron-sulfur-protein complex (Fe 4 S 4 ). S ,acid-labile sulfur; Pr, apoprotein; Cys, cysteine residue.Some iron-sulfur proteins contain two iron atoms andtwo sulfur atoms (Fe 2 S 2 ).FeFeSPrMANY POISONS INHIBIT THERESPIRATORY CHAINMuch information about the respiratory chain has beenobtained by the use of inhibitors, and, conversely, thishas provided knowledge about the mechanism of actionof several poisons (Figure 12–7). They may be classifiedas inhibitors of the respiratory chain, inhibitors of oxidativephosphorylation, and uncouplers of oxidativephosphorylation.Barbiturates such as amobarbital inhibit NADlinkeddehydrogenases by blocking the transfer fromFeS to Q. At sufficient dosage, they are fatal in vivo.Antimycin A and dimercaprol inhibit the respiratorychain between cytochrome b and cytochrome c. Theclassic poisons H 2 S, carbon monoxide, and cyanideinhibit cytochrome oxidase and can therefore totally arrestrespiration. Malonate is a competitive inhibitor ofsuccinate dehydrogenase.Atractyloside inhibits oxidative phosphorylation byinhibiting the transporter of ADP into and ATP out ofthe mitochondrion (Figure 12–10).The action of uncouplers is to dissociate oxidationin the respiratory chain from phosphorylation. Thesecompounds are toxic in vivo, causing respiration to becomeuncontrolled, since the rate is no longer limitedby the concentration of ADP or P i . The uncoupler thathas been used most frequently is 2,4-dinitrophenol,but other compounds act in a similar manner. The antibioticoligomycin completely blocks oxidation andphosphorylation by acting on a step in phosphorylation(Figures 12–7 and 12–8).THE CHEMIOSMOTIC THEORY EXPLAINSTHE MECHANISM OF OXIDATIVEPHOSPHORYLATIONMitchell’s chemiosmotic theory postulates that theenergy from oxidation of components in the respiratorychain is coupled to the translocation of hydrogen ions(protons, H + ) from the inside to the outside of theinner mitochondrial membrane. The electrochemicalpotential difference resulting from the asymmetric dis-

96 / CHAPTER 12NADHSuccinateComplex IFMN, FeSUncouplers –Malonate–Complex II––FADFeSPiericidin AAmobarbitalRotenoneCarboxinTTFAH 2 SCOCN –BALAntimycin AComplex IIIComplex IVQ Cyt b, FeS, Cyt c 1 Cyt cCyt a Cyt a 3Cu CuO 2–––Uncouplers–Oligomycin––Oligomycin–ADP + P i ATP ADP + P i ATP ADP + P i ATPFigure 12–7. Proposed sites of inhibition (− ) of the respiratory chain by specific drugs, chemicals, and antibiotics.The sites that appear to support phosphorylation are indicated. BAL, dimercaprol. TTFA, an Fe-chelatingagent. Complex I, NADH:ubiquinone oxidoreductase; complex II, succinate:ubiquinone oxidoreductase; complexIII, ubiquinol:ferricytochrome c oxidoreductase; complex IV, ferrocytochrome c:oxygen oxidoreductase. Other abbreviationsas in Figure 12–4.tribution of the hydrogen ions is used to drive themechanism responsible for the formation of ATP (Figure12–8).The Respiratory Chain Is a Proton PumpEach of the respiratory chain complexes I, III, and IV(Figures 12–7 and 12–8) acts as a proton pump. Theinner membrane is impermeable to ions in general butparticularly to protons, which accumulate outside themembrane, creating an electrochemical potential differenceacross the membrane (∆µ + H ).This consists of achemical potential (difference in pH) and an electricalpotential.A Membrane-Located ATP SynthaseFunctions as a Rotary Motor to Form ATPThe electrochemical potential difference is used to drivea membrane-located ATP synthase which in the presenceof P i + ADP forms ATP (Figure 12–8). Scatteredover the surface of the inner membrane are the phosphorylatingcomplexes, ATP synthase, responsible forthe production of ATP (Figure 12–1). These consist ofseveral protein subunits, collectively known as F 1 ,which project into the matrix and which contain thephosphorylation mechanism (Figure 12–8). These subunitsare attached to a membrane protein complexknown as F 0 , which also consists of several protein subunits.F 0 spans the membrane and forms the protonchannel. The flow of protons through F 0 causes it to rotate,driving the production of ATP in the F 1 complex(Figure 12–9). Estimates suggest that for each NADHoxidized, complex I translocates four protons and complexesIII and IV translocate 6 between them. As fourprotons are taken into the mitochondrion for each ATPexported, the P:O ratio would not necessarily be a completeinteger, ie, 3, but possibly 2.5. However, for simplicity,a value of 3 for the oxidation of NADH + H +and 2 for the oxidation of FADH 2 will continue to beused throughout this text.Experimental Findings Supportthe Chemiosmotic Theory(1) Addition of protons (acid) to the externalmedium of intact mitochondria leads to the generationof ATP.(2) Oxidative phosphorylation does not occur in solublesystems where there is no possibility of a vectorialATP synthase. A closed membrane must be present inorder to achieve oxidative phosphorylation (Figure 12–8).(3) The respiratory chain contains components organizedin a sided manner (transverse asymmetry) as requiredby the chemiosmotic theory.

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION / 97PhospholipidbilayerOligomycinF 0–ProtoncircuitMitochondrialinner (coupling)membraneH +Uncoupling agentsH +ATPSYNTHASEADP + P iINSIDEH + H +F 1ATP + H 2 OH + IIIH +–translocationProtonINADH+ H + NAD +H 2 O1 /2O 2QCIVpH gradient (∆pH)H +Respiratory(electrontransport)chainElectricalpotentialOUTSIDE+(∆Ψ)Figure 12–8. Principles of the chemiosmotic theory of oxidative phosphorylation. The main proton circuitis created by the coupling of oxidation in the respiratory chain to proton translocation from the inside to theoutside of the membrane, driven by the respiratory chain complexes I, III, and IV, each of which acts as a protonpump. Q, ubiquinone; C, cytochrome c; F 1 , F 0 , protein subunits which utilize energy from the proton gradientto promote phosphorylation. Uncoupling agents such as dinitrophenol allow leakage of H + across themembrane, thus collapsing the electrochemical proton gradient. Oligomycin specifically blocks conductionof H + through F 0 .Table 12–1. States of respiratory control.Conditions Limiting the Rate of RespirationState 1 Availability of ADP and substrateState 2 Availability of substrate onlyState 3 The capacity of the respiratory chain itself, whenall substrates and components are present insaturating amountsState 4 Availability of ADP onlyState 5 Availability of oxygen onlyThe Chemiosmotic Theory Can Accountfor Respiratory Control and the Actionof UncouplersThe electrochemical potential difference across the membrane,once established as a result of proton translocation,inhibits further transport of reducing equivalentsthrough the respiratory chain unless discharged by backtranslocationof protons across the membrane throughthe vectorial ATP synthase. This in turn depends onavailability of ADP and P i .Uncouplers (eg, dinitrophenol) are amphipathic(Chapter 14) and increase the permeability of the lipoidinner mitochondrial membrane to protons (Figure12–8), thus reducing the electrochemical potential andshort-circuiting the ATP synthase. In this way, oxidationcan proceed without phosphorylation.

98 / CHAPTER 12ADP+PiβαβATPγααβATPing electrical and osmotic equilibrium. The innerbilipoid mitochondrial membrane is freely permeableto uncharged small molecules, such as oxygen, water,CO 2 , and NH 3 , and to monocarboxylic acids, such as3-hydroxybutyric, acetoacetic, and acetic. Long-chainfatty acids are transported into mitochondria via thecarnitine system (Figure 22–1), and there is also a specialcarrier for pyruvate involving a symport that utilizesthe H + gradient from outside to inside the mitochondrion(Figure 12–10). However, dicarboxylate and tri-γInsideCOutside CCH +CCCH + MitochondrialinnermembraneFigure 12–9. Mechanism of ATP production by ATPsynthase. The enzyme complex consists of an F 0 subcomplexwhich is a disk of “C” protein subunits. Attachedis a γ-subunit in the form of a “bent axle.” Protonspassing through the disk of “C” units cause it andthe attached γ-subunit to rotate. The γ-subunit fits insidethe F 1 subcomplex of three α- and three β-subunits,which are fixed to the membrane and do not rotate.ADP and P i are taken up sequentially by theβ-subunits to form ATP, which is expelled as the rotatingγ-subunit squeezes each β-subunit in turn. Thus,three ATP molecules are generated per revolution. Forclarity, not all the subunits that have been identified areshown—eg, the “axle” also contains an ε-subunit.OUTSIDEN-EthylmaleimideH 2 PO– 4N-EthylmaleimideHydroxycinnamatePyruvate –H +Innermitochondrialmembrane12345OH –HPO 42–Malate 2– Malate 2–Citrate 3–+ H +α-Ketoglutarate 2–Malate 2–ADP 3– 6ATP 4––––INSIDEAtractylosideTHE RELATIVE IMPERMEABILITYOF THE INNER MITOCHONDRIALMEMBRANE NECESSITATESEXCHANGE TRANSPORTERSExchange diffusion systems are present in the membranefor exchange of anions against OH − ions andcations against H + ions. Such systems are necessary foruptake and output of ionized metabolites while preserv-Figure 12–10. Transporter systems in the inner mitochondrialmembrane. 1 , phosphate transporter;2 , pyruvate symport; 3 , dicarboxylate transporter;4 , tricarboxylate transporter; 5 , α-ketoglutarate transporter;6 , adenine nucleotide transporter. N-Ethylmaleimide,hydroxycinnamate, and atractyloside inhibit(− ) the indicated systems. Also present (but notshown) are transporter systems for glutamate/aspartate(Figure 12–13), glutamine, ornithine, neutral aminoacids, and carnitine (Figure 22–1).

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION / 99carboxylate anions and amino acids require specifictransporter or carrier systems to facilitate their passageacross the membrane. Monocarboxylic acids penetratemore readily in their undissociated and more lipid-solubleform.The transport of di- and tricarboxylate anions isclosely linked to that of inorganic phosphate, whichpenetrates readily as the H 2 PO 4 − ion in exchange forOH − . The net uptake of malate by the dicarboxylatetransporter requires inorganic phosphate for exchangein the opposite direction. The net uptake of citrate,isocitrate, or cis-aconitate by the tricarboxylate transporterrequires malate in exchange. α-Ketoglutaratetransport also requires an exchange with malate. Theadenine nucleotide transporter allows the exchange ofATP and ADP but not AMP. It is vital in allowingATP exit from mitochondria to the sites of extramitochondrialutilization and in allowing the return of ADPfor ATP production within the mitochondrion (Figure12–11). Na + can be exchanged for H + , driven by theproton gradient. It is believed that active uptake of Ca 2+by mitochondria occurs with a net charge transfer of 1(Ca + uniport), possibly through a Ca 2+ /H + antiport.Calcium release from mitochondria is facilitated by exchangewith Na + .OUTSIDEP i–InnermitochondrialmembraneF 13H + ATP 4–21INSIDEH +ATP SYNTHASEADP 3–Figure 12–11. Combination of phosphate transporter(1 ) with the adenine nucleotide transporter (2 )in ATP synthesis. The H + /P i symport shown is equivalentto the P i /OH − antiport shown in Figure 12–10. Fourprotons are taken into the mitochondrion for each ATPexported. However, one less proton would be taken inwhen ATP is used inside the mitochondrion.Ionophores Permit Specific Cationsto Penetrate MembranesIonophores are lipophilic molecules that complex specificcations and facilitate their transport through biologicmembranes, eg, valinomycin (K + ). The classicuncouplers such as dinitrophenol are, in fact, protonionophores.A Proton-Translocating TranshydrogenaseIs a Source of Intramitochondrial NADPHEnergy-linked transhydrogenase, a protein in the innermitochondrial membrane, couples the passage of protonsdown the electrochemical gradient from outside toinside the mitochondrion with the transfer of H fromintramitochondrial NADH to NADPH for intramitochondrialenzymes such as glutamate dehydrogenaseand hydroxylases involved in steroid synthesis.Oxidation of Extramitochondrial NADHIs Mediated by Substrate ShuttlesNADH cannot penetrate the mitochondrial membrane,but it is produced continuously in the cytosol by3-phosphoglyceraldehyde dehydrogenase, an enzyme inthe glycolysis sequence (Figure 17–2). However, underaerobic conditions, extramitochondrial NADH does notaccumulate and is presumed to be oxidized by the respiratorychain in mitochondria. The transfer of reducingequivalents through the mitochondrial membrane requiressubstrate pairs, linked by suitable dehydrogenaseson each side of the mitochondrial membrane. Themechanism of transfer using the glycerophosphateshuttle is shown in Figure 12–12). Since the mitochondrialenzyme is linked to the respiratory chain via aflavoprotein rather than NAD, only 2 mol rather than3 mol of ATP are formed per atom of oxygen consumed.Although this shuttle is present in some tissues(eg, brain, white muscle), in others (eg, heart muscle) itis deficient. It is therefore believed that the malateshuttle system (Figure 12–13) is of more universalutility. The complexity of this system is due to the impermeabilityof the mitochondrial membrane to oxaloacetate,which must react with glutamate and transaminateto aspartate and α-ketoglutarate before transportthrough the mitochondrial membrane and reconstitutionto oxaloacetate in the cytosol.Ion Transport in MitochondriaIs Energy-LinkedMitochondria maintain or accumulate cations such asK + , Na + , Ca 2+ , and Mg 2+ , and P i . It is assumed that aprimary proton pump drives cation exchange.

100 / CHAPTER 12OUTERMEMBRANEINNERMEMBRANECYTOSOLMITOCHONDRIONNAD +Glycerol 3-phosphateGlycerol 3-phosphateFADNADH + H +GLYCEROL-3-PHOSPHATEDEHYDROGENASE(CYTOSOLIC)DihydroxyacetonephosphateGLYCEROL-3-PHOSPHATEDEHYDROGENASE(MITOCHONDRIAL)DihydroxyacetonephosphateFADH 2Respiratory chainFigure 12–12.mitochondrion.Glycerophosphate shuttle for transfer of reducing equivalents from the cytosol into theThe Creatine Phosphate ShuttleFacilitates Transport of High-EnergyPhosphate From MitochondriaThis shuttle (Figure 12–14) augments the functions ofcreatine phosphate as an energy buffer by acting as adynamic system for transfer of high-energy phosphatefrom mitochondria in active tissues such as heart andskeletal muscle. An isoenzyme of creatine kinase (CK m )is found in the mitochondrial intermembrane space,catalyzing the transfer of high-energy phosphate to creatinefrom ATP emerging from the adenine nucleotidetransporter. In turn, the creatine phosphate is transportedinto the cytosol via protein pores in the outermitochondrial membrane, becoming available for generationof extramitochondrial ATP.CYTOSOLINNERMEMBRANEMITOCHONDRIONCLINICAL ASPECTSThe condition known as fatal infantile mitochondrialmyopathy and renal dysfunction involves severe diminutionor absence of most oxidoreductases of the respiratorychain. MELAS (mitochondrial encephalopathy,lactic acidosis, and stroke) is an inherited condition dueto NADH:ubiquinone oxidoreductase (complex I) orcytochrome oxidase deficiency. It is caused by a muta-Malate NAD +NAD +Malate+ H + + H +1MALATE DEHYDROGENASEMALATE DEHYDROGENASENADHOxaloacetateα-KGα-KGOxaloacetateNADHTRANSAMINASETRANSAMINASEGlutamateAspAspGlutamate2H + H +Figure 12–13. Malate shuttle for transfer of reducing equivalents from the cytosol into the mitochondrion.1 Ketoglutarate transporter; 2 , glutamate/aspartate transporter (note the proton symport with glutamate).

THE RESPIRATORY CHAIN & OXIDATIVE PHOSPHORYLATION / 101ATPEnergy-requiringprocesses(eg, muscle contraction)CK aADPATPADPCreatineCK cCreatine-PATPCK gADPFigure 12–14. The creatine phosphate shuttle ofheart and skeletal muscle. The shuttle allows rapidtransport of high-energy phosphate from the mitochondrialmatrix into the cytosol. CK a , creatine kinaseconcerned with large requirements for ATP, eg, muscularcontraction; CK c , creatine kinase for maintainingequilibrium between creatine and creatine phosphateand ATP/ADP; CK g , creatine kinase coupling glycolysisto creatine phosphate synthesis; CK m , mitochondrialcreatine kinase mediating creatine phosphate productionfrom ATP formed in oxidative phosphorylation; P,pore protein in outer mitochondrial membrane.PATPGlycolysisOuter mitochondrialmembraneCK mADPAdeninenucleotidetransporterOxidativephosphorylationMatrixPmitochondrialmembraneCytosolInter-membranespaceInnertion in mitochondrial DNA and may be involved inAlzheimer’s disease and diabetes mellitus. A number ofdrugs and poisons act by inhibition of oxidative phosphorylation(see above).SUMMARY• Virtually all energy released from the oxidation ofcarbohydrate, fat, and protein is made available inmitochondria as reducing equivalents (⎯H or e − ).These are funneled into the respiratory chain, wherethey are passed down a redox gradient of carriers totheir final reaction with oxygen to form water.• The redox carriers are grouped into respiratory chaincomplexes in the inner mitochondrial membrane.These use the energy released in the redox gradient topump protons to the outside of the membrane, creatingan electrochemical potential across the membrane.• Spanning the membrane are ATP synthase complexesthat use the potential energy of the proton gradientto synthesize ATP from ADP and P i . In thisway, oxidation is closely coupled to phosphorylationto meet the energy needs of the cell.• Because the inner mitochondrial membrane is impermeableto protons and other ions, special exchangetransporters span the membrane to allow passage ofions such as OH – , P i − , ATP 4− , ADP 3− , and metabolites,without discharging the electrochemical gradientacross the membrane.• Many well-known poisons such as cyanide arrest respirationby inhibition of the respiratory chain.REFERENCESBalaban RS: Regulation of oxidative phosphorylation in the mammaliancell. Am J Physiol 1990;258:C377.Hinkle PC et al: Mechanistic stoichiometry of mitochondrial oxidativephosphorylation. Biochemistry 1991;30:3576.Mitchell P: Keilin’s respiratory chain concept and its chemiosmoticconsequences. Science 1979;206:1148.Smeitink J et al: The genetics and pathology of oxidative phosphorylation.Nat Rev Genet 2001;2:342.Tyler DD: The Mitochondrion in Health and Disease. VCH Publishers,1992.Wallace DC: Mitochondrial DNA in aging and disease. Sci Am1997;277(2):22.Yoshida M et al: ATP synthase—a marvellous rotary engine of thecell. Nat Rev Mol Cell Biol 2001;2:669.

Carbohydrates ofPhysiologic Significance13Peter A. Mayes, PhD, DSc, & David A. Bender, PhDBIOMEDICAL IMPORTANCECarbohydrates are widely distributed in plants and animals;they have important structural and metabolicroles. In plants, glucose is synthesized from carbondioxide and water by photosynthesis and stored asstarch or used to synthesize cellulose of the plant framework.Animals can synthesize carbohydrate from lipidglycerol and amino acids, but most animal carbohydrateis derived ultimately from plants. Glucose is themost important carbohydrate; most dietary carbohydrateis absorbed into the bloodstream as glucose, andother sugars are converted into glucose in the liver.Glucose is the major metabolic fuel of mammals (exceptruminants) and a universal fuel of the fetus. It isthe precursor for synthesis of all the other carbohydratesin the body, including glycogen for storage; riboseand deoxyribose in nucleic acids; and galactosein lactose of milk, in glycolipids, and in combinationwith protein in glycoproteins and proteoglycans. Diseasesassociated with carbohydrate metabolism includediabetes mellitus, galactosemia, glycogen storagediseases, and lactose intolerance.CARBOHYDRATES ARE ALDEHYDEOR KETONE DERIVATIVES OFPOLYHYDRIC ALCOHOLS(1) Monosaccharides are those carbohydrates thatcannot be hydrolyzed into simpler carbohydrates: Theymay be classified as trioses, tetroses, pentoses, hexoses,or heptoses, depending upon the number of carbonatoms; and as aldoses or ketoses depending uponwhether they have an aldehyde or ketone group. Examplesare listed in Table 13–1.(2) Disaccharides are condensation products of twomonosaccharide units. Examples are maltose and sucrose.(3) Oligosaccharides are condensation products oftwo to ten monosaccharides; maltotriose* is an example.*Note that this is not a true triose but a trisaccharide containingthree α-glucose residues.102(4) Polysaccharides are condensation products ofmore than ten monosaccharide units; examples are thestarches and dextrins, which may be linear or branchedpolymers. Polysaccharides are sometimes classified ashexosans or pentosans, depending upon the identity ofthe constituent monosaccharides.BIOMEDICALLY, GLUCOSE IS THE MOSTIMPORTANT MONOSACCHARIDEThe Structure of Glucose Can BeRepresented in Three WaysThe straight-chain structural formula (aldohexose;Figure 13–1A) can account for some of the propertiesof glucose, but a cyclic structure is favored on thermodynamicgrounds and accounts for the remainder of itschemical properties. For most purposes, the structuralformula is represented as a simple ring in perspective asproposed by Haworth (Figure 13–1B). In this representation,the molecule is viewed from the side and abovethe plane of the ring. By convention, the bonds nearestto the viewer are bold and thickened. The six-memberedring containing one oxygen atom is in the formof a chair (Figure 13–1C).Sugars Exhibit Various Forms of IsomerismGlucose, with four asymmetric carbon atoms, can form16 isomers. The more important types of isomerismfound with glucose are as follows.(1) D and L isomerism: The designation of a sugarisomer as the D form or of its mirror image as the L formTable 13–1. Classification of important sugars.Aldoses KetosesTrioses (C 3 H 6 O 3 ) Glycerose DihydroxyacetoneTetroses (C 4 H 8 O 4 ) Erythrose ErythrulosePentoses (C 5 H 10 O 5 ) Ribose RibuloseHexoses (C 6 H 12 O 6 ) Glucose Fructose

CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE / 103AO1CHOOH2COHHOH3 C H4 C OHPyranFuranH5 C OH6 CH 2 OHHOCH 2BCHOH46HOCH 24HHO6HOCH 25H5HOHHHO3HOH3 2OHOH1OHH2OHH1OHFigure 13–1. D-Glucose. A: straight chain form.B: α-D-glucose; Haworth projection. C: α-D-glucose;chair form.HOCH 2OH H HHO OHHHOHOHHCOHOH OHHHH OHOHα-D-Glucopyranoseα-D-GlucofuranoseFigure 13–3. Pyranose and furanose forms of glucose.HOO1 C2 CHH3 CH 2 OHL-Glycerose(L-glyceraldehyde)HOCCHOHCH 2 OHD-Glycerose(D-glyceraldehyde)H6H H5HOHOHOHO4 31HOCH 2H2OHH6 OH H OH52HO H HO14 3 COHOH H H 2OOα-D-Fructopyranoseβ-D-FructopyranoseHOH1 C2 C3 CHHOHHHOCCCHOHH6HOCH 25O1HOCH 226HOCH 25OOH2HO4 CHHCOHH HHO OHH HHOHO5 C HH C OH6 CH 2 OHCH 2 OHL-GlucoseD-GlucoseFigure 13–2. D- and L-isomerism of glycerose andglucose.4343 1COHOH HOH H H 2α-D-Fructofuranoseβ-D-FructofuranoseFigure 13–4. Pyranose and furanose forms of fructose.

104 / CHAPTER 13HOCH 2HO HOHHOCH 2H HOHHOCH 2H HOH4HOHHOH4OHOHHOHOHOHHOOH22H OHH OHH Hα-D-Galactose α-D-Glucose α-D-MannoseFigure 13–5.glucose.Epimerization ofis determined by its spatial relationship to the parentcompound of the carbohydrates, the three-carbonsugar glycerose (glyceraldehyde). The L and D forms ofthis sugar, and of glucose, are shown in Figure 13–2.The orientation of the ⎯H and ⎯ OH groups aroundthe carbon atom adjacent to the terminal primary alcoholcarbon (carbon 5 in glucose) determines whetherthe sugar belongs to the D or L series. When the ⎯OHgroup on this carbon is on the right (as seen in Figure13–2), the sugar is the D-isomer; when it is on theleft, it is the L-isomer. Most of the monosaccharidesoccurring in mammals are D sugars, and the enzymesresponsible for their metabolism are specific for thisconfiguration. In solution, glucose is dextrorotatory—hence the alternative name dextrose, often used inclinical practice.The presence of asymmetric carbon atoms also confersoptical activity on the compound. When a beamof plane-polarized light is passed through a solution ofan optical isomer, it will be rotated either to the right,dextrorotatory (+); or to the left, levorotatory (−). Thedirection of rotation is independent of the stereochemistryof the sugar, so it may be designated D(−), D(+),L(−), or L(+). For example, the naturally occurring formof fructose is the D(−) isomer.(2) Pyranose and furanose ring structures: Thestable ring structures of monosaccharides are similar tothe ring structures of either pyran (a six-memberedring) or furan (a five-membered ring) (Figures 13–3and 13–4). For glucose in solution, more than 99% isin the pyranose form.(3) Alpha and beta anomers: The ring structure ofan aldose is a hemiacetal, since it is formed by combinationof an aldehyde and an alcohol group. Similarly, thering structure of a ketose is a hemiketal. Crystalline glucoseis α-D-glucopyranose. The cyclic structure is retainedin solution, but isomerism occurs about position1, the carbonyl or anomeric carbon atom, to give amixture of α-glucopyranose (38%) and β-glucopyranose(62%). Less than 0.3% is represented by α and βanomers of glucofuranose.(4) Epimers: Isomers differing as a result of variationsin configuration of the ⎯ OH and ⎯H on carbonatoms 2, 3, and 4 of glucose are known as epimers.Biologically, the most important epimers of glucose aremannose and galactose, formed by epimerization at carbons2 and 4, respectively (Figure 13–5).(5) Aldose-ketose isomerism: Fructose has thesame molecular formula as glucose but differs in itsstructural formula, since there is a potential keto groupin position 2, the anomeric carbon of fructose (Figures13–4 and 13–7), whereas there is a potential aldehydegroup in position 1, the anomeric carbon of glucose(Figures 13–2 and 13–6).Many Monosaccharides ArePhysiologically ImportantDerivatives of trioses, tetroses, and pentoses and of aseven-carbon sugar (sedoheptulose) are formed as metabolicintermediates in glycolysis and the pentose phosphatepathway. Pentoses are important in nucleotides,CHOCHOCHOCHOCHOCHOCHOHCOHHOCHHCOHHCHOCOHCH 2 OHD-Glycerose(D-glyceraldehyde)HHCHOCCOHOHCH 2 OHD-ErythroseHOHOHCCCHHOHCH 2 OHD-LyxoseHHOHCCCOHHOHCH 2 OHD-XyloseHOHHCCCHOHOHCH 2 OHD-ArabinoseH C OHH C OHH C OHCH 2 OHD-RiboseHO C HHO C HH C OHCH 2 OHD-GalactoseHO C HH C OHH C OHCH 2 OHD-MannoseHO C HH C OHH C OHCH 2 OHD-GlucoseFigure 13–6.Examples of aldoses of physiologic significance.

Table 13–2. Pentoses of physiologic importance.CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE / 105Sugar Where Found Biochemical Importance Clinical SignificanceD-Ribose Nucleic acids. Structural elements of nucleic acids andcoenzymes, eg, ATP, NAD, NADP, flavoproteins.Ribose phosphates are intermediatesin pentose phosphate pathway.D-Ribulose Formed in metabolic processes. Ribulose phosphate is an intermediate inpentose phosphate pathway.D-Arabinose Gum arabic. Plum and cherry gums. Constituent of glycoproteins.D-Xylose Wood gums, proteoglycans, Constituent of glycoproteins.glycosaminoglycans.D-Lyxose Heart muscle. A constituent of a lyxoflavin isolated fromhuman heart muscle.L-Xylulose Intermediate in uronic acid pathway. Found in urine in essentialpentosuria.nucleic acids, and several coenzymes (Table 13–2).Glucose, galactose, fructose, and mannose are physiologicallythe most important hexoses (Table 13–3). Thebiochemically important aldoses are shown in Figure13–6, and important ketoses in Figure 13–7.In addition, carboxylic acid derivatives of glucose areimportant, including D-glucuronate (for glucuronideformation and in glycosaminoglycans) and its metabolicderivative, L-iduronate (in glycosaminoglycans)(Figure 13–8) and L-gulonate (an intermediate in theuronic acid pathway; see Figure 20–4).Sugars Form Glycosides With OtherCompounds & With Each OtherGlycosides are formed by condensation between the hydroxylgroup of the anomeric carbon of a monosaccharide,or monosaccharide residue, and a second compoundthat may—or may not (in the case of an aglycone)—beanother monosaccharide. If the second group is a hydroxyl,the O-glycosidic bond is an acetal link because itresults from a reaction between a hemiacetal group(formed from an aldehyde and an ⎯OH group) and an-Table 13–3. Hexoses of physiologic importance.Sugar Source Importance Clinical SignificanceD-Glucose Fruit juices. Hydrolysis of starch, cane The “sugar” of the body. The sugar carried Present in the urine (glycosuria)sugar, maltose, and lactose. by the blood, and the principal one used in diabetes mellitus owing toby the tissues.raised blood glucose (hyperglycemia).D-Fructose Fruit juices. Honey. Hydrolysis of Can be changed to glucose in the liver Hereditary fructose intolerancecane sugar and of inulin (from the and so used in the body. leads to fructose accumulationJerusalem artichoke).and hypoglycemia.D-Galactose Hydrolysis of lactose. Can be changed to glucose in the liver Failure to metabolize leadsand metabolized. Synthesized in the to galactosemia and cataract.mammary gland to make the lactose ofmilk. A constituent of glycolipids andglycoproteins.D-Mannose Hydrolysis of plant mannans and A constituent of many glycoproteins.gums.

106 / CHAPTER 13CH 2 OHCH 2 OHCOCH 2 OHCH 2 OHCOHOCHCOCOHOCHHCOHCH 2 OHHOCHHCOHHCOHHCOHCOHCOHHCOHHCOHHCOHCH 2 OHCH 2 OHCH 2 OHCH 2 OHDihydroxyacetone D-XyluloseD-RibuloseD-FructoseFigure 13–7. Examples of ketoses of physiologic significance.CH 2 OHD-Sedoheptuloseother ⎯OH group. If the hemiacetal portion is glucose,the resulting compound is a glucoside; if galactose, agalactoside; and so on. If the second group is an amine,an N-glycosidic bond is formed, eg, between adenine andribose in nucleotides such as ATP (Figure 10–4).Glycosides are widely distributed in nature; the aglyconemay be methanol, glycerol, a sterol, a phenol, or abase such as adenine. The glycosides that are importantin medicine because of their action on the heart (cardiacglycosides) all contain steroids as the aglycone.These include derivatives of digitalis and strophanthussuch as ouabain, an inhibitor of the Na + -K + ATPase ofcell membranes. Other glycosides include antibioticssuch as streptomycin.Deoxy Sugars Lack an Oxygen AtomDeoxy sugars are those in which a hydroxyl group hasbeen replaced by hydrogen. An example is deoxyribose(Figure 13–9) in DNA. The deoxy sugar L-fucose (Figure13–15) occurs in glycoproteins; 2-deoxyglucose is usedexperimentally as an inhibitor of glucose metabolism.Amino Sugars (Hexosamines) AreComponents of Glycoproteins,Gangliosides, & GlycosaminoglycansThe amino sugars include D-glucosamine, a constituentof hyaluronic acid (Figure 13–10), D-galactosamine(chondrosamine), a constituent of chondroitin; andD-mannosamine. Several antibiotics (eg, erythromycin)contain amino sugars believed to be important for theirantibiotic activity.MALTOSE, SUCROSE, & LACTOSE AREIMPORTANT DISACCHARIDESThe physiologically important disaccharides are maltose,sucrose, and lactose (Table 13–4; Figure 13–11).Hydrolysis of sucrose yields a mixture of glucose andHHOCOO – HOOH HH COO –OHHHOHOHHOOHFigure 13–8. α-D-Glucuronate (left) andβ-L-iduronate (right).Figure 13–9.5HOCH 2HOCH 2HHO4H H 3OHHOHHOH1H HOHH+ NH3Figure 13–10. Glucosamine (2-amino-D-glucopyranose)(α form). Galactosamine is 2-amino-D-galactopyranose.Both glucosamine and galactosamine occur asN-acetyl derivatives in more complex carbohydrates,eg, glycoproteins.O2HOHHHOHHOH2-Deoxy-D-ribofuranose (β form).

CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE / 107Table 13–4. Disaccharides.Sugar Source Clinical SignificanceMaltose Digestion by amylase or hydrolysis of starch.Germinating cereals and malt.Lactose Milk. May occur in urine during pregnancy. In lactase deficiency, malabsorption leads to diarrhea and flatulence.Sucrose Cane and beet sugar. Sorghum. Pineapple. In sucrase deficiency, malabsorption leads to diarrhea and flatulence.Carrot roots.Trehalose 1 Fungi and yeasts. The major sugar of insecthemolymph.1 O-α-D-Glucopyranosyl-(1 → 1)-α-D-glucopyranoside.fructose which is called “invert sugar” because thestrongly levorotatory fructose changes (inverts) the previousdextrorotatory action of sucrose.POLYSACCHARIDES SERVE STORAGE& STRUCTURAL FUNCTIONSPolysaccharides include the following physiologicallyimportant carbohydrates.Starch is a homopolymer of glucose forming an α-glucosidic chain, called a glucosan or glucan. It is themost abundant dietary carbohydrate in cereals, potatoes,legumes, and other vegetables. The two main constituentsare amylose (15–20%), which has a nonbranchinghelical structure (Figure 13–12); and amylopectin(80–85%), which consists of branched chainscomposed of 24–30 glucose residues united by 1 → 4linkages in the chains and by 1 → 6 linkages at thebranch points.Glycogen (Figure 13–13) is the storage polysaccharidein animals. It is a more highly branched structurethan amylopectin, with chains of 12–14 α-D-glucopyranoseresidues (in α[1 → 4]-glucosidic linkage), withbranching by means of α(1 → 6)-glucosidic bonds.MaltoseLactose6HOCH 25H H4HO OH36HOCH 2O5 OH H H H141* *HOH H OH2 3 2O6HOCH 25HO H4H OH36HOCH 2O5 OH H OH1 O 41* *H HOH H H2 3 2HOHHOHHOHHOHO-α-D-Glucopyranosyl-(1 → 4)-α-D-glucopyranoseO-β-D-Galactopyranosyl-(1 → 4)-β-D-glucopyranose6HOCH 2HHO45HOH3HOH2OH1HSucrose*O1HOCH 2O-α-D-Glucopyranosyl-(1 → 2)-β-D-fructofuranoside*OHOHH2 56H HO COH3 4 H 2Figure 13–11. Structures of important disaccharides. The α and βrefer to the configuration at the anomeric carbon atom (asterisk). Whenthe anomeric carbon of the second residue takes part in the formationof the glycosidic bond, as in sucrose, the residue becomes a glycosideknown as a furanoside or pyranoside. As the disaccharide no longer hasan anomeric carbon with a free potential aldehyde or ketone group, itno longer exhibits reducing properties. The configuration of theβ-fructofuranose residue in sucrose results from turning the β-fructofuranosemolecule depicted in Figure 13–4 through 180 degrees and invertingit.

O108 / CHAPTER 136HOCH 2O6HOCH 2O4 1 4 1O OAOOOOOOBOOO6HOCH 26HOCH 26CH 26HOCH 2O OO O4 1 4 14 14 1OOOO OO OFigure 13–12. Structure of starch. A: Amylose, showing helical coil structure. B: Amylopectin, showing 1 → 6branch point.O4HOC6 H 2O1OO1O4O46CH 2O1O4HOCH 211OHOCH 2G 2 34O4HOC6 H 21OAFigure 13–13. The glycogen molecule. A: General structure. B: Enlargement of structure at a branch point. Themolecule is a sphere approximately 21 nm in diameter that can be visualized in electron micrographs. It has a molecularmass of 10 7 Da and consists of polysaccharide chains each containing about 13 glucose residues. Thechains are either branched or unbranched and are arranged in 12 concentric layers (only four are shown in thefigure). The branched chains (each has two branches) are found in the inner layers and the unbranched chainsin the outer layer. (G, glycogenin, the primer molecule for glycogen synthesis.)BO

CARBOHYDRATES OF PHYSIOLOGIC SIGNIFICANCE / 109OOOHOCH 2H HOHHChitinHOCH 2OH H1 O 4HHNHCO CH 3OHHOOH HHN CO CH 3N-Acetylglucosamine N-AcetylglucosamineHyaluronic acidHOCH 2OCOO –H HO1 OH HHO O H H413OH H HH HN CO CH 3H OHβ-Glucuronic acid N-AcetylglucosamineChondroitin 4-sulfate(Note: There is also a 6-sulfate)HOCH 2OCOO –– SO 3 O HO1 OH HH O H H341OH H HH HN CO CH 3nnInulin is a polysaccharide of fructose (and hence a fructosan)found in tubers and roots of dahlias, artichokes,and dandelions. It is readily soluble in water and is usedto determine the glomerular filtration rate. Dextrins areintermediates in the hydrolysis of starch. Cellulose isthe chief constituent of the framework of plants. It is insolubleand consists of β-D-glucopyranose units linkedby β(1 → 4) bonds to form long, straight chainsstrengthened by cross-linked hydrogen bonds. Cellulosecannot be digested by mammals because of the absenceof an enzyme that hydrolyzes the β linkage. It is an importantsource of “bulk” in the diet. Microorganisms inthe gut of ruminants and other herbivores can hydrolyzethe β linkage and ferment the products to short-chainfatty acids as a major energy source. There is limitedbacterial metabolism of cellulose in the human colon.Chitin is a structural polysaccharide in the exoskeletonof crustaceans and insects and also in mushrooms. Itconsists of N-acetyl-D-glucosamine units joined byβ (1 → 4)-glycosidic linkages (Figure 13–14).Glycosaminoglycans (mucopolysaccharides) arecomplex carbohydrates characterized by their contentof amino sugars and uronic acids. When these chainsare attached to a protein molecule, the result is a proteoglycan.Proteoglycans provide the ground or packingsubstance of connective tissues. Their property ofholding large quantities of water and occupying space,thus cushioning or lubricating other structures, is dueto the large number of ⎯OH groups and negativecharges on the molecules, which, by repulsion, keep thecarbohydrate chains apart. Examples are hyaluronicacid, chondroitin sulfate, and heparin (Figure13–14).Glycoproteins (mucoproteins) occur in many differentsituations in fluids and tissues, including the cellmembranes (Chapters 41 and 47). They are proteinsH OHnβ-Glucuronic acid N-Acetylgalactosamine sulfateHeparinCOSO –3HOOH H H H COO–O1 4OOH HOH H HOH NH SO – H OSO –3 3 nSulfated glucosamine Sulfated iduronic acidFigure 13–14. Structure of some complex polysaccharidesand glycosaminoglycans.Table 13–5. Carbohydrates found inglycoproteins.HexosesMannose (Man)Galactose (Gal)Acetyl hexosamines N-Acetylglucosamine (GlcNAc)N-Acetylgalactosamine (GalNAc)PentosesArabinose (Ara)Xylose (Xyl)Methyl pentose L-Fucose (Fuc; see Figure 13–15)Sialic acidsN-Acyl derivatives of neuraminic acid,eg, N-acetylneuraminic acid (NeuAc; seeFigure 13–16), the predominant sialicacid.

110 / CHAPTER 13Figure 13–15.containing branched or unbranched oligosaccharidechains (see Table 13–5). The sialic acids are N- orO-acyl derivatives of neuraminic acid (Figure 13–16).Neuraminic acid is a nine-carbon sugar derived frommannosamine (an epimer of glucosamine) and pyruvate.Sialic acids are constituents of both glycoproteinsand gangliosides (Chapters 14 and 47).CARBOHYDRATES OCCUR IN CELLMEMBRANES & IN LIPOPROTEINSIn addition to the lipid of cell membranes (see Chapters14 and 41), approximately 5% is carbohydrate in glycoproteinsand glycolipids. Carbohydrates are also presentin apo B of lipoproteins. Their presence on the outersurface of the plasma membrane (the glycocalyx) hasbeen shown with the use of plant lectins, protein agglutininsthat bind with specific glycosyl residues. Forexample, concanavalin A binds α-glucosyl and α-mannosylresidues. Glycophorin is a major integral membraneglycoprotein of human erythrocytes and spansthe lipid membrane, having free polypeptide portionsAcNH CHOHCHOHHHHOHCH 3HOHHCH 2 OHHOHOHHCOO —Figure 13–16. Structure of N-acetylneuraminic acid,a sialic acid (Ac = CH 3 ⎯ CO ⎯).OHOHHOHβ-L-Fucose (6-deoxy-β-L-galactose).OHoutside both the external and internal (cytoplasmic)surfaces. Carbohydrate chains are only attached to theamino terminal portion outside the external surface(Chapter 41).SUMMARY• Carbohydrates are major constituents of animal foodand animal tissues. They are characterized by thetype and number of monosaccharide residues in theirmolecules.• Glucose is the most important carbohydrate in mammalianbiochemistry because nearly all carbohydratein food is converted to glucose for metabolism.• Sugars have large numbers of stereoisomers becausethey contain several asymmetric carbon atoms.• The monosaccharides include glucose, the “bloodsugar”; and ribose, an important constituent of nucleotidesand nucleic acids.• The disaccharides include maltose (glucosyl glucose),an intermediate in the digestion of starch; sucrose(glucosyl fructose), important as a dietary constituentcontaining fructose; and lactose (galactosyl glucose),in milk.• Starch and glycogen are storage polymers of glucosein plants and animals, respectively. Starch is themajor source of energy in the diet.• Complex carbohydrates contain other sugar derivativessuch as amino sugars, uronic acids, and sialicacids. They include proteoglycans and glycosaminoglycans,associated with structural elements of the tissues;and glycoproteins, proteins containing attachedoligosaccharide chains. They are found in many situationsincluding the cell membrane.REFERENCESBinkley RW: Modern Carbohydrate Chemistry. Marcel Dekker,1988.Collins PM (editor): Carbohydrates. Chapman & Hall, 1988.El-Khadem HS: Carbohydrate Chemistry: Monosaccharides andTheir Oligomers. Academic Press, 1988.Lehman J (editor) (translated by Haines A.): Carbohydrates: Structureand Biology. Thieme, 1998.Lindahl U, Höök M: Glycosaminoglycans and their binding to biologicalmacromolecules. Annu Rev Biochem 1978;47:385.Melendes-Hevia E, Waddell TG, Shelton ED: Optimization ofmolecular design in the evolution of metabolism: the glycogenmolecule. Biochem J 1993;295:477.

Lipids of Physiologic Significance 14Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEThe lipids are a heterogeneous group of compounds,including fats, oils, steroids, waxes, and related compounds,which are related more by their physical thanby their chemical properties. They have the commonproperty of being (1) relatively insoluble in water and(2) soluble in nonpolar solvents such as ether andchloroform. They are important dietary constituentsnot only because of their high energy value but also becauseof the fat-soluble vitamins and the essential fattyacids contained in the fat of natural foods. Fat is storedin adipose tissue, where it also serves as a thermal insulatorin the subcutaneous tissues and around certain organs.Nonpolar lipids act as electrical insulators, allowingrapid propagation of depolarization waves alongmyelinated nerves. Combinations of lipid and protein(lipoproteins) are important cellular constituents, occurringboth in the cell membrane and in the mitochondria,and serving also as the means of transportinglipids in the blood. Knowledge of lipid biochemistry isnecessary in understanding many important biomedicalareas, eg, obesity, diabetes mellitus, atherosclerosis,and the role of various polyunsaturated fatty acids innutrition and health.LIPIDS ARE CLASSIFIED AS SIMPLEOR COMPLEX1. Simple lipids: Esters of fatty acids with various alcohols.a. Fats: Esters of fatty acids with glycerol. Oilsare fats in the liquid state.b. Waxes: Esters of fatty acids with higher molecularweight monohydric alcohols.2. Complex lipids: Esters of fatty acids containinggroups in addition to an alcohol and a fatty acid.a. Phospholipids: Lipids containing, in additionto fatty acids and an alcohol, a phosphoricacid residue. They frequently have nitrogencontainingbases and other substituents, eg, inglycerophospholipids the alcohol is glyceroland in sphingophospholipids the alcohol issphingosine.b. Glycolipids (glycosphingolipids): Lipidscontaining a fatty acid, sphingosine, and carbohydrate.111c. Other complex lipids: Lipids such as sulfolipidsand aminolipids. Lipoproteins mayalso be placed in this category.3. Precursor and derived lipids: These include fattyacids, glycerol, steroids, other alcohols, fatty aldehydes,and ketone bodies (Chapter 22), hydrocarbons,lipid-soluble vitamins, and hormones.Because they are uncharged, acylglycerols (glycerides),cholesterol, and cholesteryl esters are termedneutral lipids.FATTY ACIDS ARE ALIPHATICCARBOXYLIC ACIDSFatty acids occur mainly as esters in natural fats and oilsbut do occur in the unesterified form as free fattyacids, a transport form found in the plasma. Fatty acidsthat occur in natural fats are usually straight-chain derivativescontaining an even number of carbon atoms.The chain may be saturated (containing no doublebonds) or unsaturated (containing one or more doublebonds).Fatty Acids Are Named AfterCorresponding HydrocarbonsThe most frequently used systematic nomenclaturenames the fatty acid after the hydrocarbon with thesame number and arrangement of carbon atoms, with-oic being substituted for the final -e (Genevan system).Thus, saturated acids end in -anoic, eg, octanoicacid, and unsaturated acids with double bonds end in-enoic, eg, octadecenoic acid (oleic acid).Carbon atoms are numbered from the carboxyl carbon(carbon No. 1). The carbon atoms adjacent to thecarboxyl carbon (Nos. 2, 3, and 4) are also known asthe α, β, and γ carbons, respectively, and the terminalmethyl carbon is known as the ω or n-carbon.Various conventions use ∆ for indicating the numberand position of the double bonds (Figure 14–1); eg,∆ 9 indicates a double bond between carbons 9 and 10of the fatty acid; ω9 indicates a double bond on theninth carbon counting from the ω- carbon. In animals,additional double bonds are introduced only betweenthe existing double bond (eg, ω9, ω6, or ω3) and the

112 / CHAPTER 1418:1;9 or ∆ 9 18:1ω9,C18:1 or n–9, 18:118 10 91CH 3 (CH 2 ) 7 CH CH(CH 2 ) 7 COOHω 2 3 4 5 6 7 8 9 10 18CH 3 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH CH(CH 2 ) 7 COOHn 1710 91Oleic acid. n − 9 (n minus 9) is equiva-Figure 14–1.lent to ω9.Table 14–1. Saturated fatty acids.Common Number ofName C AtomsAcetic 2 Major end product of carbohydratefermentation by rumenorganisms 1Propionic 3 An end product of carbohydratefermentation by rumenorganisms 1Butyric 4 In certain fats in small amountsValeric 5(especially butter). An end productof carbohydrate fermentation byCaproic 6 rumen organisms 1Lauric 12 Spermaceti, cinnamon, palm kernel,coconut oils, laurels, butterMyristic 14 Nutmeg, palm kernel, coconut oils,myrtles, butterPalmitic 16 Common in all animal and plantStearic 18fats1 Also formed in the cecum of herbivores and to a lesser extent inthe colon of humans.orcarboxyl carbon, leading to three series of fatty acidsknown as the ω9, ω6, and ω3 families, respectively.Saturated Fatty Acids ContainNo Double BondsSaturated fatty acids may be envisaged as based onacetic acid (CH 3 ⎯COOH) as the first member of theseries in which ⎯CH 2 ⎯ is progressively added betweenthe terminal CH 3 ⎯ and ⎯ COOH groups. Examplesare shown in Table 14–1. Other higher membersof the series are known to occur, particularly inwaxes. A few branched-chain fatty acids have also beenisolated from both plant and animal sources.Unsaturated Fatty Acids Contain Oneor More Double Bonds(Table 14–2)Fatty acids may be further subdivided as follows:(1) Monounsaturated (monoethenoid, monoenoic)acids, containing one double bond.(2) Polyunsaturated (polyethenoid, polyenoic)acids, containing two or more double bonds.(3) Eicosanoids: These compounds, derived fromeicosa- (20-carbon) polyenoic fatty acids, comprisethe prostanoids, leukotrienes (LTs), andlipoxins (LXs). Prostanoids include prostaglandins(PGs), prostacyclins (PGIs), andthromboxanes (TXs).Prostaglandins exist in virtually every mammaliantissue, acting as local hormones; they have importantphysiologic and pharmacologic activities. They are synthesizedin vivo by cyclization of the center of the carbonchain of 20-carbon (eicosanoic) polyunsaturatedfatty acids (eg, arachidonic acid) to form a cyclopentanering (Figure 14–2). A related series of compounds, thethromboxanes, have the cyclopentane ring interruptedwith an oxygen atom (oxane ring) (Figure 14–3). Threedifferent eicosanoic fatty acids give rise to three groupsof eicosanoids characterized by the number of doublebonds in the side chains, eg, PG 1 , PG 2 , PG 3 . Differentsubstituent groups attached to the rings give rise to seriesof prostaglandins and thromboxanes, labeled A, B,etc—eg, the “E” type of prostaglandin (as in PGE 2 ) hasa keto group in position 9, whereas the “F” type has ahydroxyl group in this position. The leukotrienes andlipoxins are a third group of eicosanoid derivativesformed via the lipoxygenase pathway (Figure 14–4).They are characterized by the presence of three or fourconjugated double bonds, respectively. Leukotrienescause bronchoconstriction as well as being potentproinflammatory agents and play a part in asthma.Most Naturally Occurring UnsaturatedFatty Acids Have cis Double BondsThe carbon chains of saturated fatty acids form a zigzagpattern when extended, as at low temperatures. Athigher temperatures, some bonds rotate, causing chainshortening, which explains why biomembranes becomethinner with increases in temperature. A type of geometricisomerism occurs in unsaturated fatty acids, dependingon the orientation of atoms or groups aroundthe axes of double bonds, which do not allow rotation.If the acyl chains are on the same side of the bond, itis cis-, as in oleic acid; if on opposite sides, it is trans-, asin elaidic acid, the trans isomer of oleic acid (Fig-

Table 14–2. Unsaturated fatty acids of physiologic and nutritional significance.LIPIDS OF PHYSIOLOGIC SIGNIFICANCE / 113Number of CAtoms and Numberand Position ofCommonDouble Bonds Family Name Systematic Name OccurrenceMonoenoic acids (one double bond)16:1;9 ω7 Palmitoleic cis-9-Hexadecenoic In nearly all fats.18:1;9 ω9 Oleic cis-9-Octadecenoic Possibly the most common fatty acid innatural fats.18:1;9 ω9 Elaidic trans-9-Octadecenoic Hydrogenated and ruminant fats.Dienoic acids (two double bonds)18:2;9,12 ω6 Linoleic all-cis-9,12-Octadecadienoic Corn, peanut, cottonseed, soybean,and many plant oils.Trienoic acids (three double bonds)18:3;6,9,12 ω6 γ-Linolenic all-cis-6,9,12-Octadecatrienoic Some plants, eg, oil of evening primrose,borage oil; minor fatty acid inanimals.18:3;9,12,15 ω3 α-Linolenic all-cis-9,12,15-Octadecatrienoic Frequently found with linoleic acid butparticularly in linseed oil.Tetraenoic acids (four double bonds)20:4;5,8,11,14 ω6 Arachidonic all-cis-5,8,11,14-Eicosatetraenoic Found in animal fats and in peanut oil;important component of phospholipidsin animals.Pentaenoic acids (five double bonds)20:5;5,8,11,14,17 ω3 Timnodonic all-cis-5,8,11,14,17-Eicosapentaenoic Important component of fish oils, eg,cod liver, mackerel, menhaden, salmonoils.Hexaenoic acids (six double bonds)22:6;4,7,10,13,16,19 ω3 Cervonic all-cis-4,7,10,13,16,19-Docosahexaenoic Fish oils, phospholipids in brain.ure 14–5). Naturally occurring unsaturated long-chainfatty acids are nearly all of the cis configuration, themolecules being “bent” 120 degrees at the doublebond. Thus, oleic acid has an L shape, whereas elaidicacid remains “straight.” Increase in the number of cisdouble bonds in a fatty acid leads to a variety of possiblespatial configurations of the molecule—eg, arachidonicacid, with four cis double bonds, has “kinks” or aU shape. This has profound significance on molecularpacking in membranes and on the positions occupiedby fatty acids in more complex molecules such as phospholipids.Trans double bonds alter these spatial relationships.Trans fatty acids are present in certain foods,arising as a by-product of the saturation of fatty acidsduring hydrogenation, or “hardening,” of natural oilsin the manufacture of margarine. An additional smallO10911OH5OHCOO —OOOHCOO —Figure 14–2. Prostaglandin E 2 (PGE 2 ).Figure 14–3. Thromboxane A 2 (TXA 2 ).

H114 / CHAPTER 14OCOO –more unsaturated than storage lipids. Lipids in tissuesthat are subject to cooling, eg, in hibernators or in theextremities of animals, are more unsaturated.Figure 14–4. Leukotriene A 4 (LTA 4 ).contribution comes from the ingestion of ruminant fatthat contains trans fatty acids arising from the action ofmicroorganisms in the rumen.Physical and Physiologic Propertiesof Fatty Acids Reflect Chain Lengthand Degree of UnsaturationThe melting points of even-numbered-carbon fattyacids increase with chain length and decrease accordingto unsaturation. A triacylglycerol containing three saturatedfatty acids of 12 carbons or more is solid at bodytemperature, whereas if the fatty acid residues are 18:2,it is liquid to below 0 °C. In practice, natural acylglycerolscontain a mixture of fatty acids tailored to suittheir functional roles. The membrane lipids, whichmust be fluid at all environmental temperatures, areTRIACYLGLYCEROLS (TRIGLYCERIDES)*ARE THE MAIN STORAGE FORMS OFFATTY ACIDSThe triacylglycerols (Figure 14–6) are esters of the trihydricalcohol glycerol and fatty acids. Mono- and diacylglycerolswherein one or two fatty acids are esterifiedwith glycerol are also found in the tissues. Theseare of particular significance in the synthesis and hydrolysisof triacylglycerols.Carbons 1 & 3 of Glycerol AreNot IdenticalTo number the carbon atoms of glycerol unambiguously,the -sn- (stereochemical numbering) system isused. It is important to realize that carbons 1 and 3 ofglycerol are not identical when viewed in three dimensions(shown as a projection formula in Figure 14–7).Enzymes readily distinguish between them and arenearly always specific for one or the other carbon; eg,glycerol is always phosphorylated on sn-3 by glycerolkinase to give glycerol 3-phosphate and not glycerol1-phosphate.12018CH 3 CH 3Cis form(oleic acid)10CC9HHTrans form(elaidic acid)CCH110PHOSPHOLIPIDS ARE THE MAIN LIPIDCONSTITUENTS OF MEMBRANESPhospholipids may be regarded as derivatives of phosphatidicacid (Figure 14–8), in which the phosphate isesterified with the ⎯ OH of a suitable alcohol. Phosphatidicacid is important as an intermediate in the synthesisof triacylglycerols as well as phosphoglycerols butis not found in any great quantity in tissues.Phosphatidylcholines (Lecithins)Occur in Cell MembranesPhosphoacylglycerols containing choline (Figure 14–8)are the most abundant phospholipids of the cell mem-1COO – COO –Figure 14–5. Geometric isomerism of ∆ 9 , 18:1 fattyacids (oleic and elaidic acids).* According to the standardized terminology of the InternationalUnion of Pure and Applied Chemistry (IUPAC) and the InternationalUnion of Biochemistry (IUB), the monoglycerides, diglycerides,and triglycerides should be designated monoacylglycerols,diacylglycerols, and triacylglycerols, respectively. However, theolder terminology is still widely used, particularly in clinical medicine.

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE / 115Figure 14–6.O1 CH 22R 2 C O CH O3 CH 2 O C R 2Triacylglycerol.brane and represent a large proportion of the body’sstore of choline. Choline is important in nervous transmission,as acetylcholine, and as a store of labile methylgroups. Dipalmitoyl lecithin is a very effective surfaceactiveagent and a major constituent of the surfactantpreventing adherence, due to surface tension, of theinner surfaces of the lungs. Its absence from the lungsof premature infants causes respiratory distress syndrome.Most phospholipids have a saturated acyl radicalin the sn-1 position but an unsaturated radical in thesn-2 position of glycerol.Phosphatidylethanolamine (cephalin) and phosphatidylserine(found in most tissues) differ fromphosphatidylcholine only in that ethanolamine or serine,respectively, replaces choline (Figure 14–8).Phosphatidylinositol Is a Precursorof Second MessengersThe inositol is present in phosphatidylinositol as thestereoisomer, myoinositol (Figure 14–8). Phosphatidylinositol4,5-bisphosphate is an important constituentof cell membrane phospholipids; upon stimulationby a suitable hormone agonist, it is cleaved intodiacylglycerol and inositol trisphosphate, both ofwhich act as internal signals or second messengers.OOCR 1ABCDO1 CH 2 O CR 2 C O2 CH OCH 3+O CH 2 CH 2 N CH 3O CH 2 CH 2 NH 3O CH 2NH 3+OHCholineEthanolamineOH2H3 CH 21 4H6OHCH COO –SerineOH3HOH5HOPhosphatidic acidMyoinositol+HOOHCH 3R 1P O –O –Cardiolipin Is a Major Lipidof Mitochondrial MembranesCH 2OO –POOPhosphatidic acid is a precursor of phosphatidylglycerolwhich, in turn, gives rise to cardiolipin (Figure14–8).EOHCCH 2OHCH 2O H C O C R 3R 4 C O CH 2OO CR 3O1H 2 CR 13H 2 C O CR 2OC O 2 COHPhosphatidylglycerolFigure 14–8. Phosphatidic acid and its derivatives.The O − shown shaded in phosphatidic acid is substitutedby the substituents shown to form in (A) 3-phosphatidylcholine,(B) 3-phosphatidylethanolamine,(C) 3-phosphatidylserine, (D) 3-phosphatidylinositol,and (E) cardiolipin (diphosphatidylglycerol).Figure 14–7.Triacyl-sn-glycerol.

116 / CHAPTER 14Lysophospholipids Are Intermediates inthe Metabolism of PhosphoglycerolsThese are phosphoacylglycerols containing only oneacyl radical, eg, lysophosphatidylcholine (lysolecithin),important in the metabolism and interconversionof phospholipids (Figure 14–9).It is also found inoxidized lipoproteins and has been implicated in someof their effects in promoting atherosclerosis.Plasmalogens Occur in Brain & MuscleThese compounds constitute as much as 10% of thephospholipids of brain and muscle. Structurally, theplasmalogens resemble phosphatidylethanolamine butpossess an ether link on the sn-1 carbon instead of theester link found in acylglycerols. Typically, the alkylradical is an unsaturated alcohol (Figure 14–10). Insome instances, choline, serine, or inositol may be substitutedfor ethanolamine.Sphingomyelins Are Foundin the Nervous SystemSphingomyelins are found in large quantities in brainand nerve tissue. On hydrolysis, the sphingomyelinsyield a fatty acid, phosphoric acid, choline, and a complexamino alcohol, sphingosine (Figure 14–11). Noglycerol is present. The combination of sphingosineplus fatty acid is known as ceramide, a structure alsofound in the glycosphingolipids (see below).GLYCOLIPIDS (GLYCOSPHINGOLIPIDS)ARE IMPORTANT IN NERVE TISSUES& IN THE CELL MEMBRANEGlycolipids are widely distributed in every tissue of thebody, particularly in nervous tissue such as brain. Theyoccur particularly in the outer leaflet of the plasmamembrane, where they contribute to cell surface carbohydrates.The major glycolipids found in animal tissues areglycosphingolipids. They contain ceramide and one ormore sugars. Galactosylceramide is a major glyco-HO1CH 22CH3 CH 2OOOCOPO –ROCH 3+CH 2 CH 2 N CH 3CH 31 CH 2R 2OO CH CH R 1C O2 CH O3 CH 2 O P O CH 2 CH 2+NH 3O –EthanolamineFigure 14–10.Plasmalogen.sphingolipid of brain and other nervous tissue, found inrelatively low amounts elsewhere. It contains a numberof characteristic C 24 fatty acids, eg, cerebronic acid.Galactosylceramide (Figure 14–12) can be converted tosulfogalactosylceramide (sulfatide), present in highamounts in myelin. Glucosylceramide is the predominantsimple glycosphingolipid of extraneural tissues,also occurring in the brain in small amounts. Gangliosidesare complex glycosphingolipids derived from glucosylceramidethat contain in addition one or moremolecules of a sialic acid. Neuraminic acid (NeuAc;see Chapter 13) is the principal sialic acid found inhuman tissues. Gangliosides are also present in nervoustissues in high concentration. They appear to have receptorand other functions. The simplest gangliosidefound in tissues is G M3 , which contains ceramide, onemolecule of glucose, one molecule of galactose, and onemolecule of NeuAc. In the shorthand nomenclatureused, G represents ganglioside; M is a monosialocontainingspecies; and the subscript 3 is a number assignedon the basis of chromatographic migration. G M1(Figure 14–13), a more complex ganglioside derivedfrom G M3 , is of considerable biologic interest, as it isknown to be the receptor in human intestine forcholera toxin. Other gangliosides can contain anywherefrom one to five molecules of sialic acid, giving rise todi-, trisialogangliosides, etc.CeramideSphingosineOHCH CH CHOOHCH N C RCH 2 Fatty acidOP O – +O CH 2 CH 2 N(CH 3 ) 3CholineCH 3 (CH 2 ) 12CholinePhosphoric acidFigure 14–9.Lysophosphatidylcholine (lysolecithin).Figure 14–11.A sphingomyelin.

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE / 117SphingosineCeramideOHOCH (CH2 ) 3 12 CH CH CH CH N H C CH(OH) (CH 2 ) 21 CH 3CH2 OH OFatty acid(eg, cerebronic acid)GalactoseHOHOCH 2Figure 14–12. Structure of galactosylceramide(galactocerebroside, R = H), and sulfogalactosylceramide(a sulfatide, R = SO 2− 4 ).HOR3HHOHHSTEROIDS PLAY MANYPHYSIOLOGICALLY IMPORTANT ROLESCholesterol is probably the best known steroid becauseof its association with atherosclerosis. However, biochemicallyit is also of significance because it is the precursorof a large number of equally important steroidsthat include the bile acids, adrenocortical hormones,sex hormones, D vitamins, cardiac glycosides, sitosterolsof the plant kingdom, and some alkaloids.All of the steroids have a similar cyclic nucleus resemblingphenanthrene (rings A, B, and C) to which acyclopentane ring (D) is attached. The carbon positionson the steroid nucleus are numbered as shown in Figure14–14. It is important to realize that in structural formulasof steroids, a simple hexagonal ring denotes acompletely saturated six-carbon ring with all valencessatisfied by hydrogen bonds unless shown otherwise; ie,it is not a benzene ring. All double bonds are shown assuch. Methyl side chains are shown as single bonds unattachedat the farther (methyl) end. These occur typicallyat positions 10 and 13 (constituting C atoms 19and 18). A side chain at position 17 is usual (as in cholesterol).If the compound has one or more hydroxylgroups and no carbonyl or carboxyl groups, it is asterol, and the name terminates in -ol.Because of Asymmetry in the SteroidMolecule, Many StereoisomersAre PossibleEach of the six-carbon rings of the steroid nucleus is capableof existing in the three-dimensional conformationeither of a “chair” or a “boat” (Figure 14–15). In naturallyoccurring steroids, virtually all the rings are in the“chair” form, which is the more stable conformation.With respect to each other, the rings can be either cis ortrans (Figure 14–16). The junction between the A andB rings can be cis or trans in naturally occurringsteroids. That between B and C is trans, as is usually theC/D junction. Bonds attaching substituent groupsabove the plane of the rings (β bonds) are shown withbold solid lines, whereas those bonds attaching groupsbelow (α bonds) are indicated with broken lines. The Aring of a 5α steroid is always trans to the B ring,whereas it is cis in a 5β steroid. The methyl groups attachedto C 10 and C 13 are invariably in the β configuration.Ceramide Glucose Galactose N-Acetylgalactosamine(Acylsphingosine)NeuAcGalactoseorCer Glc Gal GalNAc GalNeuAcFigure 14–13. G M1 ganglioside, a monosialoganglioside,the receptor in human intestine for cholera toxin.Figure 14–14.231812 171113 1619C D1 9151410 8A B754 6The steroid nucleus.

118 / CHAPTER 14“Chair” form“Boat” formFigure 14–15. Conformations of stereoisomers ofthe steroid nucleus.Cholesterol Is a Significant Constituentof Many TissuesCholesterol (Figure 14–17) is widely distributed in allcells of the body but particularly in nervous tissue. It isa major constituent of the plasma membrane and ofplasma lipoproteins. It is often found as cholesterylester, where the hydroxyl group on position 3 is esterifiedwith a long-chain fatty acid. It occurs in animalsbut not in plants.Ergosterol Is a Precursor of Vitamin DErgosterol occurs in plants and yeast and is importantas a precursor of vitamin D (Figure 14–18). When irradiatedwith ultraviolet light, it acquires antirachiticproperties consequent to the opening of ring B.Polyprenoids Share the Same ParentCompound as CholesterolAlthough not steroids, these compounds are related becausethey are synthesized, like cholesterol (Figure26–2), from five-carbon isoprene units (Figure 14–19).They include ubiquinone (Chapter 12), a member ofthe respiratory chain in mitochondria, and the longchainalcohol dolichol (Figure 14–20), which takespart in glycoprotein synthesis by transferring carbohydrateresidues to asparagine residues of the polypeptide(Chapter 47). Plant-derived isoprenoid compounds includerubber, camphor, the fat-soluble vitamins A, D,E, and K, and β-carotene (provitamin A).LIPID PEROXIDATION IS A SOURCEOF FREE RADICALSPeroxidation (auto-oxidation) of lipids exposed tooxygen is responsible not only for deterioration of foods(rancidity) but also for damage to tissues in vivo,where it may be a cause of cancer, inflammatory diseases,atherosclerosis, and aging. The deleterious effectsare considered to be caused by free radicals (ROO • ,RO • , OH • ) produced during peroxide formation fromfatty acids containing methylene-interrupted doublebonds, ie, those found in the naturally occurringpolyunsaturated fatty acids (Figure 14–21). Lipid peroxidationis a chain reaction providing a continuoussupply of free radicals that initiate further peroxidation.The whole process can be depicted as follows:(1) Initiation:( n) + • ( n– 1)+ +ROOH+ Metal → ROO + Metal + H• •X + RH→ R + XH(2) Propagation:• •R + O2→ROO• •ROO + RH → ROOH+R , etc3AorA3H510B9HC1 9 HA10 H 85 BHH813 17D14HCH1413DB33A1AH 10510B5Figure 14–16. Generalized steroid nucleus, showing (A) an all-trans configuration betweenadjacent rings and (B) a cis configuration between rings A and B.HorB

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE / 119CH 317Figure 14–19.CH C CHIsoprene unit.CHHOFigure 14–17.cholestene.(3) Termination:356Cholesterol, 3-hydroxy-5,6-• •ROO + ROO → ROOR + O2• •ROO + R →ROOR• •R + R →RRSince the molecular precursor for the initiationprocess is generally the hydroperoxide product ROOH,lipid peroxidation is a chain reaction with potentiallydevastating effects. To control and reduce lipid peroxidation,both humans in their activities and nature invokethe use of antioxidants. Propyl gallate, butylatedhydroxyanisole (BHA), and butylated hydroxytoluene(BHT) are antioxidants used as food additives. Naturallyoccurring antioxidants include vitamin E (tocopherol),which is lipid-soluble, and urate and vitamin C,which are water-soluble. Beta-carotene is an antioxidantat low PO 2 . Antioxidants fall into two classes: (1) preventiveantioxidants, which reduce the rate of chain initiation;and (2) chain-breaking antioxidants, which interferewith chain propagation. Preventive antioxidantsinclude catalase and other peroxidases that react withROOH and chelators of metal ions such as EDTA(ethylenediaminetetraacetate) and DTPA (diethylenetriaminepentaacetate).In vivo, the principal chainbreakingantioxidants are superoxide dismutase, whichacts in the aqueous phase to trap superoxide free radicals(O 2− • ); perhaps urate; and vitamin E, which acts inthe lipid phase to trap ROO • radicals (Figure 45–6).Peroxidation is also catalyzed in vivo by heme compoundsand by lipoxygenases found in platelets andleukocytes. Other products of auto-oxidation or enzymicoxidation of physiologic significance includeoxysterols (formed from cholesterol) and isoprostanes(prostanoids).AMPHIPATHIC LIPIDS SELF-ORIENTAT OIL:WATER INTERFACESThey Form Membranes, Micelles,Liposomes, & EmulsionsIn general, lipids are insoluble in water since theycontain a predominance of nonpolar (hydrocarbon)groups. However, fatty acids, phospholipids, sphingolipids,bile salts, and, to a lesser extent, cholesterolcontain polar groups. Therefore, part of the molecule ishydrophobic, or water-insoluble; and part is hydrophilic,or water-soluble. Such molecules are describedas amphipathic (Figure 14–22). They become orientedat oil:water interfaces with the polar group in the waterphase and the nonpolar group in the oil phase. A bilayerof such amphipathic lipids has been regarded as abasic structure in biologic membranes (Chapter 41).When a critical concentration of these lipids is presentin an aqueous medium, they form micelles. Aggregationsof bile salts into micelles and liposomes and theformation of mixed micelles with the products of fat digestionare important in facilitating absorption of lipidsfrom the intestine. Liposomes may be formed by sonicatingan amphipathic lipid in an aqueous medium.They consist of spheres of lipid bilayers that enclosepart of the aqueous medium. They are of potential clinicaluse—particularly when combined with tissuespecificantibodies—as carriers of drugs in the circulation,targeted to specific organs, eg, in cancer therapy.In addition, they are being used for gene transfer intovascular cells and as carriers for topical and transdermalCH 2 OHBHO16Figure 14–18.Ergosterol.Figure 14–20.Dolichol—a C 95 alcohol.

120 / CHAPTER 14RH R• R• ROO•X• XH HO 2 H O O••HHHOOHH•OO•HRHOOHMalondialdehyde Endoperoxide HydroperoxideROOHFigure 14–21. Lipid peroxidation. The reaction is initiated by an existing free radical (X • ), by light, or bymetal ions. Malondialdehyde is only formed by fatty acids with three or more double bonds and is used as ameasure of lipid peroxidation together with ethane from the terminal two carbons of ω3 fatty acids and pentanefrom the terminal five carbons of ω6 fatty acids.+R•AMPHIPATHIC LIPIDANonpolar orhydrophobic groupsPolar orhydrophiIic groupsAqueous phaseAqueous phase“Oil” or nonpolar phaseAqueous phaseNonpolarphase“Oil” ornonpolar phaseAqueous phaseLIPID BILAYERBMICELLECOIL IN WATER EMULSIONDNonpolarphaseAqueousphaseAqueousphaseLipidbilayerAqueouscompartmentsLipidbilayersLIPOSOME(UNILAMELLAR)ELIPOSOME(MULTILAMELLAR)FFigure 14–22. Formation of lipid membranes, micelles, emulsions, and liposomes from amphipathiclipids, eg, phospholipids.

LIPIDS OF PHYSIOLOGIC SIGNIFICANCE / 121delivery of drugs and cosmetics. Emulsions are muchlarger particles, formed usually by nonpolar lipids in anaqueous medium. These are stabilized by emulsifyingagents such as amphipathic lipids (eg, lecithin), whichform a surface layer separating the main bulk of thenonpolar material from the aqueous phase (Figure14–22).SUMMARY• Lipids have the common property of being relativelyinsoluble in water (hydrophobic) but soluble in nonpolarsolvents. Amphipathic lipids also contain oneor more polar groups, making them suitable as constituentsof membranes at lipid:water interfaces.• The lipids of major physiologic significance are fattyacids and their esters, together with cholesterol andother steroids.• Long-chain fatty acids may be saturated, monounsaturated,or polyunsaturated, according to the numberof double bonds present. Their fluidity decreaseswith chain length and increases according to degreeof unsaturation.• Eicosanoids are formed from 20-carbon polyunsaturatedfatty acids and make up an important group ofphysiologically and pharmacologically active compoundsknown as prostaglandins, thromboxanes,leukotrienes, and lipoxins.• The esters of glycerol are quantitatively the most significantlipids, represented by triacylglycerol (“fat”),a major constituent of lipoproteins and the storageform of lipid in adipose tissue. Phosphoacylglycerolsare amphipathic lipids and have important roles—asmajor constituents of membranes and the outer layerof lipoproteins, as surfactant in the lung, as precursorsof second messengers, and as constituents of nervoustissue.• Glycolipids are also important constituents of nervoustissue such as brain and the outer leaflet of thecell membrane, where they contribute to the carbohydrateson the cell surface.• Cholesterol, an amphipathic lipid, is an importantcomponent of membranes. It is the parent moleculefrom which all other steroids in the body, includingmajor hormones such as the adrenocortical and sexhormones, D vitamins, and bile acids, are synthesized.• Peroxidation of lipids containing polyunsaturatedfatty acids leads to generation of free radicals thatmay damage tissues and cause disease.REFERENCESBenzie IFF: Lipid peroxidation: a review of causes, consequences,measurement and dietary influences. Int J Food Sci Nutr1996;47:233.Christie WW: Lipid Analysis, 2nd ed. Pergamon Press, 1982.Cullis PR, Fenske DB, Hope MJ: Physical properties and functionalroles of lipids in membranes. In: Biochemistry of Lipids,Lipoproteins and Membranes. Vance DE, Vance JE (editors).Elsevier, 1996.Gunstone FD, Harwood JL, Padley FB: The Lipid Handbook.Chapman & Hall, 1986.Gurr MI, Harwood JL: Lipid Biochemistry: An Introduction, 4th ed.Chapman & Hall, 1991.

Overview of Metabolism 15Peter A. Mayes, PhD, DSc, & David A. Bender, PhDBIOMEDICAL IMPORTANCEThe fate of dietary components after digestion and absorptionconstitutes metabolism—the metabolic pathwaystaken by individual molecules, their interrelationships,and the mechanisms that regulate the flow ofmetabolites through the pathways. Metabolic pathwaysfall into three categories: (1) Anabolic pathways arethose involved in the synthesis of compounds. Proteinsynthesis is such a pathway, as is the synthesis of fuelreserves of triacylglycerol and glycogen. Anabolic pathwaysare endergonic. (2) Catabolic pathways are involvedin the breakdown of larger molecules, commonlyinvolving oxidative reactions; they are exergonic,producing reducing equivalents and, mainly via the respiratorychain, ATP. (3) Amphibolic pathways occurat the “crossroads” of metabolism, acting as links betweenthe anabolic and catabolic pathways, eg, the citricacid cycle.A knowledge of normal metabolism is essential foran understanding of abnormalities underlying disease.Normal metabolism includes adaptation to periods ofstarvation, exercise, pregnancy, and lactation. Abnormalmetabolism may result from nutritional deficiency,enzyme deficiency, abnormal secretion of hormones, orthe actions of drugs and toxins. An important exampleof a metabolic disease is diabetes mellitus.PATHWAYS THAT PROCESS THE MAJORPRODUCTS OF DIGESTIONThe nature of the diet sets the basic pattern of metabolism.There is a need to process the products of digestionof dietary carbohydrate, lipid, and protein. Theseare mainly glucose, fatty acids and glycerol, and aminoacids, respectively. In ruminants (and to a lesser extentin other herbivores), dietary cellulose is fermented bysymbiotic microorganisms to short-chain fatty acids(acetic, propionic, butyric), and metabolism in theseanimals is adapted to use these fatty acids as major substrates.All the products of digestion are metabolized toa common product, acetyl-CoA, which is then oxidizedby the citric acid cycle (Figure 15–1).Carbohydrate Metabolism Is Centeredon the Provision & Fate of Glucose(Figure 15–2)Glucose is metabolized to pyruvate by the pathway ofglycolysis, which can occur anaerobically (in the absenceof oxygen), when the end product is lactate. Aerobictissues metabolize pyruvate to acetyl-CoA, whichcan enter the citric acid cycle for complete oxidationto CO 2 and H 2 O, linked to the formation of ATPin the process of oxidative phosphorylation (Figure16–2). Glucose is the major fuel of most tissues.CarbohydrateSimple sugars(mainly glucose)ProteinDigestion and absorptionAmino acidsCatabolismAcetyl-CoACitricacidcycleFatFatty acids+ glycerol2CO 22H ATPFigure 15–1. Outline of the pathways for the catabolismof dietary carbohydrate, protein, and fat. All thepathways lead to the production of acetyl-CoA, which isoxidized in the citric acid cycle, ultimately yielding ATPin the process of oxidative phosphorylation.122

ifiyGlycolysisldAminoacidsDietGlucoseGlucosephosphatesTriosephosphatesPyruvatePentose phosphatepathwayLactateCO 2Glycogen3CO 2RibosephosphateAcylglycerols(fat)RNADNAOVERVIEW OF METABOLISM / 123Glucose and its metabolites also take part in otherprocesses. Examples: (1) Conversion to the storagepolymer glycogen in skeletal muscle and liver. (2) Thepentose phosphate pathway, an alternative to part ofthe pathway of glycolysis, is a source of reducing equivalents(NADPH) for biosynthesis and the source of ribosefor nucleotide and nucleic acid synthesis.(3) Triose phosphate gives rise to the glycerol moietyof triacylglycerols. (4) Pyruvate and intermediates ofthe citric acid cycle provide the carbon skeletons forthe synthesis of amino acids; and acetyl-CoA, the precursorof fatty acids and cholesterol (and hence of allsteroids synthesized in the body). Gluconeogenesis isthe process of forming glucose from noncarbohydrateprecursors, eg, lactate, amino acids, and glycerol.Lipid Metabolism Is Concerned MainlyWith Fatty Acids & Cholesterol(Figure 15–3)The source of long-chain fatty acids is either dietarylipid or de novo synthesis from acetyl-CoA derived fromcarbohydrate. Fatty acids may be oxidized to acetyl-CoA (β-oxidation) or esterified with glycerol, formingtriacylglycerol (fat) as the body’s main fuel reserve.Acetyl-CoA formed by β-oxidation may undergoseveral fates:(1) As with acetyl-CoA arising from glycolysis, it isoxidized to CO 2 + H 2 O via the citric acid cycle.ProteinAcetyl-CoAFattyacidstc ai o nTriacylglycerol(fat)sisSteroidsAminoacidsCitricacidcycleCholesterolDietrt eE ssi p o g e n esiFatty acidsLnoiopita- OixSteroidogenesisCholesterolLβ2CO 2Figure 15–2. Overview of carbohydrate metabolismshowing the major pathways and end products. Gluconeogenesisis not shown.CarbohydrateAmino acidsAcetyl-CoACholesterologenesisKetogenesisKetonebodiesCitricacidcycle2CO 2Figure 15–3. Overview of fatty acid metabolismshowing the major pathways and end products. Ketonebodies comprise the substances acetoacetate, 3-hydroxybutyrate,and acetone.

124 / CHAPTER 15(2) It is the precursor for synthesis of cholesterol andother steroids.(3) In the liver, it forms ketone bodies (acetone, acetoacetate,and 3-hydroxybutyrate) that are importantfuels in prolonged starvation.Much of Amino Acid MetabolismInvolves Transamination(Figure 15–4)The amino acids are required for protein synthesis.Some must be supplied in the diet (the essential aminoacids) since they cannot be synthesized in the body.The remainder are nonessential amino acids that aresupplied in the diet but can be formed from metabolicintermediates by transamination, using the amino nitrogenfrom other amino acids. After deamination,amino nitrogen is excreted as urea, and the carbonskeletons that remain after transamination (1) are oxidizedto CO 2 via the citric acid cycle, (2) form glucose(gluconeogenesis), or (3) form ketone bodies.Several amino acids are also the precursors of othercompounds, eg, purines, pyrimidines, hormones suchas epinephrine and thyroxine, and neurotransmitters.METABOLIC PATHWAYS MAY BESTUDIED AT DIFFERENT LEVELSOF ORGANIZATIONIn addition to studies in the whole organism, the locationand integration of metabolic pathways is revealedby studies at several levels of organization. At the tissueand organ level, the nature of the substrates enteringand metabolites leaving tissues and organs is defined. Atthe subcellular level, each cell organelle (eg, the mitochondrion)or compartment (eg, the cytosol) has specificroles that form part of a subcellular pattern ofmetabolic pathways.At the Tissue and Organ Level, the BloodCirculation Integrates MetabolismAmino acids resulting from the digestion of dietaryprotein and glucose resulting from the digestion of carbohydrateare absorbed and directed to the liver via thehepatic portal vein. The liver has the role of regulatingthe blood concentration of most water-soluble metabolites(Figure 15–5). In the case of glucose, this isachieved by taking up glucose in excess of immediaterequirements and converting it to glycogen (glycogene-Diet proteinTissue proteinAmino acidsNonproteinnitrogen derivativesT R A N S A M I N A T I O NCarbohydrate(glucose)Ketone bodiesAmino nitrogen inglutamateAcetyl-CoADEAMINATIONCitricacidcycleNH 32CO 2UreaFigure 15–4.Overview of amino acid metabolism showing the major pathways and end products.

OVERVIEW OF METABOLISM / 125Plasma proteinsUreaProteinLIVERAmino acidsGlucoseGlycogenCO 2AminoacidsProteinUreaLactateAmino acidsAlanine, etcKIDNEYUrineBLOOD PLASMAHepaticportal veinGlucoseAmino acidsGlucoseERYTHROCYTESDietCarbohydrateProteinGlucosephosphateGlycogenMUSCLECO 2SMALL INTESTINEFigure 15–5. Transport and fate of major carbohydrate and amino acid substrates and metabolites. Note thatthere is little free glucose in muscle, since it is rapidly phosphorylated upon entry.sis) or to fat (lipogenesis). Between meals, the liveracts to maintain the blood glucose concentration fromglycogen (glycogenolysis) and, together with the kidney,by converting noncarbohydrate metabolites suchas lactate, glycerol, and amino acids to glucose (gluconeogenesis).Maintenance of an adequate concentrationof blood glucose is vital for those tissues in which itis the major fuel (the brain) or the only fuel (the erythrocytes).The liver also synthesizes the major plasmaproteins (eg, albumin) and deaminates amino acidsthat are in excess of requirements, forming urea, whichis transported to the kidney and excreted.Skeletal muscle utilizes glucose as a fuel, formingboth lactate and CO 2 . It stores glycogen as a fuel for itsuse in muscular contraction and synthesizes muscleprotein from plasma amino acids. Muscle accounts forapproximately 50% of body mass and consequentlyrepresents a considerable store of protein that can bedrawn upon to supply amino acids for gluconeogenesisin starvation.Lipids in the diet (Figure 15–6) are mainly triacylglyceroland are hydrolyzed to monoacylglycerols andfatty acids in the gut, then reesterified in the intestinalmucosa. Here they are packaged with protein and secretedinto the lymphatic system and thence into theblood stream as chylomicrons, the largest of the plasmalipoproteins. Chylomicrons also contain other lipidsolublenutrients, eg, vitamins. Unlike glucose andamino acids, chylomicron triacylglycerol is not taken updirectly by the liver. It is first metabolized by tissues thathave lipoprotein lipase, which hydrolyzes the triacylglycerol,releasing fatty acids that are incorporated intotissue lipids or oxidized as fuel. The other major sourceof long-chain fatty acid is synthesis (lipogenesis) fromcarbohydrate, mainly in adipose tissue and the liver.Adipose tissue triacylglycerol is the main fuel reserveof the body. On hydrolysis (lipolysis) free fatty acids arereleased into the circulation. These are taken up by mosttissues (but not brain or erythrocytes) and esterified toacylglycerols or oxidized as a fuel. In the liver, triacylglycerolarising from lipogenesis, free fatty acids, andchylomicron remnants (see Figures 25–3 and 25–4) is secretedinto the circulation as very low density lipoprotein(VLDL). This triacylglycerol undergoes a fate similarto that of chylomicrons. Partial oxidation of fattyacids in the liver leads to ketone body production (keto-

126 / CHAPTER 15FFAGlucosei sl y si poLFattyacidsEs t er i f i cat i onCO 2KetonebodiesBLOODPLASMATGLIVERLPLCO 2FattyacidsGlucosei sl y si poLFattyacidsEs t er i f i cat i onLPLVLDLLipoproteinTGChylomicronsi sl y si poLTGMUSCLEEs t er i f i cat i onTGADIPOSETISSUEDietTGMG +fatty acidsTGSMALL INTESTINEFigure 15–6. Transport and fate of major lipid substrates and metabolites. (FFA, free fatty acids; LPL, lipoproteinlipase; MG, monoacylglycerol; TG, triacylglycerol; VLDL, very low density lipoprotein.)genesis). Ketone bodies are transported to extrahepatictissues, where they act as a fuel source in starvation.At the Subcellular Level, Glycolysis Occursin the Cytosol & the Citric Acid Cyclein the MitochondriaCompartmentation of pathways in separate subcellularcompartments or organelles permits integration andregulation of metabolism. Not all pathways are of equalimportance in all cells. Figure 15–7 depicts the subcellularcompartmentation of metabolic pathways in a hepaticparenchymal cell.The central role of the mitochondrion is immediatelyapparent, since it acts as the focus of carbohydrate,lipid, and amino acid metabolism. It contains the enzymesof the citric acid cycle, β-oxidation of fatty acids,and ketogenesis, as well as the respiratory chain andATP synthase.Glycolysis, the pentose phosphate pathway, and fattyacid synthesis are all found in the cytosol. In gluconeogenesis,substrates such as lactate and pyruvate, whichare formed in the cytosol, enter the mitochondrion toyield oxaloacetate before formation of glucose.The membranes of the endoplasmic reticulum containthe enzyme system for acylglycerol synthesis, andthe ribosomes are responsible for protein synthesis.THE FLUX OF METABOLITES INMETABOLIC PATHWAYS MUST BEREGULATED IN A CONCERTED MANNERRegulation of the overall flux through a pathway is importantto ensure an appropriate supply, when required,of the products of that pathway. Regulation isachieved by control of one or more key reactions inthe pathway, catalyzed by “regulatory enzymes.” Thephysicochemical factors that control the rate of an

OVERVIEW OF METABOLISM / 127GlycogenCYTOSOLAARibosomeProteinGlucosePentosephosphatepathwayENDOPLASMICRETICULUMTriose phosphateGlycerol phosphateTriacylglycerolFatty acidsGlycolysisGlycerolPhosphoenolpyruvateLactateGluconeogenesisAAPyruvateAAPyruvateCO 2β-OxidationLipogenesisOxaloacetateAcetyl-CoAKetonebodiesAAFumarateAACitrateAASuccinyl-CoACitric acidcycleCO 2α-KetoglutarateCO 2AAAAMITOCHONDRIONFigure 15–7. Intracellular location and overview of major metabolic pathways in a liver parenchymalcell. (AA →, metabolism of one or more essential amino acids; AA ↔, metabolism of one or morenonessential amino acids.)AAAA

128 / CHAPTER 15enzyme-catalyzed reaction, eg, substrate concentration,are of primary importance in the control of the overallrate of a metabolic pathway (Chapter 9).“Nonequilibrium” Reactions ArePotential Control PointsIn a reaction at equilibrium, the forward and reverse reactionsoccur at equal rates, and there is therefore nonet flux in either direction:A ↔B↔ C↔DIn vivo, under “steady-state” conditions, there is a netflux from left to right because there is a continuous supplyof A and removal of D. In practice, there are invariablyone or more nonequilibrium reactions in a metabolicpathway, where the reactants are present inconcentrations that are far from equilibrium. In attemptingto reach equilibrium, large losses of free energyoccur as heat, making this type of reaction essentiallyirreversible, eg,HeatA ↔ B→ C ↔DInactiveEnz 12+ +2Ca 2+ /calmodulincAMPCellmembraneX Y ActiveEnz 1A A B C D+Enz 2–1+or–Positive allostericfeed-forwardactivationNegative allostericfeed-backinhibition+3or–Ribosomal synthesisof new enzyme proteinNuclear productionof mRNA+–4 5InductionRepressionFigure 15–8. Mechanisms of control of an enzyme-catalyzed reaction. Circlednumbers indicate possible sites of action of hormones. 1 , Alteration of membranepermeability; 2 , conversion of an inactive to an active enzyme, usually involvingphosphorylation/dephosphorylation reactions; 3 , alteration of the rateof translation of mRNA at the ribosomal level; 4 , induction of new mRNA formation;and 5 , repression of mRNA formation. 1 and 2 are rapid, whereas 3 –5are slower ways of regulating enzyme activity.

OVERVIEW OF METABOLISM / 129Such a pathway has both flow and direction. Theenzymes catalyzing nonequilibrium reactions are usuallypresent in low concentrations and are subject to avariety of regulatory mechanisms. However, many ofthe reactions in metabolic pathways cannot be classifiedas equilibrium or nonequilibrium but fall somewherebetween the two extremes.The Flux-Generating ReactionIs the First Reaction in a PathwayThat Is Saturated With SubstrateIt may be identified as a nonequilibrium reaction inwhich the K m of the enzyme is considerably lower thanthe normal substrate concentration. The first reactionin glycolysis, catalyzed by hexokinase (Figure 17–2), issuch a flux-generating step because its K m for glucose of0.05 mmol/L is well below the normal blood glucoseconcentration of 5 mmol/L.ALLOSTERIC & HORMONALMECHANISMS ARE IMPORTANTIN THE METABOLIC CONTROL OFENZYME-CATALYZED REACTIONSA hypothetical metabolic pathway is shown in Figure15–8, in which reactions A ↔ B and C ↔ D are equilibriumreactions and B → C is a nonequilibrium reaction.The flux through such a pathway can be regulatedby the availability of substrate A. This depends on itssupply from the blood, which in turn depends on eitherfood intake or key reactions that maintain and releasesubstrates from tissue reserves to the blood, eg, theglycogen phosphorylase in liver (Figure 18–1) and hormone-sensitivelipase in adipose tissue (Figure 25–7).The flux also depends on the transport of substrate Aacross the cell membrane. Flux is also determined bythe removal of the end product D and the availabilityof cosubstrate or cofactors represented by X and Y. Enzymescatalyzing nonequilibrium reactions are often allostericproteins subject to the rapid actions of “feedback”or “feed-forward” control by allosteric modifiersin immediate response to the needs of the cell (Chapter9). Frequently, the product of a biosynthetic pathwaywill inhibit the enzyme catalyzing the first reactionin the pathway. Other control mechanisms depend onthe action of hormones responding to the needs of thebody as a whole; they may act rapidly, by altering theactivity of existing enzyme molecules, or slowly, by alteringthe rate of enzyme synthesis.SUMMARY• The products of digestion provide the tissues withthe building blocks for the biosynthesis of complexmolecules and also with the fuel to power the livingprocesses.• Nearly all products of digestion of carbohydrate, fat,and protein are metabolized to a common metabolite,acetyl-CoA, before final oxidation to CO 2 in thecitric acid cycle.• Acetyl-CoA is also used as the precursor for biosynthesisof long-chain fatty acids; steroids, includingcholesterol; and ketone bodies.• Glucose provides carbon skeletons for the glycerolmoiety of fat and of several nonessential amino acids.• Water-soluble products of digestion are transporteddirectly to the liver via the hepatic portal vein. Theliver regulates the blood concentrations of glucoseand amino acids.• Pathways are compartmentalized within the cell.Glycolysis, glycogenesis, glycogenolysis, the pentosephosphate pathway, and lipogenesis occur in the cytosol.The mitochondrion contains the enzymes ofthe citric acid cycle, β-oxidation of fatty acids, and ofoxidative phosphorylation. The endoplasmic reticulumalso contains the enzymes for many otherprocesses, including protein synthesis, glycerolipidformation, and drug metabolism.• Metabolic pathways are regulated by rapid mechanismsaffecting the activity of existing enzymes, eg,allosteric and covalent modification (often in responseto hormone action); and slow mechanisms affectingthe synthesis of enzymes.REFERENCESCohen P: Control of Enzyme Activity, 2nd ed. Chapman & Hall,1983.Fell D: Understanding the Control of Metabolism. Portland Press,1997.Frayn KN: Metabolic Regulation—A Human Perspective. PortlandPress, 1996.Newsholme EA, Crabtree B: Flux-generating and regulatory stepsin metabolic control. Trends Biochem Sci 1981;6:53.

The Citric Acid Cycle:The Catabolism of Acetyl-CoA 16Peter A. Mayes, PhD, DSc, & David A. Bender, PhDBIOMEDICAL IMPORTANCEThe citric acid cycle (Krebs cycle, tricarboxylic acidcycle) is a series of reactions in mitochondria that oxidizeacetyl residues (as acetyl-CoA) and reduce coenzymesthat upon reoxidation are linked to the formationof ATP.The citric acid cycle is the final common pathwayfor the aerobic oxidation of carbohydrate, lipid, andprotein because glucose, fatty acids, and most aminoacids are metabolized to acetyl-CoA or intermediates ofthe cycle. It also has a central role in gluconeogenesis,lipogenesis, and interconversion of amino acids. Manyof these processes occur in most tissues, but the liver isthe only tissue in which all occur to a significant extent.The repercussions are therefore profound when, for example,large numbers of hepatic cells are damaged as inacute hepatitis or replaced by connective tissue (as incirrhosis). Very few, if any, genetic abnormalities ofcitric acid cycle enzymes have been reported; such abnormalitieswould be incompatible with life or normaldevelopment.THE CITRIC ACID CYCLE PROVIDESSUBSTRATE FOR THERESPIRATORY CHAINThe cycle starts with reaction between the acetyl moietyof acetyl-CoA and the four-carbon dicarboxylic acid oxaloacetate,forming a six-carbon tricarboxylic acid, citrate.In the subsequent reactions, two molecules of CO 2are released and oxaloacetate is regenerated (Figure16–1). Only a small quantity of oxaloacetate is neededfor the oxidation of a large quantity of acetyl-CoA; oxaloacetatemay be considered to play a catalytic role.The citric acid cycle is an integral part of the processby which much of the free energy liberated during theoxidation of fuels is made available. During oxidationof acetyl-CoA, coenzymes are reduced and subsequentlyreoxidized in the respiratory chain, linked to the formationof ATP (oxidative phosphorylation; see Figure16–2 and also Chapter 12). This process is aerobic, requiringoxygen as the final oxidant of the reducedcoenzymes. The enzymes of the citric acid cycle are lo-130cated in the mitochondrial matrix, either free or attachedto the inner mitochondrial membrane, wherethe enzymes of the respiratory chain are also found.REACTIONS OF THE CITRIC ACIDCYCLE LIBERATE REDUCINGEQUIVALENTS & CO 2(Figure 16–3)*The initial reaction between acetyl-CoA and oxaloacetateto form citrate is catalyzed by citrate synthasewhich forms a carbon-carbon bond between the methylcarbon of acetyl-CoA and the carbonyl carbon of oxaloacetate.The thioester bond of the resultant citryl-CoA is hydrolyzed, releasing citrate and CoASH—anexergonic reaction.Citrate is isomerized to isocitrate by the enzymeaconitase (aconitate hydratase); the reaction occurs intwo steps: dehydration to cis-aconitate, some of whichremains bound to the enzyme; and rehydration to isocitrate.Although citrate is a symmetric molecule, aconitasereacts with citrate asymmetrically, so that the twocarbon atoms that are lost in subsequent reactions ofthe cycle are not those that were added from acetyl-CoA. This asymmetric behavior is due to channeling—transfer of the product of citrate synthase directly ontothe active site of aconitase without entering free solution.This provides integration of citric acid cycle activityand the provision of citrate in the cytosol as a sourceof acetyl-CoA for fatty acid synthesis. The poison fluoroacetateis toxic because fluoroacetyl-CoA condenseswith oxaloacetate to form fluorocitrate, which inhibitsaconitase, causing citrate to accumulate.Isocitrate undergoes dehydrogenation catalyzed byisocitrate dehydrogenase to form, initially, oxalosuccinate,which remains enzyme-bound and undergoes decarboxylationto α-ketoglutarate. The decarboxylation*From Circular No. 200 of the Committee of Editors of BiochemicalJournals Recommendations (1975): “According to standardbiochemical convention, the ending ate in, eg, palmitate, denotesany mixture of free acid and the ionized form(s) (according to pH)in which the cations are not specified.” The same convention isadopted in this text for all carboxylic acids.

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA / 131Acetyl-CoA(C 2 )CarbohydrateProteinLipidsCoAAcetyl-CoA(C 2 )Oxaloacetate(C 4 )CO 2CO 2Citrate(C 6 )Figure 16–1. Citric acid cycle, illustrating the catalyticrole of oxaloacetate.requires Mg 2+ or Mn 2+ ions. There are three isoenzymesof isocitrate dehydrogenase. One, which uses NAD + , isfound only in mitochondria. The other two use NADP +and are found in mitochondria and the cytosol. Respiratorychain-linked oxidation of isocitrate proceeds almostcompletely through the NAD + -dependent enzyme.α-Ketoglutarate undergoes oxidative decarboxylationin a reaction catalyzed by a multi-enzyme complexsimilar to that involved in the oxidative decarboxylationof pyruvate (Figure 17–5). The -ketoglutarate dehydrogenasecomplex requires the same cofactors as thepyruvate dehydrogenase complex—thiamin diphosphate,lipoate, NAD + , FAD, and CoA—and results inthe formation of succinyl-CoA. The equilibrium of thisreaction is so much in favor of succinyl-CoA formationthat it must be considered physiologically unidirectional.As in the case of pyruvate oxidation (Chapter17), arsenite inhibits the reaction, causing the substrate,-ketoglutarate, to accumulate.Succinyl-CoA is converted to succinate by the enzymesuccinate thiokinase (succinyl-CoA synthetase).This is the only example in the citric acid cycle ofsubstrate-level phosphorylation. Tissues in which gluconeogenesisoccurs (the liver and kidney) contain twoisoenzymes of succinate thiokinase, one specific forGDP and the other for ADP. The GTP formed isused for the decarboxylation of oxaloacetate to phosphoenolpyruvatein gluconeogenesis and provides aregulatory link between citric acid cycle activity andthe withdrawal of oxaloacetate for gluconeogenesis.Nongluconeogenic tissues have only the isoenzyme thatuses ADP.H 2 OMalate(C 4 )Fumarate(C4)F pCytP2HHOxaloacetate 2 O(C 4 )Citric acidcycleCytochromeSuccinate(C 4 )Respiratory chainFlavoprotein2HHigh-energy phosphateCitrate(C 6 )2HH 2 OCis-aconitate(C 6 )H 2 OIsocitrate(C 6 )CO 2α-Ketoglutarate(C 5 )NAD2HCO 2Succinyl-CoA(C 4 )P F pH 2 OPQCyt b P OxidativephosphorylationCyt cCyt aa 3 P1/2 O 2–Anaerobiosis(hypoxia, anoxia)H 2 OFigure 16–2. The citric acid cycle: the major catabolicpathway for acetyl-CoA in aerobic organisms. Acetyl-CoA, the product of carbohydrate, protein, and lipid catabolism,is taken into the cycle, together with H 2 O, andoxidized to CO 2 with the release of reducing equivalents(2H). Subsequent oxidation of 2H in the respiratorychain leads to coupled phosphorylation of ADP to ATP.For one turn of the cycle, 11~P are generated via oxidativephosphorylation and one ~P arises at substratelevel from the conversion of succinyl-CoA to succinate.

HO CH COO –CH 2 COO –L-MalateMALATEDEHYDROGENASE**OC COO –CH 2 COO –NADH + H + OxaloacetateNAD +*CH 3 CO S CoAAcetyl-CoACITRATE SYNTHASECoA SHCH 2 COO * –H 2 OHO C COO –CH 2 COO –CitrateACONITASEFUMARASEH– OOC * C HFumarateH 2 OC COO * –FluoroacetateH 2 O*CH 2 COO –C COO –CH COO –Cis-aconitateFe 2+ Fe 2+FADH 2SUCCINATEDEHYDROGENASEFADCH 2 COO * – Malonate*CH 2 COO –SuccinateATPH 2 OACONITASE*CH 2 COO –CH COO –HO CH COO –NAD + IsocitrateCoASHSUCCINATETHIOKINASEMg 2+ADP + PiCH 2 COO –CH 2O C S CoASuccinyl-CoAα-KETOGLUTARATEDEHYDROGENASE COMPLEX*CoASHNADH + H +NAD +CO 2ArseniteCO 2CH 2 COO * –CH 2Mn 2+O C COO –α-KetoglutarateNADH + H +CH 2 COO –CH*COO –O C COO –OxalosuccinateISOCITRATEDEHYDROGENASEISOCITRATEDEHYDROGENASEFigure 16–3. Reactions of the citric acid (Krebs) cycle. Oxidation of NADH and FADH 2 in the respiratory chainleads to the generation of ATP via oxidative phosphorylation. In order to follow the passage of acetyl-CoA throughthe cycle, the two carbon atoms of the acetyl radical are shown labeled on the carboxyl carbon (designated by asterisk)and on the methyl carbon (using the designation • ). Although two carbon atoms are lost as CO 2 in one revolutionof the cycle, these atoms are not derived from the acetyl-CoA that has immediately entered the cycle butfrom that portion of the citrate molecule that was derived from oxaloacetate. However, on completion of a singleturn of the cycle, the oxaloacetate that is regenerated is now labeled, which leads to labeled CO 2 being evolvedduring the second turn of the cycle. Because succinate is a symmetric compound and because succinate dehydrogenasedoes not differentiate between its two carboxyl groups, “randomization” of label occurs at this step suchthat all four carbon atoms of oxaloacetate appear to be labeled after one turn of the cycle. During gluconeogenesis,some of the label in oxaloacetate is incorporated into glucose and glycogen (Figure 19–1). For a discussion ofthe stereochemical aspects of the citric acid cycle, see Greville (1968). The sites of inhibition ( − ) by fluoroacetate,malonate, and arsenite are indicated.132

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA / 133When ketone bodies are being metabolized in extrahepatictissues there is an alternative reaction catalyzedby succinyl-CoA–acetoacetate-CoA transferase (thiophorase)—involvingtransfer of CoA from succinyl-CoA to acetoacetate, forming acetoacetyl-CoA (Chapter22).The onward metabolism of succinate, leading to theregeneration of oxaloacetate, is the same sequence ofchemical reactions as occurs in the β-oxidation of fattyacids: dehydrogenation to form a carbon-carbon doublebond, addition of water to form a hydroxyl group, anda further dehydrogenation to yield the oxo- group ofoxaloacetate.The first dehydrogenation reaction, forming fumarate,is catalyzed by succinate dehydrogenase, whichis bound to the inner surface of the inner mitochondrialmembrane. The enzyme contains FAD and iron-sulfur(Fe:S) protein and directly reduces ubiquinone in therespiratory chain. Fumarase (fumarate hydratase) catalyzesthe addition of water across the double bond offumarate, yielding malate. Malate is converted to oxaloacetateby malate dehydrogenase, a reaction requiringNAD + . Although the equilibrium of this reactionstrongly favors malate, the net flux is toward the directionof oxaloacetate because of the continual removal ofoxaloacetate (either to form citrate, as a substrate forgluconeogenesis, or to undergo transamination to aspartate)and also because of the continual reoxidationof NADH.TWELVE ATP ARE FORMED PER TURNOF THE CITRIC ACID CYCLEAs a result of oxidations catalyzed by the dehydrogenasesof the citric acid cycle, three molecules of NADHand one of FADH 2 are produced for each molecule ofacetyl-CoA catabolized in one turn of the cycle. Thesereducing equivalents are transferred to the respiratorychain (Figure 16–2), where reoxidation of each NADHresults in formation of 3 ATP and reoxidation ofFADH 2 in formation of 2 ATP. In addition, 1 ATP(or GTP) is formed by substrate-level phosphorylationcatalyzed by succinate thiokinase.VITAMINS PLAY KEY ROLESIN THE CITRIC ACID CYCLEFour of the B vitamins are essential in the citric acidcycle and therefore in energy-yielding metabolism: (1)riboflavin, in the form of flavin adenine dinucleotide(FAD), a cofactor in the α-ketoglutarate dehydrogenasecomplex and in succinate dehydrogenase; (2) niacin, inthe form of nicotinamide adenine dinucleotide (NAD),the coenzyme for three dehydrogenases in the cycle—isocitrate dehydrogenase, α-ketoglutarate dehydrogenase,and malate dehydrogenase; (3) thiamin (vitaminB 1 ), as thiamin diphosphate, the coenzyme for decarboxylationin the α-ketoglutarate dehydrogenase reaction;and (4) pantothenic acid, as part of coenzyme A,the cofactor attached to “active” carboxylic acid residuessuch as acetyl-CoA and succinyl-CoA.THE CITRIC ACID CYCLE PLAYS APIVOTAL ROLE IN METABOLISMThe citric acid cycle is not only a pathway for oxidationof two-carbon units—it is also a major pathway for interconversionof metabolites arising from transaminationand deamination of amino acids. It also providesthe substrates for amino acid synthesis by transamination,as well as for gluconeogenesis and fatty acid synthesis.Because it functions in both oxidative and syntheticprocesses, it is amphibolic (Figure 16–4).The Citric Acid Cycle Takes Part inGluconeogenesis, Transamination,& DeaminationAll the intermediates of the cycle are potentially glucogenic,since they can give rise to oxaloacetate and thusnet production of glucose (in the liver and kidney, theorgans that carry out gluconeogenesis; see Chapter 19).The key enzyme that catalyzes net transfer out of thecycle into gluconeogenesis is phosphoenolpyruvatecarboxykinase, which decarboxylates oxaloacetate tophosphoenolpyruvate, with GTP acting as the donorphosphate (Figure 16–4).Net transfer into the cycle occurs as a result of severaldifferent reactions. Among the most important ofsuch anaplerotic reactions is the formation of oxaloacetateby the carboxylation of pyruvate, catalyzed bypyruvate carboxylase. This reaction is important inmaintaining an adequate concentration of oxaloacetatefor the condensation reaction with acetyl-CoA. If acetyl-CoA accumulates, it acts both as an allosteric activatorof pyruvate carboxylase and as an inhibitor of pyruvatedehydrogenase, thereby ensuring a supply of oxaloacetate.Lactate, an important substrate for gluconeogenesis,enters the cycle via oxidation to pyruvate and thencarboxylation to oxaloacetate.Aminotransferase (transaminase) reactions formpyruvate from alanine, oxaloacetate from aspartate, andα-ketoglutarate from glutamate. Because these reactionsare reversible, the cycle also serves as a source ofcarbon skeletons for the synthesis of these amino acids.Other amino acids contribute to gluconeogenesis becausetheir carbon skeletons give rise to citric acid cycle

134 / CHAPTER 16HydroxyprolineSerineCysteineThreonineGlycineTryptophanGlucoseAlaninePhosphoenolpyruvateTRANSAMINASEPHOSPHOENOLPYRUVATECARBOXYKINASELactatePyruvateOxaloacetatePYRUVATECARBOXYLASEAcetyl-CoATyrosinePhenylalanineFumarateAspartateTRANSAMINASECitrateIsoleucineMethionineValineSuccinyl-CoACO 2Propionateα-KetoglutarateHistidineProlineGlutamineArginineCO 2GlutamateTRANSAMINASEFigure 16–4. Involvement of the citric acid cycle in transamination and gluconeogenesis.The bold arrows indicate the main pathway of gluconeogenesis.intermediates. Alanine, cysteine, glycine, hydroxyproline,serine, threonine, and tryptophan yield pyruvate;arginine, histidine, glutamine, and proline yield α-ketoglutarate;isoleucine, methionine, and valine yieldsuccinyl-CoA; and tyrosine and phenylalanine yield fumarate(Figure 16–4).In ruminants, whose main metabolic fuel is shortchainfatty acids formed by bacterial fermentation, theconversion of propionate, the major glucogenic productof rumen fermentation, to succinyl-CoA via themethylmalonyl-CoA pathway (Figure 19–2) is especiallyimportant.The Citric Acid Cycle Takes Partin Fatty Acid Synthesis(Figure 16–5)Acetyl-CoA, formed from pyruvate by the action ofpyruvate dehydrogenase, is the major building block forlong-chain fatty acid synthesis in nonruminants. (In ruminants,acetyl-CoA is derived directly from acetate.)Pyruvate dehydrogenase is a mitochondrial enzyme,and fatty acid synthesis is a cytosolic pathway, but themitochondrial membrane is impermeable to acetyl-CoA. Acetyl-CoA is made available in the cytosol fromcitrate synthesized in the mitochondrion, transportedinto the cytosol and cleaved in a reaction catalyzed byATP-citrate lyase.Regulation of the Citric Acid CycleDepends Primarily on a Supplyof Oxidized CofactorsIn most tissues, where the primary role of the citric acidcycle is in energy-yielding metabolism, respiratorycontrol via the respiratory chain and oxidative phosphorylationregulates citric acid cycle activity (Chapter14). Thus, activity is immediately dependent on thesupply of NAD + , which in turn, because of the tightcoupling between oxidation and phosphorylation, is dependenton the availability of ADP and hence, ulti-

THE CITRIC ACID CYCLE: THE CATABOLISM OF ACETYL-CoA / 135OxaloacetatePyruvateAcetyl-CoACitricacidcyclePYRUVATEDEHYDROGENASECO 2 CO 2MITOCHONDRIALMEMBRANECitrateGlucoseFattyacidsAcetyl-CoACitrateOxaloacetateATP-CITRATELYASEFigure 16–5. Participation of the citric acid cycle infatty acid synthesis from glucose. See also Figure 21–5.mately, on the rate of utilization of ATP in chemicaland physical work. In addition, individual enzymes ofthe cycle are regulated. The most likely sites for regulationare the nonequilibrium reactions catalyzed bypyruvate dehydrogenase, citrate synthase, isocitrate dehydrogenase,and α-ketoglutarate dehydrogenase. Thedehydrogenases are activated by Ca 2+ , which increasesin concentration during muscular contraction and secretion,when there is increased energy demand. In atissue such as brain, which is largely dependent on carbohydrateto supply acetyl-CoA, control of the citricacid cycle may occur at pyruvate dehydrogenase. Severalenzymes are responsive to the energy status, asshown by the [ATP]/[ADP] and [NADH]/[NAD + ] ratios.Thus, there is allosteric inhibition of citrate synthaseby ATP and long-chain fatty acyl-CoA. Allostericactivation of mitochondrial NAD-dependent isocitratedehydrogenase by ADP is counteracted by ATP andNADH. The α-ketoglutarate dehydrogenase complex isregulated in the same way as is pyruvate dehydrogenase(Figure 17–6). Succinate dehydrogenase is inhibited byoxaloacetate, and the availability of oxaloacetate, ascontrolled by malate dehydrogenase, depends on the[NADH]/[NAD + ] ratio. Since the K m for oxaloacetateof citrate synthase is of the same order of magnitude asthe intramitochondrial concentration, it is likely thatthe concentration of oxaloacetate controls the rate ofcitrate formation. Which of these mechanisms are importantin vivo has still to be resolved.SUMMARY• The citric acid cycle is the final pathway for the oxidationof carbohydrate, lipid, and protein whosecommon end-metabolite, acetyl-CoA, reacts with oxaloacetateto form citrate. By a series of dehydrogenationsand decarboxylations, citrate is degraded,releasing reduced coenzymes and 2CO 2 and regeneratingoxaloacetate.• The reduced coenzymes are oxidized by the respiratorychain linked to formation of ATP. Thus, thecycle is the major route for the generation of ATPand is located in the matrix of mitochondria adjacentto the enzymes of the respiratory chain and oxidativephosphorylation.• The citric acid cycle is amphibolic, since in additionto oxidation it is important in the provision of carbonskeletons for gluconeogenesis, fatty acid synthesis,and interconversion of amino acids.REFERENCESBaldwin JE, Krebs HA: The evolution of metabolic cycles. Nature1981;291:381.Goodwin TW (editor): The Metabolic Roles of Citrate. AcademicPress, 1968.Greville GD: Vol 1, p 297, in: Carbohydrate Metabolism and ItsDisorders. Dickens F, Randle PJ, Whelan WJ (editors). AcademicPress, 1968.Kay J, Weitzman PDJ (editors): Krebs’ Citric Acid Cycle—Half aCentury and Still Turning. Biochemical Society, London,1987.Srere PA: The enzymology of the formation and breakdown of citrate.Adv Enzymol 1975;43:57.Tyler DD: The Mitochondrion in Health and Disease. VCH Publishers,1992.

Glycolysis & the Oxidationof Pyruvate 17Peter A. Mayes, PhD, DSc, & David A. Bender, PhDBIOMEDICAL IMPORTANCEMost tissues have at least some requirement for glucose.In brain, the requirement is substantial. Glycolysis, themajor pathway for glucose metabolism, occurs in thecytosol of all cells. It is unique in that it can function eitheraerobically or anaerobically. Erythrocytes, whichlack mitochondria, are completely reliant on glucose astheir metabolic fuel and metabolize it by anaerobic glycolysis.However, to oxidize glucose beyond pyruvate(the end product of glycolysis) requires both oxygenand mitochondrial enzyme systems such as the pyruvatedehydrogenase complex, the citric acid cycle, and therespiratory chain.Glycolysis is both the principal route for glucosemetabolism and the main pathway for the metabolismof fructose, galactose, and other carbohydrates derivedfrom the diet. The ability of glycolysis to provide ATPin the absence of oxygen is especially important becauseit allows skeletal muscle to perform at very high levelswhen oxygen supply is insufficient and because it allowstissues to survive anoxic episodes. However, heart muscle,which is adapted for aerobic performance, has relativelylow glycolytic activity and poor survival underconditions of ischemia. Diseases in which enzymes ofglycolysis (eg, pyruvate kinase) are deficient are mainlyseen as hemolytic anemias or, if the defect affectsskeletal muscle (eg, phosphofructokinase), as fatigue.In fast-growing cancer cells, glycolysis proceeds at ahigher rate than is required by the citric acid cycle,forming large amounts of pyruvate, which is reduced tolactate and exported. This produces a relatively acidiclocal environment in the tumor which may have implicationsfor cancer therapy. The lactate is used for gluconeogenesisin the liver, an energy-expensive process responsiblefor much of the hypermetabolism seen incancer cachexia. Lactic acidosis results from severalcauses, including impaired activity of pyruvate dehydrogenase.136GLYCOLYSIS CAN FUNCTION UNDERANAEROBIC CONDITIONSWhen a muscle contracts in an anaerobic medium, ie,one from which oxygen is excluded, glycogen disappearsand lactate appears as the principal end product.When oxygen is admitted, aerobic recovery takes placeand lactate disappears. However, if contraction occursunder aerobic conditions, lactate does not accumulateand pyruvate is the major end product of glycolysis.Pyruvate is oxidized further to CO 2 and water (Figure17–1). When oxygen is in short supply, mitochondrialreoxidation of NADH formed from NAD + during glycolysisis impaired, and NADH is reoxidized by reducingpyruvate to lactate, so permitting glycolysis to proceed(Figure 17–1). While glycolysis can occur underanaerobic conditions, this has a price, for it limits theamount of ATP formed per mole of glucose oxidized,so that much more glucose must be metabolized underanaerobic than under aerobic conditions.THE REACTIONS OF GLYCOLYSISCONSTITUTE THE MAIN PATHWAYOF GLUCOSE UTILIZATIONThe overall equation for glycolysis from glucose to lactateis as follows:Glucos e + 2ADP + 2Pi→ 2L( + ) − Lactate + 2ATP + 2H 2 OAll of the enzymes of glycolysis (Figure 17–2) arefound in the cytosol. Glucose enters glycolysis by phosphorylationto glucose 6-phosphate, catalyzed by hexokinase,using ATP as the phosphate donor. Underphysiologic conditions, the phosphorylation of glucoseto glucose 6-phosphate can be regarded as irreversible.Hexokinase is inhibited allosterically by its product,glucose 6-phosphate. In tissues other than the liver andpancreatic B islet cells, the availability of glucose for

GLYCOLYSIS & THE OXIDATION OF PYRUVATE / 137CO 2+H 2 OGlucoseC 6Glycogen(C 6 ) nHexose phosphatesC 6Triose phosphateC 3PyruvateC 3Triose phosphateC 3NAD +O 2 NADH+ H + 1 /2O 2H 2 OLactateC 3Figure 17–1. Summary of glycolysis. − , blocked byanaerobic conditions or by absence of mitochondriacontaining key respiratory enzymes, eg, as in erythrocytes.glycolysis (or glycogen synthesis in muscle and lipogenesisin adipose tissue) is controlled by transport into thecell, which in turn is regulated by insulin. Hexokinasehas a high affinity (low K m ) for its substrate, glucose,and in the liver and pancreatic B islet cells is saturatedunder all normal conditions and so acts at a constantrate to provide glucose 6-phosphate to meet the cell’sneed. Liver and pancreatic B islet cells also contain anisoenzyme of hexokinase, glucokinase, which has a K mvery much higher than the normal intracellular concentrationof glucose. The function of glucokinase in theliver is to remove glucose from the blood following ameal, providing glucose 6-phosphate in excess of requirementsfor glycolysis, which will be used for glycogensynthesis and lipogenesis. In the pancreas, theglucose 6-phosphate formed by glucokinase signals increasedglucose availability and leads to the secretion ofinsulin.Glucose 6-phosphate is an important compound atthe junction of several metabolic pathways (glycolysis,gluconeogenesis, the pentose phosphate pathway, glycogenesis,and glycogenolysis). In glycolysis, it is convertedto fructose 6-phosphate by phosphohexoseisomerase,which involves an aldose-ketose isomerization.This reaction is followed by another phosphorylationwith ATP catalyzed by the enzyme phosphofructokinase(phosphofructokinase-1), forming fructose 1,6-bisphosphate. The phosphofructokinase reaction maybe considered to be functionally irreversible underphysiologic conditions; it is both inducible and subjectto allosteric regulation and has a major role in regulatingthe rate of glycolysis. Fructose 1,6-bisphosphate iscleaved by aldolase (fructose 1,6-bisphosphate aldolase)into two triose phosphates, glyceraldehyde 3-phosphateand dihydroxyacetone phosphate. Glyceraldehyde3-phosphate and dihydroxyacetone phosphate are interconvertedby the enzyme phosphotriose isomerase.Glycolysis continues with the oxidation of glyceraldehyde3-phosphate to 1,3-bisphosphoglycerate. Theenzyme catalyzing this oxidation, glyceraldehyde3-phosphate dehydrogenase, is NAD-dependent.Structurally, it consists of four identical polypeptides(monomers) forming a tetramer. ⎯SH groups arepresent on each polypeptide, derived from cysteineresidues within the polypeptide chain. One of the⎯SH groups at the active site of the enzyme (Figure17–3) combines with the substrate forming a thiohemiacetalthat is oxidized to a thiol ester; the hydrogens removedin this oxidation are transferred to NAD + . Thethiol ester then undergoes phosphorolysis; inorganicphosphate (P i ) is added, forming 1,3-bisphosphoglycerate,and the ⎯SH group is reconstituted.In the next reaction, catalyzed by phosphoglyceratekinase, phosphate is transferred from 1,3-bisphosphoglycerateonto ADP, forming ATP (substrate-levelphosphorylation) and 3-phosphoglycerate. Since twomolecules of triose phosphate are formed per moleculeof glucose, two molecules of ATP are generated at thisstage per molecule of glucose undergoing glycolysis.The toxicity of arsenic is due to competition of arsenatewith inorganic phosphate (P i ) in the above reactions togive 1-arseno-3-phosphoglycerate, which hydrolyzesspontaneously to give 3-phosphoglycerate plus heat,without generating ATP. 3-Phosphoglycerate is isomerizedto 2-phosphoglycerate by phosphoglycerate mutase.It is likely that 2,3-bisphosphoglycerate (diphosphoglycerate;DPG) is an intermediate in this reaction.The subsequent step is catalyzed by enolase and involvesa dehydration, forming phosphoenolpyruvate.Enolase is inhibited by fluoride. To prevent glycolysisin the estimation of glucose, blood is collected intubes containing fluoride. The enzyme is also dependenton the presence of either Mg 2+ or Mn 2+ . Thephosphate of phosphoenolpyruvate is transferred toADP by pyruvate kinase to generate, at this stage,two molecules of ATP per molecule of glucose oxidized.The product of the enzyme-catalyzed reaction,enolpyruvate, undergoes spontaneous (nonenzymic)isomerization to pyruvate and so is not available to

GlycogenGlucose 1-phosphateHEXOKINASECH 2 OHHHOHOHHOHOHGLUCOKINASEMg 2+HHOHHOCH 2 O POHHOHPHOSPHOHEXOSEISOMERASECH 2 O POCH 2 OHHH HOOHHOHα-D-GlucoseATPADPH OHα-D-Glucose 6-phosphateATPOHHD-Fructose 6-phosphateHCOO –COHPHOSPHOGLYCERATEKINASEMg 2+D-Fructose 1,6-bisphosphate HHHOCCCH 2 O POCH * 2HOOHIodoacetate HO HGLYCERALDEHYDE-3-PHOSPHATEDEHYDROGENASEO PH CPiOHH CMg 2+ADPOOHOPPHOSPHOFRUCTO-KINASEALDOLASECH * 2 O PCOCH 2 OHDihydroxyacetone phosphateNAD + CH 2 O PATP ADPCH 2 O PNADH+ H + CH 2 O P3-Phosphoglycerate 1,3-Bisphosphoglycerate Glyceraldehyde3-phosphatePHOSPHOGLYCERATE MUTASECOO –H C O P 2-Phosphoglycerate3ADP+ P iMg 2+CH 2 OHFluorideH 2 OENOLASEAnaerobiosisMitochondrialrespiratory chain3ATPPHOSPHOTRIOSEISOMERASE1 /2O 2H 2 OMg 2+COO –C OCH 2ATPCOO –CADPOHPPhosphoenolpyruvatePYRUVATEKINASESpontaneousOxidationin citricacid cycleCOO –CONADH + H +NAD +HOCOO –C HCH 2CH 3LACTATEDEHYDROGENASECH 3(Enol)Pyruvate(Keto)PyruvateL(+)-LactateFigure 17–2. The pathway of glycolysis. ( P , ⎯PO 2− 3 ; P i , HOPO 2− 3 ; − , inhibition.) At asterisk: Carbonatoms 1–3 of fructose bisphosphate form dihydroxyacetone phosphate, whereas carbons 4–6 formglyceraldehyde 3-phosphate. The term “bis-,” as in bisphosphate, indicates that the phosphate groupsare separated, whereas diphosphate, as in adenosine diphosphate, indicates that they are joined.138

GLYCOLYSIS & THE OXIDATION OF PYRUVATE / 139S EnzH C OH C OHH C OHNAD +H C OHCH 2 O PCH 2 O PGlyceraldehyde 3-phosphateEnzyme-substrate complexHS EnzNAD +OCOPBoundcoenzymeSubstrateoxidationby boundNAD +H C OHCH 2 O P1,3-BisphosphoglyceratePiSEnzSEnzHC OC OHCH 2 OC ONADH + H +H C OHNADH + H + NAD +CH 2 O PNAD * +*PEnergy-rich intermediateFigure 17–3. Mechanism of oxidation of glyceraldehyde 3-phosphate. (Enz, glyceraldehyde-3-phosphatedehydrogenase.) The enzyme is inhibited by the ⎯SH poisoniodoacetate, which is thus able to inhibit glycolysis. The NADH produced on the enzymeis not as firmly bound to the enzyme as is NAD + . Consequently, NADH is easily displacedby another molecule of NAD + .undergo the reverse reaction. The pyruvate kinase reactionis thus also irreversible under physiologic conditions.The redox state of the tissue now determines whichof two pathways is followed. Under anaerobic conditions,the reoxidation of NADH through the respiratorychain to oxygen is prevented. Pyruvate is reducedby the NADH to lactate, the reaction being catalyzedby lactate dehydrogenase. Several tissue-specific isoenzymesof this enzyme have been described and haveclinical significance (Chapter 7). The reoxidation ofNADH via lactate formation allows glycolysis to proceedin the absence of oxygen by regenerating sufficientNAD + for another cycle of the reaction catalyzed byglyceraldehyde-3-phosphate dehydrogenase. Under aerobicconditions, pyruvate is taken up into mitochondriaand after conversion to acetyl-CoA is oxidized toCO 2 by the citric acid cycle. The reducing equivalentsfrom the NADH + H + formed in glycolysis are takenup into mitochondria for oxidation via one of the twoshuttles described in Chapter 12.Tissues That Function Under HypoxicCircumstances Tend to Produce Lactate(Figure 17–2)This is true of skeletal muscle, particularly the whitefibers, where the rate of work output—and thereforethe need for ATP formation—may exceed the rate atwhich oxygen can be taken up and utilized. Glycolysisin erythrocytes, even under aerobic conditions, alwaysterminates in lactate, because the subsequent reactionsof pyruvate are mitochondrial, and erythrocytes lackmitochondria. Other tissues that normally derive muchof their energy from glycolysis and produce lactate includebrain, gastrointestinal tract, renal medulla, retina,and skin. The liver, kidneys, and heart usually take up

140 / CHAPTER 17lactate and oxidize it but will produce it under hypoxicconditions.Glycolysis Is Regulated at Three StepsInvolving Nonequilibrium ReactionsAlthough most of the reactions of glycolysis are reversible,three are markedly exergonic and must thereforebe considered physiologically irreversible. These reactions,catalyzed by hexokinase (and glucokinase),phosphofructokinase, and pyruvate kinase, are themajor sites of regulation of glycolysis. Cells that are capableof reversing the glycolytic pathway (gluconeogenesis)have different enzymes that catalyze reactionswhich effectively reverse these irreversible reactions.The importance of these steps in the regulation of glycolysisand gluconeogenesis is discussed in Chapter 19.In Erythrocytes, the First Site in Glycolysisfor ATP Generation May Be BypassedIn the erythrocytes of many mammals, the reaction catalyzedby phosphoglycerate kinase may be bypassedby a process that effectively dissipates as heat the freeenergy associated with the high-energy phosphate of1,3-bisphosphoglycerate (Figure 17–4). Bisphosphoglyceratemutase catalyzes the conversion of 1,3-bisphosphoglycerateto 2,3-bisphosphoglycerate, which isconverted to 3-phosphoglycerate by 2,3-bisphosphoglyceratephosphatase (and possibly also phosphoglyceratemutase). This alternative pathway involves no netyield of ATP from glycolysis. However, it does serve toprovide 2,3-bisphosphoglycerate, which binds to hemoglobin,decreasing its affinity for oxygen and so makingoxygen more readily available to tissues (see Chapter 6).THE OXIDATION OF PYRUVATE TOACETYL-CoA IS THE IRREVERSIBLEROUTE FROM GLYCOLYSIS TO THECITRIC ACID CYCLEPyruvate, formed in the cytosol, is transported into themitochondrion by a proton symporter (Figure 12–10).Inside the mitochondrion, pyruvate is oxidatively decarboxylatedto acetyl-CoA by a multienzyme complex thatis associated with the inner mitochondrial membrane.This pyruvate dehydrogenase complex is analogous tothe α-ketoglutarate dehydrogenase complex of the citricacid cycle (Figure 16–3). Pyruvate is decarboxylated bythe pyruvate dehydrogenase component of the enzymecomplex to a hydroxyethyl derivative of the thiazole ringof enzyme-bound thiamin diphosphate, which in turnreacts with oxidized lipoamide, the prosthetic group ofdihydrolipoyl transacetylase, to form acetyl lipoamide(Figure 17–5). Thiamin is vitamin B 1 (Chapter 45), andCH 2 O PGlyceraldehyde 3-phosphatePiNAD +ADPATPHCOH C OHHOCCCH 2NADH + H +GlucoseGLYCERALDEHYDE-3-PHOSPHATEDEHYDROGENASEOOHOPP1,3-BisphosphoglycerateHCOO –C OHPHOSPHOGLYCERATEKINASECH 2 O P3-PhosphoglycerateFigure 17–4.erythrocytes.BISPHOSPHOGLYCERATEMUTASEHCOO –C OCH 2OPP2,3-BisphosphoglyceratePi2,3-BISPHOSPHOGLYCERATEPHOSPHATASEPyruvate2,3-Bisphosphoglycerate pathway inin thiamin deficiency glucose metabolism is impairedand there is significant (and potentially life-threatening)lactic and pyruvic acidosis. Acetyl lipoamide reacts withcoenzyme A to form acetyl-CoA and reduced lipoamide.The cycle of reaction is completed when the reducedlipoamide is reoxidized by a flavoprotein, dihydrolipoyldehydrogenase, containing FAD. Finally, the reducedflavoprotein is oxidized by NAD + , which in turn transfersreducing equivalents to the respiratory chain.++Pyruvate + NAD + CoA → Acetyl − CoA + NADH + H + CO2The pyruvate dehydrogenase complex consists of anumber of polypeptide chains of each of the three componentenzymes, all organized in a regular spatial configuration.Movement of the individual enzymes appearsto be restricted, and the metabolic intermediatesdo not dissociate freely but remain bound to the enzymes.Such a complex of enzymes, in which the sub-

CHCHOCNGLYCOLYSIS & THE OXIDATION OF PYRUVATE / 141OCH 3 C COO – + H + TDPPyruvatePYRUVATEDEHYDROGENASECO 2C OAcetyllipoamideHSCoA-SHCH 2 CH 2HH3 C STDPOCHNH 3 C C OHHydroxyethylHOxidized lipoamide 2 CCHH 2 CS SLipoic acidHNCOLysineside chainDIHYDROLIPOYLTRANSACETYLASENAD + NADH + H +FADH 2SHCH 2 CH 2 SHDIHYDROLIPOYLDEHYDROGENASECH 3 CO SAcetyl-CoACoADihydrolipoamideFADFigure 17–5. Oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase complex. Lipoic acid isjoined by an amide link to a lysine residue of the transacetylase component of the enzyme complex. It forms a longflexible arm, allowing the lipoic acid prosthetic group to rotate sequentially between the active sites of each of theenzymes of the complex. (NAD + , nicotinamide adenine dinucleotide; FAD, flavin adenine dinucleotide; TDP, thiamindiphosphate.)strates are handed on from one enzyme to the next, increasesthe reaction rate and eliminates side reactions,increasing overall efficiency.Pyruvate Dehydrogenase Is Regulatedby End-Product Inhibition& Covalent ModificationPyruvate dehydrogenase is inhibited by its products,acetyl-CoA and NADH (Figure 17–6). It is also regulatedby phosphorylation by a kinase of three serineresidues on the pyruvate dehydrogenase component ofthe multienzyme complex, resulting in decreased activity,and by dephosphorylation by a phosphatase thatcauses an increase in activity. The kinase is activated byincreases in the [ATP]/[ADP], [acetyl-CoA]/[CoA],and [NADH]/[NAD + ] ratios. Thus, pyruvate dehydrogenase—andtherefore glycolysis—is inhibited not onlyby a high-energy potential but also when fatty acids arebeing oxidized. Thus, in starvation, when free fatty acid

142 / CHAPTER 17[ Acetyl-CoA ][ CoA ][ NADH ][ NAD + ][ ATP ][ ADP ]+ ++Acetyl-CoACa 2+–PDH KINASE––DichloroacetatePyruvateNADH + H +CO 2ATPMg 2+ADP––PDHPDH-a(Active DEPHOSPHO-ENZYME)PDH-b(Inactive PHOSPHO-ENZYME)PNAD +CoAP iH 2 OPyruvatePDH PHOSPHATASEAB++Mg 2+ , Ca 2+Insulin(in adipose tissue)Figure 17–6. Regulation of pyruvate dehydrogenase (PDH). Arrows with wavy shafts indicate allosteric effects.A: Regulation by end-product inhibition. B: Regulation by interconversion of active and inactive forms.concentrations increase, there is a decrease in the proportionof the enzyme in the active form, leading to asparing of carbohydrate. In adipose tissue, where glucoseprovides acetyl CoA for lipogenesis, the enzyme isactivated in response to insulin.Oxidation of Glucose Yields Up to 38 Molof ATP Under Aerobic Conditions But Only2 Mol When O 2 Is AbsentWhen 1 mol of glucose is combusted in a calorimeterto CO 2 and water, approximately 2870 kJ are liberatedas heat. When oxidation occurs in the tissues, approximately38 mol of ATP are generated per molecule ofglucose oxidized to CO 2 and water. In vivo, ∆G for theATP synthase reaction has been calculated as approximately51.6 kJ. It follows that the total energy capturedin ATP per mole of glucose oxidized is 1961 kJ, or approximately68% of the energy of combustion. Most ofthe ATP is formed by oxidative phosphorylation resultingfrom the reoxidation of reduced coenzymes by therespiratory chain. The remainder is formed by substratelevelphosphorylation (Table 17–1).CLINICAL ASPECTSInhibition of Pyruvate MetabolismLeads to Lactic AcidosisArsenite and mercuric ions react with the ⎯SH groupsof lipoic acid and inhibit pyruvate dehydrogenase, as

GLYCOLYSIS & THE OXIDATION OF PYRUVATE / 143Table 17–1. Generation of high-energy phosphate in the catabolism of glucose.Number of ~PFormed perPathway Reaction Catalyzed by Method of ~P Production Mole of GlucoseGlycolysis Glyceraldehyde-3-phosphate dehydrogenase Respiratory chain oxidation of 2 NADH 6*Phosphoglycerate kinase Phosphorylation at substrate level 2Pyruvate kinase Phosphorylation at substrate level 210Allow for consumption of ATP by reactions catalyzed by hexokinase and phosphofructokinase−2Net 8Pyruvate dehydrogenase Respiratory chain oxidation of 2 NADH 6Isocitrate dehydrogenase Respiratory chain oxidation of 2 NADH 6α-Ketoglutarate dehydrogenase Respiratory chain oxidation of 2 NADH 6Citric acid cycle Succinate thiokinase Phosphorylation at substrate level 2Succinate dehydrogenase Respiratory chain oxidation of 2 FADH 2 4Malate dehydrogenase Respiratory chain oxidation of 2 NADH 6Net 30Total per mole of glucose under aerobic conditions 38Total per mole of glucose under anaerobic conditions 2*It is assumed that NADH formed in glycolysis is transported into mitochondria via the malate shuttle (see Figure 12–13). If the glycerophosphateshuttle is used, only 2 ~P would be formed per mole of NADH, the total net production being 26 instead of 38. Thecalculation ignores the small loss of ATP due to a transport of H + into the mitochondrion with pyruvate and a similar transport of H +in the operation of the malate shuttle, totaling about 1 mol of ATP. Note that there is a substantial benefit under anaerobic conditionsif glycogen is the starting point, since the net production of high-energy phosphate in glycolysis is increased from 2 to 3, as ATPis no longer required by the hexokinase reaction.does a dietary deficiency of thiamin, allowing pyruvateto accumulate. Nutritionally deprived alcoholicsare thiamin-deficient and may develop potentially fatalpyruvic and lactic acidosis. Patients with inheritedpyruvate dehydrogenase deficiency, which can be dueto defects in one or more of the components of the enzymecomplex, also present with lactic acidosis, particularlyafter a glucose load. Because of its dependence onglucose as a fuel, brain is a prominent tissue where thesemetabolic defects manifest themselves in neurologicdisturbances.Inherited aldolase A deficiency and pyruvate kinasedeficiency in erythrocytes cause hemolytic anemia.The exercise capacity of patients with muscle phosphofructokinasedeficiency is low, particularly onhigh-carbohydrate diets. By providing an alternativelipid fuel, eg, during starvation, when blood free fattyacids and ketone bodies are increased, work capacity isimproved.SUMMARY• Glycolysis is the cytosolic pathway of all mammaliancells for the metabolism of glucose (or glycogen) topyruvate and lactate.• It can function anaerobically by regenerating oxidizedNAD + (required in the glyceraldehyde-3-phosphate dehydrogenasereaction) by reducing pyruvate to lactate.• Lactate is the end product of glycolysis under anaerobicconditions (eg, in exercising muscle) or when themetabolic machinery is absent for the further oxidationof pyruvate (eg, in erythrocytes).• Glycolysis is regulated by three enzymes catalyzingnonequilibrium reactions: hexokinase, phosphofructokinase,and pyruvate kinase.• In erythrocytes, the first site in glycolysis for generationof ATP may be bypassed, leading to the formationof 2,3-bisphosphoglycerate, which is importantin decreasing the affinity of hemoglobin for O 2 .• Pyruvate is oxidized to acetyl-CoA by a multienzymecomplex, pyruvate dehydrogenase, that is dependenton the vitamin cofactor thiamin diphosphate.• Conditions that involve an inability to metabolizepyruvate frequently lead to lactic acidosis.REFERENCESBehal RH et al: Regulation of the pyruvate dehydrogenase multienzymecomplex. Annu Rev Nutr 1993;13:497.

144 / CHAPTER 17Boiteux A, Hess B: Design of glycolysis. Phil Trans R Soc LondonB 1981;293:5.Fothergill-Gilmore LA: The evolution of the glycolytic pathway.Trends Biochem Sci 1986;11:47.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Sols A: Multimodulation of enzyme activity. Curr Top Cell Reg1981;19:77.Srere PA: Complexes of sequential metabolic enzymes. Annu RevBiochem 1987;56:89.

Metabolism of Glycogen18Peter A. Mayes, PhD, DSc, & David A. Bender, PhDBIOMEDICAL IMPORTANCEGlycogen is the major storage carbohydrate in animals,corresponding to starch in plants; it is a branched polymerof α-D-glucose. It occurs mainly in liver (up to 6%)and muscle, where it rarely exceeds 1%. However, becauseof its greater mass, muscle contains about three tofour times as much glycogen as does liver (Table 18–1).Muscle glycogen is a readily available source of glucosefor glycolysis within the muscle itself. Liver glycogenfunctions to store and export glucose to maintainblood glucose between meals. After 12–18 hours offasting, the liver glycogen is almost totally depleted.Glycogen storage diseases are a group of inherited disorderscharacterized by deficient mobilization of glycogenor deposition of abnormal forms of glycogen, leadingto muscular weakness or even death.GLYCOGENESIS OCCURS MAINLYIN MUSCLE & LIVERThe Pathway of Glycogen BiosynthesisInvolves a Special Nucleotide of Glucose(Figure 18–1)As in glycolysis, glucose is phosphorylated to glucose6-phosphate, catalyzed by hexokinase in muscle andglucokinase in liver. Glucose 6-phosphate is isomerizedto glucose 1-phosphate by phosphoglucomutase.The enzyme itself is phosphorylated, and the phosphogrouptakes part in a reversible reaction in which glucose1,6-bisphosphate is an intermediate. Next, glucose1-phosphate reacts with uridine triphosphate (UTP) toform the active nucleotide uridine diphosphate glucose(UDPGlc)* and pyrophosphate (Figure 18–2),catalyzed by UDPGlc pyrophosphorylase. Pyrophos-* Other nucleoside diphosphate sugar compounds are known, eg,UDPGal. In addition, the same sugar may be linked to differentnucleotides. For example, glucose may be linked to uridine (asshown above) as well as to guanosine, thymidine, adenosine, or cytidinenucleotides.phatase catalyzes hydrolysis of pyrophosphate to 2 molof inorganic phosphate, shifting the equilibrium of themain reaction by removing one of its products.Glycogen synthase catalyzes the formation of a glycosidebond between C 1 of the activated glucose ofUDPGlc and C 4 of a terminal glucose residue of glycogen,liberating uridine diphosphate (UDP). A preexistingglycogen molecule, or “glycogen primer,” must bepresent to initiate this reaction. The glycogen primermay in turn be formed on a primer known as glycogenin,which is a 37-kDa protein that is glycosylatedon a specific tyrosine residue by UDPGlc. Further glucoseresidues are attached in the 1→4 position to makea short chain that is a substrate for glycogen synthase.In skeletal muscle, glycogenin remains attached in thecenter of the glycogen molecule (Figure 13–15),whereas in liver the number of glycogen molecules isgreater than the number of glycogenin molecules.Branching Involves Detachmentof Existing Glycogen ChainsThe addition of a glucose residue to a preexisting glycogenchain, or “primer,” occurs at the nonreducing,outer end of the molecule so that the “branches” of theglycogen “tree” become elongated as successive 1→4linkages are formed (Figure 18–3). When the chain hasbeen lengthened to at least 11 glucose residues, branchingenzyme transfers a part of the 1→4 chain (at leastsix glucose residues) to a neighboring chain to form a1→6 linkage, establishing a branch point. Thebranches grow by further additions of 1→4-glucosylunits and further branching.GLYCOGENOLYSIS IS NOT THE REVERSEOF GLYCOGENESIS BUT IS A SEPARATEPATHWAY (Figure 18–1)Glycogen phosphorylase catalyzes the rate-limitingstep in glycogenolysis by promoting the phosphorylyticcleavage by inorganic phosphate (phosphorylysis; cf hy-145

146 / CHAPTER 18Glycogen(1→4 and 1→6 glucosyl units) xBRANCHING ENZYMEP i(1→4 Glucosyl units) x InsulinUDPGlycogenprimerGLYCOGENSYNTHASEcAMPGLYCOGENPHOSPHORYLASEGlycogeninUridinedisphosphateglucose (UDPGlc)GlucagonEpinephrineGLUCAN *TRANSFERASEDEBRANCHINGENZYMETo uronic acidpathwayINORGANICPYROPHOSPHATASEPP iUDPGlc PYROPHOSPHORYLASEFree glucose fromdebranchingenzymeUDP2 P iUridinetriphosphate (UTP)Glucose 1-phosphateMg 2+PHOSPHOGLUCOMUTASEGlucose 6-phosphateHNUCLEOSIDE2 OADPDIPHOSPHO-ATP GLUCOSE-6-KINASE ADP Mg 2+ GLUCOKINASEPHOSPHATASEP iGlucoseATPTo glycolysis and pentosephosphate pathwayFigure 18–1. Pathway of glycogenesis and of glycogenolysis in the liver. Two high-energy phosphates areused in the incorporation of 1 mol of glucose into glycogen. + , stimulation; − , inhibition. Insulin decreases thelevel of cAMP only after it has been raised by glucagon or epinephrine—ie, it antagonizes their action. Glucagonis active in heart muscle but not in skeletal muscle. At asterisk: Glucan transferase and debranching enzyme appearto be two separate activities of the same enzyme.Table 18–1. Storage of carbohydrate inpostabsorptive normal adult humans (70 kg).Liver glycogen 4.0% = 72 g 1Muscle glycogen 0.7% = 245 g 2Extracellular glucose 0.1% = 10 g 31 Liver weight 1800 g.2 Muscle mass 35 kg.3 Total volume 10 L.327 gdrolysis) of the 1→4 linkages of glycogen to yield glucose1-phosphate. The terminal glucosyl residues fromthe outermost chains of the glycogen molecule are removedsequentially until approximately four glucoseresidues remain on either side of a 1→6 branch (Figure18–4). Another enzyme (-[1v4]v-[1v4] glucantransferase) transfers a trisaccharide unit from onebranch to the other, exposing the 1→6 branch point.Hydrolysis of the 1→6 linkages requires the debranchingenzyme. Further phosphorylase action can

METABOLISM OF GLYCOGEN / 147H OHH1OH HHO6CH 2 OHGlucoseFigure 18–2.OOO P O P O CH 2H OH O – O –Diphosphatethen proceed. The combined action of phosphorylaseand these other enzymes leads to the complete breakdownof glycogen. The reaction catalyzed by phosphoglucomutaseis reversible, so that glucose 6-phosphatecan be formed from glucose 1-phosphate. In liver (andkidney), but not in muscle, there is a specific enzyme,glucose-6-phosphatase, that hydrolyzes glucose6-phosphate, yielding glucose that is exported, leadingto an increase in the blood glucose concentration.CYCLIC AMP INTEGRATES THEREGULATION OF GLYCOGENOLYSIS& GLYCOGENESISThe principal enzymes controlling glycogen metabolism—glycogenphosphorylase and glycogen synthase—are regulated by allosteric mechanisms and covalentmodifications due to reversible phosphorylation andHHHOOHNOHONHOHUridineUracilRiboseUridine diphosphate glucose (UDPGlc).dephosphorylation of enzyme protein in response tohormone action (Chapter 9).Cyclic AMP (cAMP) (Figure 18–5) is formed fromATP by adenylyl cyclase at the inner surface of cellmembranes and acts as an intracellular second messengerin response to hormones such as epinephrine, norepinephrine,and glucagon. cAMP is hydrolyzed byphosphodiesterase, so terminating hormone action. Inliver, insulin increases the activity of phosphodiesterase.Phosphorylase Differs BetweenLiver & MuscleIn liver, one of the serine hydroxyl groups of activephosphorylase a is phosphorylated. It is inactivated byhydrolytic removal of the phosphate by protein phosphatase-1to form phosphorylase b. Reactivation requiresrephosphorylation catalyzed by phosphorylasekinase.Muscle phosphorylase is distinct from that of liver. Itis a dimer, each monomer containing 1 mol of pyridoxalphosphate (vitamin B 6 ). It is present in two forms: phosphorylasea, which is phosphorylated and active in eitherthe presence or absence of 5′-AMP (its allosteric modifier);and phosphorylase b, which is dephosphorylatedand active only in the presence of 5′-AMP. This occursduring exercise when the level of 5′-AMP rises, providing,by this mechanism, fuel for the muscle. Phosphorylase a isthe normal physiologically active form of the enzyme.cAMP Activates Muscle PhosphorylasePhosphorylase in muscle is activated in response to epinephrine(Figure 18–6) acting via cAMP. Increasingthe concentration of cAMP activates cAMP-dependent1→4- Glucosidic bondUnlabeled glucose residue1→6- Glucosidic bond14 C-labeled glucose residue14 C-GlucoseaddedNew 1→6- bondGLYCOGENSYNTHASEBRANCHINGENZYMEFigure 18–3. The biosynthesis of glycogen. The mechanism of branching as revealedby adding 14 C-labeled glucose to the diet in the living animal and examining the liverglycogen at further intervals.

148 / CHAPTER 18PHOSPHORYLASEGLUCANTRANSFERASEDEBRANCHINGENZYMEtypes of subunits—α, β, γ, and δ—in a structure representedas (αβγδ) 4 . The α and β subunits contain serineresidues that are phosphorylated by cAMP-dependentprotein kinase. The δ subunit binds four Ca 2+ and isidentical to the Ca 2+ -binding protein calmodulin(Chapter 43). The binding of Ca 2+ activates the catalyticsite of the γ subunit while the molecule remainsin the dephosphorylated b configuration. However, thephosphorylated a form is only fully activated in thepresence of Ca 2+ . A second molecule of calmodulin, orTpC (the structurally similar Ca 2+ -binding protein inmuscle), can interact with phosphorylase kinase, causingfurther activation. Thus, activation of muscle contractionand glycogenolysis are carried out by the sameCa 2+ -binding protein, ensuring their synchronization.Figure 18–4.Figure 18–5.– OGlucose residues joined by1 → 4- glucosidic bondsGlucose residues joined by1 → 6- glucosidic bondsSteps in glycogenolysis.protein kinase, which catalyzes the phosphorylation byATP of inactive phosphorylase kinase b to activephosphorylase kinase a, which in turn, by means of afurther phosphorylation, activates phosphorylase b tophosphorylase a.Ca 2+ Synchronizes the Activation ofPhosphorylase With Muscle ContractionGlycogenolysis increases in muscle several hundred-foldimmediately after the onset of contraction. This involvesthe rapid activation of phosphorylase by activationof phosphorylase kinase by Ca 2+ , the same signal asthat which initiates contraction in response to nervestimulation. Muscle phosphorylase kinase has fourOPON5′CH 2HNH 2NHO 3′OHNNHOH3′,5′-Adenylic acid (cyclic AMP; cAMP).Glycogenolysis in Liver CanBe cAMP-IndependentIn addition to the action of glucagon in causing formationof cAMP and activation of phosphorylase in liver, 1 -adrenergic receptors mediate stimulation of glycogenolysisby epinephrine and norepinephrine. This involvesa cAMP-independent mobilization of Ca 2+from mitochondria into the cytosol, followed by thestimulation of a Ca 2+ /calmodulin-sensitive phosphorylasekinase. cAMP-independent glycogenolysis is alsocaused by vasopressin, oxytocin, and angiotensin II actingthrough calcium or the phosphatidylinositol bisphosphatepathway (Figure 43–7).Protein Phosphatase-1Inactivates PhosphorylaseBoth phosphorylase a and phosphorylase kinase a aredephosphorylated and inactivated by protein phosphatase-1.Protein phosphatase-1 is inhibited by aprotein, inhibitor-1, which is active only after it hasbeen phosphorylated by cAMP-dependent protein kinase.Thus, cAMP controls both the activation and inactivationof phosphorylase (Figure 18–6). Insulin reinforcesthis effect by inhibiting the activation ofphosphorylase b. It does this indirectly by increasinguptake of glucose, leading to increased formation ofglucose 6-phosphate, which is an inhibitor of phosphorylasekinase.Glycogen Synthase & PhosphorylaseActivity Are Reciprocally Regulated(Figure 18–7)Like phosphorylase, glycogen synthase exists in either aphosphorylated or nonphosphorylated state. However,unlike phosphorylase, the active form is dephosphorylated(glycogen synthase a) and may be inactivated to

Epinephrineβ Receptor+InactiveadenylylcyclaseActiveadenylylcyclaseATP+cAMPPHOSPHODIESTERASE5′-AMPGlycogen (n+1)Glycogen (n)+Glucose 1-phosphate+149Inhibitor-1(inactive)InactivecAMP-DEPENDENTPROTEIN KINASEATPActivecAMP-DEPENDENTPROTEIN KINASECALMODULINCOMPONENT OFPHOSPHORYLASEKINASEADPP iADPPHOSPHORYLASE a(active)H 2 OATPPHOSPHORYLASE KINASE b(inactive)Ca 2+PHOSPHORYLASE KINASE a(active)–G6P+InsulinPROTEINPHOSPHATASE-1ADP+P i–Ca 2+PROTEINPHOSPHATASE-1H 2 OATPPHOSPHORYLASE b(inactive)P i–Inhibitor-1-phosphate(active)–Figure 18–6. Control of phosphorylase in muscle. The sequence of reactions arranged as a cascade allows amplification of the hormonal signalat each step. (n = number of glucose residues; G6P, glucose 6-phosphate.)

150 / CHAPTER 18Epinephrineβ Receptor+InactiveadenylylcyclaseActiveadenylylcyclase+ATPcAMPPHOSPHODIESTERASE5′-AMP+PHOSPHORYLASEKINASE+Ca 2+Inhibitor-1(inactive)InactivecAMP-DEPENDENTPROTEIN KINASEADPActivecAMP-DEPENDENTPROTEIN KINASEGSKATPGlycogen (n+1)CALMODULIN-DEPENDENTPROTEIN KINASEATPADP+Inhibitor-1-phosphate(active)GLYCOGEN SYNTHASEb(inactive)H 2 O–Insulin+ GLYCOGEN SYNTHASEaCa 2+ (active)G6P++PROTEINPHOSPHATASEPROTEINPHOSPHATASE-1P iGlycogen (n)+ UDPGFigure 18–7. Control of glycogen synthase in muscle (n = number of glucose residues). The sequence of reactionsarranged in a cascade causes amplification at each step, allowing only nanomole quantities of hormone tocause major changes in glycogen concentration. (GSK, glycogen synthase kinase-3, -4, and -5; G6P, glucose6-phosphate.)glycogen synthase b by phosphorylation on serineresidues by no fewer than six different protein kinases.Two of the protein kinases are Ca 2+ /calmodulindependent(one of these is phosphorylase kinase). Anotherkinase is cAMP-dependent protein kinase, whichallows cAMP-mediated hormonal action to inhibitglycogen synthesis synchronously with the activation ofglycogenolysis. Insulin also promotes glycogenesis inmuscle at the same time as inhibiting glycogenolysis byraising glucose 6-phosphate concentrations, whichstimulates the dephosphorylation and activation ofglycogen synthase. Dephosphorylation of glycogen synthaseb is carried out by protein phosphatase-1, whichis under the control of cAMP-dependent protein kinase.REGULATION OF GLYCOGENMETABOLISM IS EFFECTED BYA BALANCE IN ACTIVITIESBETWEEN GLYCOGENSYNTHASE & PHOSPHORYLASE(Figure 18–8)Not only is phosphorylase activated by a rise in concentrationof cAMP (via phosphorylase kinase), but glycogensynthase is at the same time converted to theinactive form; both effects are mediated via cAMPdependentprotein kinase. Thus, inhibition of glycogenolysisenhances net glycogenesis, and inhibition ofglycogenesis enhances net glycogenolysis. Furthermore,

METABOLISM OF GLYCOGEN / 151Epinephrine(liver, muscle)Glucagon(liver)PHOSPHODIESTERASEcAMP5′-AMPInhibitor-1Inhibitor-1phosphateGLYCOGENSYNTHASE bPHOSPHORYLASEKINASE bPROTEINPHOSPHATASE-1cAMP-DEPENDENTPROTEIN KINASEPROTEINPHOSPHATASE-1GLYCOGENSYNTHASE aPHOSPHORYLASEKINASE aGlycogenUDPGIcGlycogencyclePHOSPHORYLASEaPHOSPHORYLASEbGlucose 1-phosphatePROTEINPHOSPHATASE-1Glucose (liver)GlucoseLactate (muscle)Figure 18–8. Coordinated control of glycogenolysis and glycogenesis by cAMP-dependent protein kinase.The reactions that lead to glycogenolysis as a result of an increase in cAMP concentrations are shownwith bold arrows, and those that are inhibited by activation of protein phosphatase-1 are shown as brokenarrows. The reverse occurs when cAMP concentrations decrease as a result of phosphodiesterase activity,leading to glycogenesis.the dephosphorylation of phosphorylase a, phosphorylasekinase a, and glycogen synthase b is catalyzed bya single enzyme of wide specificity—protein phosphatase-1.In turn, protein phosphatase-1 is inhibitedby cAMP-dependent protein kinase via inhibitor-1.Thus, glycogenolysis can be terminated and glycogenesiscan be stimulated synchronously, or vice versa, becauseboth processes are keyed to the activity of cAMP-dependentprotein kinase. Both phosphorylase kinase andglycogen synthase may be reversibly phosphorylated inmore than one site by separate kinases and phosphatases.These secondary phosphorylations modify the sensitivityof the primary sites to phosphorylation and dephosphorylation(multisite phosphorylation). What ismore, they allow insulin, via glucose 6-phosphate elevation,to have effects that act reciprocally to those ofcAMP (Figures 18–6 and 18–7).CLINICAL ASPECTSGlycogen Storage Diseases Are Inherited“Glycogen storage disease” is a generic term to describea group of inherited disorders characterized by depositionof an abnormal type or quantity of glycogen in thetissues. The principal glycogenoses are summarized inTable 18–2. Deficiencies of adenylyl kinase andcAMP-dependent protein kinase have also been re-

152 / CHAPTER 18Table 18–2. Glycogen storage diseases.Glycogenosis Name Cause of Disorder CharacteristicsType I Von Gierke’s disease Deficiency of glucose-6-phosphatase Liver cells and renal tubule cells loadedwith glycogen. Hypoglycemia, lacticacidemia,ketosis, hyperlipemia.Type II Pompe’s disease Deficiency of lysosomal α-1→4- and Fatal, accumulation of glycogen in lyso-1→6-glucosidase (acid maltase) somes, heart failure.Type III Limit dextrinosis, Forbes’ or Absence of debranching enzyme Accumulation of a characteristicCori’s diseasebranched polysaccharide.Type IV Amylopectinosis, Andersen’s Absence of branching enzyme Accumulation of a polysaccharide havdiseaseing few branch points. Death due tocardiac or liver failure in first year of life.Type V Myophosphorylase deficiency, Absence of muscle phosphorylase Diminished exercise tolerance; musclesMcArdle’s syndromehave abnormally high glycogen content(2.5–4.1%). Little or no lactate inblood after exercise.Type VI Hers’ disease Deficiency of liver phosphorylase High glycogen content in liver, tendencytoward hypoglycemia.Type VII Tarui’s disease Deficiency of phosphofructokinase As for type V but also possibility of heinmuscle and erythrocytesmolytic anemia.Type VIII Deficiency of liver phosphorylase As for type VI.kinaseported. Some of the conditions described have benefitedfrom liver transplantation.SUMMARY• Glycogen represents the principal storage form ofcarbohydrate in the mammalian body, mainly in theliver and muscle.• In the liver, its major function is to provide glucosefor extrahepatic tissues. In muscle, it serves mainly asa ready source of metabolic fuel for use in muscle.• Glycogen is synthesized from glucose by the pathwayof glycogenesis. It is broken down by a separate pathwayknown as glycogenolysis. Glycogenolysis leads toglucose formation in liver and lactate formation inmuscle owing to the respective presence or absence ofglucose-6-phosphatase.• Cyclic AMP integrates the regulation of glycogenolysisand glycogenesis by promoting the simultaneousactivation of phosphorylase and inhibition of glycogensynthase. Insulin acts reciprocally by inhibitingglycogenolysis and stimulating glycogenesis.• Inherited deficiencies in specific enzymes of glycogenmetabolism in both liver and muscle are the causes ofglycogen storage diseases.REFERENCESBollen M, Keppens S, Stalmans W: Specific features of glycogenmetabolism in the liver. Biochem J 1998;336:19.Cohen P: The role of protein phosphorylation in the hormonalcontrol of enzyme activity. Eur J Biochem 1985;151:439.Ercan N, Gannon MC, Nuttall FQ: Incorporation of glycogenininto a hepatic proteoglycogen after oral glucose administration.J Biol Chem 1994;269:22328.Geddes R: Glycogen: a metabolic viewpoint. Bioscience Rep1986;6:415.McGarry JD et al: From dietary glucose to liver glycogen: the fullcircle round. Annu Rev Nutr 1987;7:51.Meléndez-Hevia E, Waddell TG, Shelton ED: Optimization ofmolecular design in the evolution of metabolism: the glycogenmolecule. Biochem J 1993;295:477.Raz I, Katz A, Spencer MK: Epinephrine inhibits insulin-mediatedglycogenesis but enhances glycolysis in human skeletal muscle.Am J Physiol 1991;260:E430.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Shulman GI, Landau BR: Pathways of glycogen repletion. PhysiolRev 1992;72:1019.Villar-Palasi C: On the mechanism of inactivation of muscle glycogenphosphorylase by insulin. Biochim Biophys Acta 1994;1224:384.

Gluconeogenesis & Controlof the Blood Glucose 19Peter A. Mayes, PhD, DSc, & David A. Bender, PhDBIOMEDICAL IMPORTANCEGluconeogenesis is the term used to include all pathwaysresponsible for converting noncarbohydrate precursorsto glucose or glycogen. The major substrates arethe glucogenic amino acids and lactate, glycerol, andpropionate. Liver and kidney are the major gluconeogenictissues. Gluconeogenesis meets the needs ofthe body for glucose when carbohydrate is not availablein sufficient amounts from the diet or from glycogenreserves. A supply of glucose is necessary especially forthe nervous system and erythrocytes. Failure of gluconeogenesisis usually fatal. Hypoglycemia causes braindysfunction, which can lead to coma and death. Glucoseis also important in maintaining the level of intermediatesof the citric acid cycle even when fatty acidsare the main source of acetyl-CoA in the tissues. In addition,gluconeogenesis clears lactate produced by muscleand erythrocytes and glycerol produced by adiposetissue. Propionate, the principal glucogenic fatty acidproduced in the digestion of carbohydrates by ruminants,is a major substrate for gluconeogenesis in thesespecies.GLUCONEOGENESIS INVOLVESGLYCOLYSIS, THE CITRIC ACID CYCLE,& SOME SPECIAL REACTIONS(Figure 19–1)Thermodynamic Barriers Preventa Simple Reversal of GlycolysisThree nonequilibrium reactions catalyzed by hexokinase,phosphofructokinase, and pyruvate kinase preventsimple reversal of glycolysis for glucose synthesis(Chapter 17). They are circumvented as follows:A. PYRUVATE & PHOSPHOENOLPYRUVATEMitochondrial pyruvate carboxylase catalyzes the carboxylationof pyruvate to oxaloacetate, an ATP-requiringreaction in which the vitamin biotin is the coenzyme.Biotin binds CO 2 from bicarbonate ascarboxybiotin prior to the addition of the CO 2 to pyruvate(Figure 45–17). A second enzyme, phosphoenolpyruvatecarboxykinase, catalyzes the decarboxylationand phosphorylation of oxaloacetate to phosphoenolpyruvateusing GTP (or ITP) as the phosphatedonor. Thus, reversal of the reaction catalyzed by pyruvatekinase in glycolysis involves two endergonic reactions.In pigeon, chicken, and rabbit liver, phosphoenolpyruvatecarboxykinase is a mitochondrial enzyme,and phosphoenolpyruvate is transported into the cytosolfor gluconeogenesis. In the rat and the mouse, theenzyme is cytosolic. Oxaloacetate does not cross the mitochondrialinner membrane; it is converted to malate,which is transported into the cytosol, and convertedback to oxaloacetate by cytosolic malate dehydrogenase.In humans, the guinea pig, and the cow, the enzyme isequally distributed between mitochondria and cytosol.The main source of GTP for phosphoenolpyruvatecarboxykinase inside the mitochondrion is the reactionof succinyl-CoA synthetase (Chapter 16). This providesa link and limit between citric acid cycle activity andthe extent of withdrawal of oxaloacetate for gluconeogenesis.B. FRUCTOSE 1,6-BISPHOSPHATE& FRUCTOSE 6-PHOSPHATEThe conversion of fructose 1,6-bisphosphate to fructose6-phosphate, to achieve a reversal of glycolysis, is catalyzedby fructose-1,6-bisphosphatase. Its presencedetermines whether or not a tissue is capable of synthesizingglycogen not only from pyruvate but also fromtriosephosphates. It is present in liver, kidney, andskeletal muscle but is probably absent from heart andsmooth muscle.C. GLUCOSE 6-PHOSPHATE & GLUCOSEThe conversion of glucose 6-phosphate to glucose iscatalyzed by glucose-6-phosphatase. It is present inliver and kidney but absent from muscle and adiposetissue, which, therefore, cannot export glucose into thebloodstream.153

PiGLUCOSE-6-PHOSPHATASEH 2 OGlucoseGlucose 6-phosphateATPGLUCOKINASEHEXOKINASEADPAMPGlycogenPiFRUCTOSE-1,6-BISPHOSPHATASEH 2 OFructose 6-phosphateFructose 1,6-bisphosphateATPPHOSPHOFRUCTOKINASEADPFructose2,6-bisphosphateAMPcAMP(glucagon)Fructose2,6-bisphosphatecAMP(glucagon)Glyceraldehyde 3-phosphateNAD + PiNADH + H +1,3-BisphosphoglycerateADPATP3-Phosphoglycerate2-PhosphoglycerateDihydroxyacetone phosphateNADH + H +GLYCEROL 3-PHOSPHATEDEHYDROGENASENAD +Glycerol 3-phosphateADPGLYCEROL KINASEATPGlycerolPhosphoenolpyruvatecAMP(glucagon)OxaloacetateGDP + CO 2PHOSPHOENOLPYRUVATECARBOXYKINASEGTPNADH + H +NAD +CYTOSOLMITOCHONDRIONADPPYRUVATE KINASEATPPyruvateLactateNADH + H + NAD +PyruvateMg 2+CO 2 + ATPADP + PiPYRUVATEDEHYDROGENASEPYRUVATE CARBOXYLASEAlanineFattyacidsAcetyl-CoACitrateNADH + H +NAD +OxaloacetateMalateMalateCitrateCitric acid cycleα-KetoglutarateFumarateSuccinyl-CoAPropionateFigure 19–1. Major pathways and regulation of gluconeogenesis and glycolysis in the liver. Entrypoints of glucogenic amino acids after transamination are indicated by arrows extended from circles.(See also Figure 16–4.) The key gluconeogenic enzymes are enclosed in double-bordered boxes. TheATP required for gluconeogenesis is supplied by the oxidation of long-chain fatty acids. Propionate isof quantitative importance only in ruminants. Arrows with wavy shafts signify allosteric effects; dashshaftedarrows, covalent modification by reversible phosphorylation. High concentrations of alanineact as a “gluconeogenic signal” by inhibiting glycolysis at the pyruvate kinase step.154

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE / 155D. GLUCOSE 1-PHOSPHATE & GLYCOGENThe breakdown of glycogen to glucose 1-phosphate iscatalyzed by phosphorylase. Glycogen synthesis involvesa different pathway via uridine diphosphate glucoseand glycogen synthase (Figure 18–1).The relationships between gluconeogenesis and theglycolytic pathway are shown in Figure 19–1. Aftertransamination or deamination, glucogenic amino acidsyield either pyruvate or intermediates of the citric acidcycle. Therefore, the reactions described above can accountfor the conversion of both glucogenic aminoacids and lactate to glucose or glycogen. Propionate is amajor source of glucose in ruminants and enters gluconeogenesisvia the citric acid cycle. Propionate is esterifiedwith CoA, then propionyl-CoA, is carboxylated toD-methylmalonyl-CoA, catalyzed by propionyl-CoAcarboxylase, a biotin-dependent enzyme (Figure 19–2).Methylmalonyl-CoA racemase catalyzes the conversionof D-methylmalonyl-CoA to L-methylmalonyl-CoA, which then undergoes isomerization to succinyl-CoA catalyzed by methylmalonyl-CoA isomerase.This enzyme requires vitamin B 12 as a coenzyme, anddeficiency of this vitamin results in the excretion ofmethylmalonate (methylmalonic aciduria).C 15 and C 17 fatty acids are found particularly in thelipids of ruminants. Dietary odd-carbon fatty acidsupon oxidation yield propionate (Chapter 22), which isa substrate for gluconeogenesis in human liver.Glycerol is released from adipose tissue as a result oflipolysis, and only tissues such as liver and kidney thatpossess glycerol kinase, which catalyzes the conversionof glycerol to glycerol 3-phosphate, can utilize it. Glycerol3-phosphate may be oxidized to dihydroxyacetonephosphate by NAD + catalyzed by glycerol-3-phosphatedehydrogenase.SINCE GLYCOLYSIS & GLUCONEOGENESISSHARE THE SAME PATHWAY BUT INOPPOSITE DIRECTIONS, THEY MUSTBE REGULATED RECIPROCALLYChanges in the availability of substrates are responsiblefor most changes in metabolism either directly or indirectlyacting via changes in hormone secretion. Threemechanisms are responsible for regulating the activityof enzymes in carbohydrate metabolism: (1) changes inthe rate of enzyme synthesis, (2) covalent modificationby reversible phosphorylation, and (3) allosteric effects.Induction & Repression of Key EnzymeSynthesis Requires Several HoursThe changes in enzyme activity in the liver that occurunder various metabolic conditions are listed in Table19–1. The enzymes involved catalyze nonequilibrium(physiologically irreversible) reactions. The effects aregenerally reinforced because the activity of the enzymescatalyzing the changes in the opposite direction variesreciprocally (Figure 19–1). The enzymes involved inthe utilization of glucose (ie, those of glycolysis and lipogenesis)all become more active when there is a superfluityof glucose, and under these conditions the enzymesresponsible for gluconeogenesis all have lowactivity. The secretion of insulin, in response to increasedblood glucose, enhances the synthesis of the keyCH 2PropionateCoAATPSHMg 2+ACYL-CoASYNTHETASEAMP + PPiCH 3CO 2 + H 2 OPROPIONYL-CoACARBOXYLASECH 2BiotinCO S CoAATP ADP + PiPropionyl-CoAHCH 3CCOCOO –SCoACH 3S CoACOO – CH 2METHYLMALONYL-CoARACEMASEIntermediatesof citric acid cycleFigure 19–2.COO –CH 2COMetabolism of propionate.Succinyl-CoAMETHYLMALONYL-CoA ISOMERASEB 12 coenzyme– OOCCH 3CCOHSD-Methylmalonyl-CoAL-Methylmalonyl-CoACoA

156 / CHAPTER 19Table 19–1. Regulatory and adaptive enzymes of the rat (mainly liver).Activity InCarbo- Starvahydratetion andFeeding Diabetes Inducer Repressor Activator InhibitorEnzymes of glycogenesis, glycolysis, and pyruvate oxidationGlycogen synthase ↑ ↓ Insulin Insulin Glucagon (cAMP) phossystemGlucose 6- phorylase, glycogenphosphate 1Hexokinase Glucose 6-phosphate 1Glucokinase ↑ ↓ Insulin Glucagon(cAMP)Phosphofructokinase-1 ↑ ↓ Insulin Glucagon AMP, fructose 6- Citrate (fatty acids, ketone(cAMP) phosphate, P i, fruc- bodies), 1 ATP, 1 glucagontose 2,6-bisphos- (cAMP)phate 1Pyruvate kinase ↑ ↓ Insulin, fructose Glucagon Fructose 1,6- ATP, alanine, glucagon(cAMP) bisphosphate 1 , in- (cAMP), epinephrinesulinPyruvate dehydro- ↑ ↓ CoA, NAD + , insu- Acetyl-CoA, NADH, ATPgenase lin, 2 ADP, pyruvate (fatty acids, ketone bodies)Enzymes of gluconeogenesisPyruvate carboxylase ↓ ↑ Glucocorticoids, Insulin Acetyl-CoA 1 ADP 1glucagon, epinephrine(cAMP)Phosphoenolpyruvate ↓ ↑ Glucocorticoids, Insulin Glucagon?carboxykinaseglucagon, epinephrine(cAMP)Fructose-1,6- ↓ ↑ Glucocorticoids, Insulin Glucagon (cAMP) Fructose 1,6- bisphosphate,bisphosphatase glucagon, epi- AMP, fructose 2,6-bisphosnephrine(cAMP) phate 1Glucose-6-phosphatase ↓ ↑ Glucocorticoids, Insulinglucagon, epinephrine(cAMP)Enzymes of the pentose phosphate pathway and lipogenesisGlucose-6-phosphate ↑ ↓ Insulindehydrogenase6-Phosphogluconate ↑ ↓ Insulindehydrogenase“Malic enzyme” ↑ ↓ InsulinATP-citrate lyase ↑ ↓ InsulinAcetyl-CoA carboxylase ↑ ↓ Insulin? Citrate, 1 insulin Long-chain acyl-CoA, cAMP,glucagonFatty acid synthase ↑ ↓ Insulin?1 Allosteric.2 In adipose tissue but not in liver.

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE / 157enzymes in glycolysis. Likewise, it antagonizes the effectof the glucocorticoids and glucagon-stimulated cAMP,which induce synthesis of the key enzymes responsiblefor gluconeogenesis.Both dehydrogenases of the pentose phosphatepathway can be classified as adaptive enzymes, sincethey increase in activity in the well-fed animal andwhen insulin is given to a diabetic animal. Activity islow in diabetes or starvation. “Malic enzyme” andATP-citrate lyase behave similarly, indicating that thesetwo enzymes are involved in lipogenesis rather thangluconeogenesis (Chapter 21).Covalent Modification by ReversiblePhosphorylation Is RapidGlucagon, and to a lesser extent epinephrine, hormonesthat are responsive to decreases in blood glucose,inhibit glycolysis and stimulate gluconeogenesis in theliver by increasing the concentration of cAMP. This inturn activates cAMP-dependent protein kinase, leadingto the phosphorylation and inactivation of pyruvatekinase. They also affect the concentration of fructose2,6-bisphosphate and therefore glycolysis and gluconeogenesis,as explained below.Allosteric Modification Is InstantaneousIn gluconeogenesis, pyruvate carboxylase, which catalyzesthe synthesis of oxaloacetate from pyruvate, requiresacetyl-CoA as an allosteric activator. The presenceof acetyl-CoA results in a change in the tertiarystructure of the protein, lowering the K m value for bicarbonate.This means that as acetyl-CoA is formedfrom pyruvate, it automatically ensures the provision ofoxaloacetate and, therefore, its further oxidation in thecitric acid cycle. The activation of pyruvate carboxylaseand the reciprocal inhibition of pyruvate dehydrogenaseby acetyl-CoA derived from the oxidation of fattyacids explains the action of fatty acid oxidation in sparingthe oxidation of pyruvate and in stimulating gluconeogenesis.The reciprocal relationship between thesetwo enzymes in both liver and kidney alters the metabolicfate of pyruvate as the tissue changes from carbohydrateoxidation, via glycolysis, to gluconeogenesisduring transition from a fed to a starved state (Figure19–1). A major role of fatty acid oxidation in promotinggluconeogenesis is to supply the requirement forATP. Phosphofructokinase (phosphofructokinase-1)occupies a key position in regulating glycolysis and isalso subject to feedback control. It is inhibited by citrateand by ATP and is activated by 5′-AMP. 5′-AMPacts as an indicator of the energy status of the cell. Thepresence of adenylyl kinase in liver and many othertissues allows rapid equilibration of the reaction:ATP + AMP ↔2ADPThus, when ATP is used in energy-requiring processesresulting in formation of ADP, [AMP] increases. As[ATP] may be 50 times [AMP] at equilibrium, a smallfractional decrease in [ATP] will cause a severalfold increasein [AMP]. Thus, a large change in [AMP] acts asa metabolic amplifier of a small change in [ATP]. Thismechanism allows the activity of phosphofructokinase-1to be highly sensitive to even small changes in energystatus of the cell and to control the quantity of carbohydrateundergoing glycolysis prior to its entry into thecitric acid cycle. The increase in [AMP] can also explainwhy glycolysis is increased during hypoxia when [ATP]decreases. Simultaneously, AMP activates phosphorylase,increasing glycogenolysis. The inhibition of phosphofructokinase-1by citrate and ATP is another explanationof the sparing action of fatty acid oxidation onglucose oxidation and also of the Pasteur effect,whereby aerobic oxidation (via the citric acid cycle) inhibitsthe anaerobic degradation of glucose. A consequenceof the inhibition of phosphofructokinase-1 is anaccumulation of glucose 6-phosphate that, in turn, inhibitsfurther uptake of glucose in extrahepatic tissuesby allosteric inhibition of hexokinase.Fructose 2,6-Bisphosphate Plays a UniqueRole in the Regulation of Glycolysis &Gluconeogenesis in LiverThe most potent positive allosteric effector of phosphofructokinase-1and inhibitor of fructose-1,6-bisphosphatasein liver is fructose 2,6-bisphosphate. It relievesinhibition of phosphofructokinase-1 by ATP andincreases affinity for fructose 6-phosphate. It inhibitsfructose-1,6-bisphosphatase by increasing the K m forfructose 1,6-bisphosphate. Its concentration is underboth substrate (allosteric) and hormonal control (covalentmodification) (Figure 19–3).Fructose 2,6-bisphosphate is formed by phosphorylationof fructose 6-phosphate by phosphofructokinase-2.The same enzyme protein is also responsible forits breakdown, since it has fructose-2,6-bisphosphataseactivity. This bifunctional enzyme is underthe allosteric control of fructose 6-phosphate, whichstimulates the kinase and inhibits the phosphatase.Hence, when glucose is abundant, the concentration offructose 2,6-bisphosphate increases, stimulating glycolysisby activating phosphofructokinase-1 and inhibiting

158 / CHAPTER 19GLUCONEOGENESISP iADPH 2 OF-1,6-PaseH 2 OFructose 6-phosphateP iActiveF-2,6-PaseInactivePFK-2GlycogenGlucoseGlucagoncAMPcAMP-DEPENDENTPROTEIN KINASEPPROTEINPHOSPHATASE-2ATPP iFructose 2,6-bisphosphateFructose 1,6-bisphosphatePyruvateInactiveF-2,6-PaseActivePFK-2ADPCitrateATPPFK-1ADPFigure 19–3. Control of glycolysis and gluconeogenesisin the liver by fructose 2,6-bisphosphate and thebifunctional enzyme PFK-2/F-2,6-Pase (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase).(PFK-1,phosphofructokinase-1 [6-phosphofructo-1-kinase];F-1,6-Pase, fructose-1,6-bisphosphatase. Arrows withwavy shafts indicate allosteric effects.)fructose-1,6-bisphosphatase. When glucose is short,glucagon stimulates the production of cAMP, activatingcAMP-dependent protein kinase, which in turn inactivatesphosphofructokinase-2 and activates fructose2,6-bisphosphatase by phosphorylation. Therefore, gluconeogenesisis stimulated by a decrease in the concentrationof fructose 2,6-bisphosphate, which deactivatesphosphofructokinase-1 and deinhibits fructose-1,6-bisphosphatase.This mechanism also ensures that glucagonstimulation of glycogenolysis in liver results inglucose release rather than glycolysis.GLYCOLYSISSubstrate (Futile) Cycles Allow Fine TuningIt will be apparent that the control points in glycolysisand glycogen metabolism involve a cycle of phosphorylationand dephosphorylation catalyzed by: glucokinaseand glucose-6-phosphatase; phosphofructokinase-1 andfructose-1,6-bisphosphatase; pyruvate kinase, pyruvatecarboxylase, and phosphoenolypyruvate carboxykinase;and glycogen synthase and phosphorylase. If these wereallowed to cycle unchecked, they would amount to futilecycles whose net result was hydrolysis of ATP. Thisdoes not occur extensively due to the various controlmechanisms, which ensure that one reaction is inhibitedas the other is stimulated. However, there is a physiologicadvantage in allowing some cycling. The rate ofnet glycolysis may increase several thousand-fold in responseto stimulation, and this is more readily achievedby both increasing the activity of phosphofructokinaseand decreasing that of fructose bisphosphatase if bothare active, than by switching one enzyme “on” and theother “off” completely. This “fine tuning” of metaboliccontrol occurs at the expense of some loss of ATP.THE CONCENTRATION OF BLOODGLUCOSE IS REGULATED WITHINNARROW LIMITSIn the postabsorptive state, the concentration of bloodglucose in most mammals is maintained between 4.5and 5.5 mmol/L. After the ingestion of a carbohydratemeal, it may rise to 6.5–7.2 mmol/L, and in starvation,it may fall to 3.3–3.9 mmol/L. A sudden decrease inblood glucose will cause convulsions, as in insulin overdose,owing to the immediate dependence of the brainon a supply of glucose. However, much lower concentrationscan be tolerated, provided progressive adaptationis allowed. The blood glucose level in birds is considerablyhigher (14.0 mmol/L) and in ruminantsconsiderably lower (approximately 2.2 mmol/L insheep and 3.3 mmol/L in cattle). These lower normallevels appear to be associated with the fact that ruminantsferment virtually all dietary carbohydrate to lower(volatile) fatty acids, and these largely replace glucose asthe main metabolic fuel of the tissues in the fed condition.BLOOD GLUCOSE IS DERIVED FROMTHE DIET, GLUCONEOGENESIS,& GLYCOGENOLYSISThe digestible dietary carbohydrates yield glucose,galactose, and fructose that are transported via thehepatic portal vein to the liver where galactose andfructose are readily converted to glucose (Chapter 20).

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE / 159Glucose is formed from two groups of compoundsthat undergo gluconeogenesis (Figures 16–4 and 19–1):(1) those which involve a direct net conversion to glucosewithout significant recycling, such as some aminoacids and propionate; and (2) those which are theproducts of the metabolism of glucose in tissues. Thus,lactate, formed by glycolysis in skeletal muscle anderythrocytes, is transported to the liver and kidneywhere it re-forms glucose, which again becomes availablevia the circulation for oxidation in the tissues. Thisprocess is known as the Cori cycle, or lactic acid cycle(Figure 19–4). Triacylglycerol glycerol in adipose tissueis derived from blood glucose. This triacylglycerol iscontinuously undergoing hydrolysis to form free glycerol,which cannot be utilized by adipose tissue and isconverted back to glucose by gluconeogenic mechanismsin the liver and kidney (Figure 19–1).Of the amino acids transported from muscle to theliver during starvation, alanine predominates. The glucose-alaninecycle (Figure 19–4) transports glucosefrom liver to muscle with formation of pyruvate, followedby transamination to alanine, then transportsalanine to the liver, followed by gluconeogenesis backto glucose. A net transfer of amino nitrogen from muscleto liver and of free energy from liver to muscle is effected.The energy required for the hepatic synthesis ofglucose from pyruvate is derived from the oxidation offatty acids.Glucose is also formed from liver glycogen byglycogenolysis (Chapter 18).Metabolic & Hormonal MechanismsRegulate the Concentrationof the Blood GlucoseThe maintenance of stable levels of glucose in the bloodis one of the most finely regulated of all homeostaticmechanisms, involving the liver, extrahepatic tissues,and several hormones. Liver cells are freely permeableto glucose (via the GLUT 2 transporter), whereas cellsof extrahepatic tissues (apart from pancreatic B islets)are relatively impermeable, and their glucose transportersare regulated by insulin. As a result, uptakefrom the bloodstream is the rate-limiting step in theutilization of glucose in extrahepatic tissues. The role ofvarious glucose transporter proteins found in cell membranes,each having 12 transmembrane domains, isshown in Table 19–2.Glucokinase Is Important in RegulatingBlood Glucose After a MealHexokinase has a low K m for glucose and in the liver issaturated and acting at a constant rate under all normalconditions. Glucokinase has a considerably higher K m(lower affinity) for glucose, so that its activity increasesover the physiologic range of glucose concentrations(Figure 19–5). It promotes hepatic uptake of largeamounts of glucose at the high concentrations found inthe hepatic portal vein after a carbohydrate meal. It isabsent from the liver of ruminants, which have littleBLOODGlucoseLIVERMUSCLEGlucose 6-phosphateGlycogenGlycogenGlucose 6-phosphateUreaPyruvateLactateLactatePyruvate–NH 2AlanineTransaminationLactateBLOODPyruvateTransamination–NH 2AlanineAlanineFigure 19–4.The lactic acid (Cori) cycle and glucose-alanine cycle.

160 / CHAPTER 19Table 19–2. Glucose transporters.Tissue LocationFunctionsFacilitative bidirectional transportersGLUT 1 Brain, kidney, colon, placenta, erythrocyte Uptake of glucoseGLUT 2 Liver, pancreatic B cell, small intestine, kidney Rapid uptake and release of glucoseGLUT 3 Brain, kidney, placenta Uptake of glucoseGLUT 4 Heart and skeletal muscle, adipose tissue Insulin-stimulated uptake of glucoseGLUT 5 Small intestine Absorption of glucoseSodium-dependent unidirectional transporterSGLT 1 Small intestine and kidney Active uptake of glucose from lumen of intestine andreabsorption of glucose in proximal tubule of kidneyagainst a concentration gradientglucose entering the portal circulation from the intestines.At normal systemic-blood glucose concentrations(4.5–5.5 mmol/L), the liver is a net producer of glucose.However, as the glucose level rises, the output ofglucose ceases, and there is a net uptake.Activity10050HexokinaseGlucokinaseInsulin Plays a Central Role inRegulating Blood GlucoseIn addition to the direct effects of hyperglycemia in enhancingthe uptake of glucose into the liver, the hormoneinsulin plays a central role in regulating blood glucose.It is produced by the B cells of the islets ofLangerhans in the pancreas in response to hyperglycemia.The B islet cells are freely permeable to glucosevia the GLUT 2 transporter, and the glucose isphosphorylated by glucokinase. Therefore, increasingblood glucose increases metabolic flux through glycolysis,the citric acid cycle, and the generation of ATP. Increasein [ATP] inhibits ATP-sensitive K + channels,causing depolarization of the B cell membrane, whichincreases Ca 2+ influx via voltage-sensitive Ca 2+ channels,stimulating exocytosis of insulin. Thus, the concentrationof insulin in the blood parallels that of the bloodglucose. Other substances causing release of insulin fromthe pancreas include amino acids, free fatty acids, ketonebodies, glucagon, secretin, and the sulfonylurea drugstolbutamide and glyburide. These drugs are used tostimulate insulin secretion in type 2 diabetes mellitus(NIDDM, non-insulin-dependent diabetes mellitus);they act by inhibiting the ATP-sensitive K + channels.Epinephrine and norepinephrine block the release of insulin.Insulin lowers blood glucose immediately by enhancingglucose transport into adipose tissue and muscleby recruitment of glucose transporters (GLUT 4) fromthe interior of the cell to the plasma membrane. Althoughit does not affect glucose uptake into the liverdirectly, insulin does enhance long-term uptake as a resultof its actions on the enzymes controlling glycolysis,glycogenesis, and gluconeogenesis (Chapter 18).V max5 10015 20 25Blood glucose (mmol/L)Figure 19–5. Variation in glucose phosphorylatingactivity of hexokinase and glucokinase with increase ofblood glucose concentration. The K m for glucose ofhexokinase is 0.05 mmol/L and of glucokinase is 10mmol/L.Glucagon Opposes the Actions of InsulinGlucagon is the hormone produced by the A cells ofthe pancreatic islets. Its secretion is stimulated by hypoglycemia.In the liver, it stimulates glycogenolysis by activatingphosphorylase. Unlike epinephrine, glucagondoes not have an effect on muscle phosphorylase.Glucagon also enhances gluconeogenesis from aminoacids and lactate. In all these actions, glucagon acts viageneration of cAMP (Table 19–1). Both hepaticglycogenolysis and gluconeogenesis contribute to the

GLUCONEOGENESIS & CONTROL OF THE BLOOD GLUCOSE / 161hyperglycemic effect of glucagon, whose actions opposethose of insulin. Most of the endogenous glucagon(and insulin) is cleared from the circulation by the liver.Other Hormones Affect Blood GlucoseThe anterior pituitary gland secretes hormones thattend to elevate the blood glucose and therefore antagonizethe action of insulin. These are growth hormone,ACTH (corticotropin), and possibly other “diabetogenic”hormones. Growth hormone secretion is stimulatedby hypoglycemia; it decreases glucose uptake inmuscle. Some of this effect may not be direct, since itstimulates mobilization of free fatty acids from adiposetissue, which themselves inhibit glucose utilization. Theglucocorticoids (11-oxysteroids) are secreted by theadrenal cortex and increase gluconeogenesis. This is aresult of enhanced hepatic uptake of amino acids andincreased activity of aminotransferases and key enzymesof gluconeogenesis. In addition, glucocorticoids inhibitthe utilization of glucose in extrahepatic tissues. In allthese actions, glucocorticoids act in a manner antagonisticto insulin.Epinephrine is secreted by the adrenal medulla as aresult of stressful stimuli (fear, excitement, hemorrhage,hypoxia, hypoglycemia, etc) and leads to glycogenolysisin liver and muscle owing to stimulation of phosphorylasevia generation of cAMP. In muscle, glycogenolysisresults in increased glycolysis, whereas in liver glucose isthe main product leading to increase in blood glucose.meals or at night. Furthermore, premature and lowbirth-weightbabies are more susceptible to hypoglycemia,since they have little adipose tissue to generatealternative fuels such as free fatty acids or ketonebodies during the transition from fetal dependency tothe free-living state. The enzymes of gluconeogenesismay not be completely functional at this time, and theprocess is dependent on a supply of free fatty acids forenergy. Glycerol, which would normally be releasedfrom adipose tissue, is less available for gluconeogenesis.The Body’s Ability to Utilize GlucoseMay Be Ascertained by Measuring ItsGlucose ToleranceGlucose tolerance is the ability to regulate the bloodglucose concentration after the administration of a testdose of glucose (normally 1 g/kg body weight) (Figure19–6). Diabetes mellitus (type 1, or insulin-dependentdiabetes mellitus; IDDM) is characterized by decreasedglucose tolerance due to decreased secretion of insulinin response to the glucose challenge. Glucose toleranceis also impaired in type 2 diabetes mellitus (NIDDM),which is often associated with obesity and raised levelsof plasma free fatty acids and in conditions where theliver is damaged; in some infections; and in response tosome drugs. Poor glucose tolerance can also be expected15FURTHER CLINICAL ASPECTSGlucosuria Occurs When the RenalThreshold for Glucose Is ExceededWhen the blood glucose rises to relatively high levels,the kidney also exerts a regulatory effect. Glucose iscontinuously filtered by the glomeruli but is normallycompletely reabsorbed in the renal tubules by activetransport. The capacity of the tubular system to reabsorbglucose is limited to a rate of about 350 mg/min,and in hyperglycemia (as occurs in poorly controlled diabetesmellitus) the glomerular filtrate may containmore glucose than can be reabsorbed, resulting in glucosuria.Glucosuria occurs when the venous blood glucoseconcentration exceeds 9.5–10.0 mmol/L; this istermed the renal threshold for glucose.Hypoglycemia May Occur DuringPregnancy & in the NeonateDuring pregnancy, fetal glucose consumption increasesand there is a risk of maternal and possibly fetal hypoglycemia,particularly if there are long intervals betweenBlood glucose (mmol/L)10501Time (h)NormalDiabeticFigure 19–6. Glucose tolerance test. Blood glucosecurves of a normal and a diabetic individual after oraladministration of 50 g of glucose. Note the initial raisedconcentration in the diabetic. A criterion of normality isthe return of the curve to the initial value within 2 hours.2

162 / CHAPTER 19due to hyperactivity of the pituitary or adrenal cortexbecause of the antagonism of the hormones secreted bythese glands to the action of insulin.Administration of insulin (as in the treatment of diabetesmellitus type 1) lowers the blood glucose and increasesits utilization and storage in the liver and muscleas glycogen. An excess of insulin may cause hypoglycemia,resulting in convulsions and even in deathunless glucose is administered promptly. Increased toleranceto glucose is observed in pituitary or adrenocorticalinsufficiency—attributable to a decrease in the antagonismto insulin by the hormones normally secretedby these glands.SUMMARY• Gluconeogenesis is the process of converting noncarbohydratesto glucose or glycogen. It is of particularimportance when carbohydrate is not available fromthe diet. Significant substrates are amino acids, lactate,glycerol, and propionate.• The pathway of gluconeogenesis in the liver and kidneyutilizes those reactions in glycolysis which are reversibleplus four additional reactions that circumventthe irreversible nonequilibrium reactions.• Since glycolysis and gluconeogenesis share the samepathway but operate in opposite directions, their activitiesare regulated reciprocally.• The liver regulates the blood glucose after a meal becauseit contains the high-K m glucokinase that promotesincreased hepatic utilization of glucose.• Insulin is secreted as a direct response to hyperglycemia;it stimulates the liver to store glucose asglycogen and facilitates uptake of glucose into extrahepatictissues.• Glucagon is secreted as a response to hypoglycemiaand activates both glycogenolysis and gluconeogenesisin the liver, causing release of glucose into theblood.REFERENCESBurant CF et al: Mammalian glucose transporters: structure andmolecular regulation. Recent Prog Horm Res 1991;47:349.Krebs HA: Gluconeogenesis. Proc R Soc London (Biol) 1964;159:545.Lenzen S: Hexose recognition mechanisms in pancreatic B-cells.Biochem Soc Trans 1990;18:105.Newgard CB, McGarry JD: Metabolic coupling factors in pancreaticbeta-cell signal transduction. Annu Rev Biochem 1995;64:689.Newsholme EA, Start C: Regulation in Metabolism. Wiley, 1973.Nordlie RC, Foster JD, Lange AJ: Regulation of glucose productionby the liver. Annu Rev Nutr 1999;19:379.Pilkis SJ, El-Maghrabi MR, Claus TH: Hormonal regulation of hepaticgluconeogenesis and glycolysis. Annu Rev Biochem1988;57:755.Pilkis SJ, Granner DK: Molecular physiology of the regulation ofhepatic gluconeogenesis and glycolysis. Annu Rev Physiol1992;54:885.Yki-Jarvinen H: Action of insulin on glucose metabolism in vivo.Baillieres Clin Endocrinol Metab 1993;7:903.

The Pentose PhosphatePathway & Other Pathwaysof Hexose Metabolism20Peter A. Mayes, PhD, DSc, & David A. Bender, PhDBIOMEDICAL IMPORTANCEThe pentose phosphate pathway is an alternative routefor the metabolism of glucose. It does not generateATP but has two major functions: (1) The formation ofNADPH for synthesis of fatty acids and steroids and(2) the synthesis of ribose for nucleotide and nucleicacid formation. Glucose, fructose, and galactose are themain hexoses absorbed from the gastrointestinal tract,derived principally from dietary starch, sucrose, andlactose, respectively. Fructose and galactose are convertedto glucose, mainly in the liver.Genetic deficiency of glucose 6-phosphate dehydrogenase,the first enzyme of the pentose phosphate pathway,is a major cause of hemolysis of red blood cells, resultingin hemolytic anemia and affecting approximately100 million people worldwide. Glucuronic acid is synthesizedfrom glucose via the uronic acid pathway, of majorsignificance for the excretion of metabolites and foreignchemicals (xenobiotics) as glucuronides. A deficiency inthe pathway leads to essential pentosuria. The lack ofone enzyme of the pathway (gulonolactone oxidase) inprimates and some other animals explains why ascorbicacid (vitamin C) is a dietary requirement for humans butnot most other mammals. Deficiencies in the enzymes offructose and galactose metabolism lead to essential fructosuriaand the galactosemias.THE PENTOSE PHOSPHATE PATHWAYGENERATES NADPH & RIBOSEPHOSPHATE (Figure 20–1)The pentose phosphate pathway (hexose monophosphateshunt) is a more complex pathway than glycolysis.Three molecules of glucose 6-phosphate give rise tothree molecules of CO 2 and three five-carbon sugars.These are rearranged to regenerate two molecules ofglucose 6-phosphate and one molecule of the glycolyticintermediate, glyceraldehyde 3-phosphate. Since twomolecules of glyceraldehyde 3-phosphate can regenerateglucose 6-phosphate, the pathway can account for thecomplete oxidation of glucose.163REACTIONS OF THE PENTOSEPHOSPHATE PATHWAY OCCURIN THE CYTOSOLThe enzymes of the pentose phosphate pathway, as ofglycolysis, are cytosolic. As in glycolysis, oxidationis achieved by dehydrogenation; but NADP + and notNAD + is the hydrogen acceptor. The sequence of reactionsof the pathway may be divided into two phases: anoxidative nonreversible phase and a nonoxidative reversiblephase. In the first phase, glucose 6-phosphateundergoes dehydrogenation and decarboxylation to yielda pentose, ribulose 5-phosphate. In the second phase,ribulose 5-phosphate is converted back to glucose 6-phosphateby a series of reactions involving mainly two enzymes:transketolase and transaldolase (Figure 20–1).The Oxidative Phase Generates NADPH(Figures 20–1 and 20–2)Dehydrogenation of glucose 6-phosphate to 6-phosphogluconateoccurs via the formation of 6-phosphogluconolactone,catalyzed by glucose-6-phosphatedehydrogenase, an NADP-dependent enzyme. Thehydrolysis of 6-phosphogluconolactone is accomplishedby the enzyme gluconolactone hydrolase. A secondoxidative step is catalyzed by 6-phosphogluconate dehydrogenase,which also requires NADP + as hydrogenacceptor and involves decarboxylation followed by formationof the ketopentose, ribulose 5-phosphate.The Nonoxidative Phase GeneratesRibose PrecursorsRibulose 5-phosphate is the substrate for two enzymes.Ribulose 5-phosphate 3-epimerase alters the configurationabout carbon 3, forming another ketopentose,xylulose 5-phosphate. Ribose 5-phosphate ketoisomeraseconverts ribulose 5-phosphate to the correspondingaldopentose, ribose 5-phosphate, which is the precursorof the ribose required for nucleotide and nucleicacid synthesis. Transketolase transfers the two-carbon

164 / CHAPTER 20Glucose 6-phosphate Glucose 6-phosphate Glucose 6-phosphateC 6NADP + C 6 C 6+ H 2 ONADP + + H 2 ONADP + + H 2 OGLUCOSE-6-PHOSPHATEDEHYDROGENASENADPH + H +NADPH + H +NADPH + H +NADP +6-Phosphogluconate 6-Phosphogluconate 6-PhosphogluconateC 6NADP +C 6NADP + C 6DEHYDROGENASENADPH + H +NADPH + H + NADPH + H +6-PHOSPHO-GLUCONATECO 2 CO 2 CO 2Ribulose 5-phosphate Ribulose 5-phosphate Ribulose 5-phosphateC 5 C 5 C 53-EPIMERASEKETO-ISOMERASE3-EPIMERASEXylulose 5-phosphateC 5Ribose 5-phosphateC 5Xylulose 5-phosphateC 5TRANSKETOLASEGlyceraldehyde 3-phosphateSedoheptulose 7-phosphateSynthesis ofnucleotides,RNA, DNAC 3 C 7TRANSKETOLASETRANSALDOLASEFructose 6-phosphateErythrose 4-phosphateC 4C 6C 6Glucose 6-phosphateFructose 6-phosphateC 6Glyceraldehyde 3-phosphateC 3PHOSPHOTRIOSEALDOLASE ISOMERASEPHOSPHOHEXOSEISOMERASEPHOSPHOHEXOSEISOMERASE1 /2 Fructose 1,6-bisphosphateC 6FRUCTOSE-1,6-BISPHOSPHATASE1 /2 Fructose 6-phosphateC 6PHOSPHOHEXOSEISOMERASEGlucose 6-phosphate1 /2 Glucose 6-phosphateC 6C 6Figure 20–1. Flow chart of pentose phosphate pathway and its connections with the pathwayof glycolysis. The full pathway, as indicated, consists of three interconnected cycles in which glucose6-phosphate is both substrate and end product. The reactions above the broken line arenonreversible, whereas all reactions under that line are freely reversible apart from that catalyzedby fructose-1,6-bisphosphatase.

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM / 165HOHHOHHCCCCCHOHHOHOCH 2 O Pβ-D-Glucose 6-phosphateO+ + CH 2 ONADP NADPH + HMg 2+or Ca 2+H C OHMg 2+ , Mn 2+ ,or Ca 2+HO C HOGLUCOSE-6-PHOSPHATE H C OHGLUCONOLACTONEDEHYDROGENASEHYDROLASEH CCH 2 O P6-PhosphogluconolactoneCOO –H C OHHO C HH C OHH C OHCH 2 O P6-PhosphogluconateNADP +6-PHOSPHOGLUCONATEDEHYDROGENASEMg 2+ , Mn 2+ ,or Ca 2+NADP + + H +COO –CHOHC OHRIBOSE 5-PHOSPHATEKETOISOMERASECH 2 OHC OHCCOHOHCOHHCOHHCOHHCOHHCOHHCOHCH 2 O PCH 2 O PCO 2CH 2 O PEnediol formRibulose 5-phosphate3-Keto 6-phosphogluconateRIBULOSE 5-PHOSPHATE3-EPIMERASEHHHHHHHHHOHC OHC OHC OH OCCH 2 O PRibose 5-phosphateATPMg 2+ PRPPSYNTHETASEAMPC O P PC OHC OH OCCH 2 O PPRPPCH 2 OHC OC HC OHCH 2 O PXylulose 5-phosphate*CCH 2 OHCH 2 OHC OC OHO *C HHO C HH *C OHH C OH*CH 2 O PH C OHXylulose 5-phosphateH C OHCH 2 O PSedoheptulose 7-phosphateTRANSKETOLASEThiamin– P 2H *C OMg 2+H *C OHCH 2 OH*CH 2 O PC OGlyceraldehyde 3-phosphateHO C HTRANSALDOLASE H OHMg 2+ H C OHH C OHH C OH *C OHH C OH*CH 2 O PH C OHFructose 6-phosphateCH 2 O PErythrose 4-phosphateCH 2 OHC OTRANSKETOLASEHO C HThiamin– P 2H C OH C OHCH 2 O PGlyceraldehyde 3-phosphateCH 2 O PFructose 6-phosphateFigure 20–2. The pentose phosphate pathway. ( P , ⎯PO 2– 3 ; PRPP, 5-phosphoribosyl 1-pyrophosphate.)

166 / CHAPTER 20unit comprising carbons 1 and 2 of a ketose onto thealdehyde carbon of an aldose sugar. It therefore effectsthe conversion of a ketose sugar into an aldose with twocarbons less and simultaneously converts an aldose sugarinto a ketose with two carbons more. The reaction requiresMg 2+ and thiamin diphosphate (vitamin B 1 ) ascoenzyme. Thus, transketolase catalyzes the transfer ofthe two-carbon unit from xylulose 5-phosphate to ribose5-phosphate, producing the seven-carbon ketose sedoheptulose7-phosphate and the aldose glyceraldehyde3-phosphate. Transaldolase allows the transfer of athree-carbon dihydroxyacetone moiety (carbons 1–3)from the ketose sedoheptulose 7-phosphate onto the aldoseglyceraldehyde 3-phosphate to form the ketosefructose 6-phosphate and the four-carbon aldose erythrose4-phosphate. In a further reaction catalyzed by transketolase,xylulose 5-phosphate donates a two-carbon unitto erythrose 4-phosphate to form fructose 6-phosphateand glyceraldehyde 3-phosphate.In order to oxidize glucose completely to CO 2 viathe pentose phosphate pathway, there must be enzymespresent in the tissue to convert glyceraldehyde 3-phosphateto glucose 6-phosphate. This involves reversal ofglycolysis and the gluconeogenic enzyme fructose 1,6-bisphosphatase. In tissues that lack this enzyme, glyceraldehyde3-phosphate follows the normal pathway ofglycolysis to pyruvate.The Two Major Pathways for theCatabolism of Glucose HaveLittle in CommonAlthough glucose 6-phosphate is common to bothpathways, the pentose phosphate pathway is markedlydifferent from glycolysis. Oxidation utilizes NADPrather than NAD, and CO 2 , which is not produced inglycolysis, is a characteristic product. No ATP is generatedin the pentose phosphate pathway, whereas ATP isa major product of glycolysis.Reducing Equivalents Are Generatedin Those Tissues Specializingin Reductive SynthesesThe pentose phosphate pathway is active in liver, adiposetissue, adrenal cortex, thyroid, erythrocytes, testis, andlactating mammary gland. Its activity is low in nonlactatingmammary gland and skeletal muscle. Those tissues inwhich the pathway is active use NADPH in reductivesyntheses, eg, of fatty acids, steroids, amino acids via glutamatedehydrogenase, and reduced glutathione. Thesynthesis of glucose-6-phosphate dehydrogenase and6-phosphogluconate dehydrogenase may also be inducedby insulin during conditions associated with the “fedstate” (Table 19–1), when lipogenesis increases.Ribose Can Be Synthesized in VirtuallyAll TissuesLittle or no ribose circulates in the bloodstream, so tissuesmust synthesize the ribose required for nucleotideand nucleic acid synthesis (Chapter 34). The source ofribose 5-phosphate is the pentose phosphate pathway(Figure 20–2). Muscle has only low activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconatedehydrogenase. Nevertheless, like most other tissues, itis capable of synthesizing ribose 5-phosphate by reversalof the nonoxidative phase of the pentose phosphatepathway utilizing fructose 6-phosphate. It is not necessaryto have a completely functioning pentose phosphatepathway for a tissue to synthesize ribose phosphates.THE PENTOSE PHOSPHATE PATHWAY& GLUTATHIONE PEROXIDASE PROTECTERYTHROCYTES AGAINST HEMOLYSISIn erythrocytes, the pentose phosphate pathway providesNADPH for the reduction of oxidized glutathionecatalyzed by glutathione reductase, a flavoproteincontaining FAD. Reduced glutathione removesH 2 O 2 in a reaction catalyzed by glutathione peroxidase,an enzyme that contains the selenium analogueof cysteine (selenocysteine) at the active site (Figure20–3). This reaction is important, since accumulationof H 2 O 2 may decrease the life span of the erythrocyteby causing oxidative damage to the cell membrane,leading to hemolysis.GLUCURONATE, A PRECURSOR OFPROTEOGLYCANS & CONJUGATEDGLUCURONIDES, IS A PRODUCT OFTHE URONIC ACID PATHWAYIn liver, the uronic acid pathway catalyzes the conversionof glucose to glucuronic acid, ascorbic acid, andpentoses (Figure 20–4). It is also an alternative oxidativepathway for glucose, but—like the pentose phosphatepathway—it does not lead to the generation of ATP.Glucose 6-phosphate is isomerized to glucose 1-phosphate,which then reacts with uridine triphosphate(UTP) to form uridine diphosphate glucose (UDPGlc)in a reaction catalyzed by UDPGlc pyrophosphorylase,as occurs in glycogen synthesis (Chapter 18). UDPGlc isoxidized at carbon 6 by NAD-dependent UDPGlc dehydrogenasein a two-step reaction to yield UDP-glucuronate.UDP-glucuronate is the “active” form of glucuronatefor reactions involving incorporation ofglucuronic acid into proteoglycans or for reactions inwhich substrates such as steroid hormones, bilirubin, anda number of drugs are conjugated with glucuronate forexcretion in urine or bile (Figure 32–14).

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM / 167NADPH + H +GSSG2H 2 OPENTOSEPHOSPHATEPATHWAYFADGLUTATHIONEREDUCTASESeGLUTATHIONEPEROXIDASE2HNADP +2GSHH 2 O 2Figure 20–3. Role of the pentose phosphate pathway in the glutathione peroxidase reactionof erythrocytes. (G-S-S-G, oxidized glutathione; G-SH, reduced glutathione; Se, seleniumcofactor.)Glucuronate is reduced to L-gulonate in an NADPHdependentreaction; L-gulonate is the direct precursor ofascorbate in those animals capable of synthesizing thisvitamin. In humans and other primates as well as guineapigs, ascorbic acid cannot be synthesized because of theabsence of L-gulonolactone oxidase. L-Gulonate is metabolizedultimately to D-xylulose 5-phosphate, a constituentof the pentose phosphate pathway.INGESTION OF LARGE QUANTITIESOF FRUCTOSE HAS PROFOUNDMETABOLIC CONSEQUENCESDiets high in sucrose or in high-fructose syrups used inmanufactured foods and beverages lead to large amountsof fructose (and glucose) entering the hepatic portal vein.Fructose undergoes more rapid glycolysis in the liver thandoes glucose because it bypasses the regulatory step catalyzedby phosphofructokinase (Figure 20–5). This allowsfructose to flood the pathways in the liver, leading to enhancedfatty acid synthesis, increased esterification of fattyacids, and increased VLDL secretion, which may raiseserum triacylglycerols and ultimately raise LDL cholesterolconcentrations (Figure 25–6). A specific kinase,fructokinase, in liver (and kidney and intestine) catalyzesthe phosphorylation of fructose to fructose 1-phosphate.This enzyme does not act on glucose, and, unlike glucokinase,its activity is not affected by fasting or by insulin,which may explain why fructose is cleared from the bloodof diabetic patients at a normal rate. Fructose 1-phosphateis cleaved to D-glyceraldehyde and dihydroxyacetonephosphate by aldolase B, an enzyme found in theliver, which also functions in glycolysis by cleaving fructose1,6-bisphosphate. D-Glyceraldehyde enters glycolysisvia phosphorylation to glyceraldehyde 3-phosphate, catalyzedby triokinase. The two triose phosphates, dihydroxyacetonephosphate and glyceraldehyde 3-phosphate,may be degraded by glycolysis or may be substrates for aldolaseand hence gluconeogenesis, which is the fate ofmuch of the fructose metabolized in the liver.In extrahepatic tissues, hexokinase catalyzes thephosphorylation of most hexose sugars, including fructose.However, glucose inhibits the phosphorylation offructose since it is a better substrate for hexokinase.Nevertheless, some fructose can be metabolized in adiposetissue and muscle. Fructose, a potential fuel, isfound in seminal plasma and in the fetal circulation ofungulates and whales. Aldose reductase is found in theplacenta of the ewe and is responsible for the secretionof sorbitol into the fetal blood. The presence of sorbitoldehydrogenase in the liver, including the fetalliver, is responsible for the conversion of sorbitol intofructose. This pathway is also responsible for the occurrenceof fructose in seminal fluid.GALACTOSE IS NEEDED FOR THESYNTHESIS OF LACTOSE, GLYCOLIPIDS,PROTEOGLYCANS, & GLYCOPROTEINSGalactose is derived from intestinal hydrolysis of thedisaccharide lactose, the sugar of milk. It is readily convertedin the liver to glucose. Galactokinase catalyzesthe phosphorylation of galactose, using ATP as phosphatedonor (Figure 20–6A). Galactose 1-phosphate reactswith uridine diphosphate glucose (UDPGlc) toform uridine diphosphate galactose (UDPGal) and glucose1-phosphate, in a reaction catalyzed by galactose1-phosphate uridyl transferase. The conversion ofUDPGal to UDPGlc is catalyzed by UDPGal 4-epimerase.Epimerization involves an oxidation and reductionat carbon 4 with NAD + as coenzyme. Finally, glucoseis liberated from UDPGlc after conversion toglucose 1-phosphate, probably via incorporation intoglycogen followed by phosphorolysis (Chapter 18).Since the epimerase reaction is freely reversible, glucosecan be converted to galactose, so that galactose isnot a dietary essential. Galactose is required in the bodynot only in the formation of lactose but also as a constituentof glycolipids (cerebrosides), proteoglycans,and glycoproteins. In the synthesis of lactose in themammary gland, UDPGal condenses with glucose toyield lactose, catalyzed by lactose synthase (Figure20–6B).

168 / CHAPTER 20H *CHHOHHCCCCOHOHHOHOCH 2 O Pα-D-Glucose6-phosphatePHOSPHO-GLUCOMUTASEH *CH CHO CH CH CO POHHOOHCH 2 OHGlucose1-phosphateHUDPGlc PYRO-*C O UDPH *C O UDPUTP PP i H C2NAD + 2NADH+ H 2 OH CPHOSPHORYLASE H C OHUDPGlcDEHYDROGENASE H C OHHO C HHO C HOOH C OHH C OHCH 2 OHC O –Uridine diphosphateglucose (UDPGlc)GlucuronidesProteoglycans+ 2H + H 2 OOUridine diphosphateglucuronateHHOHHOH*CH 2 OHCCCCCCCH 2 OHXylitolOOHHCH 2 OHL-Xylulose*CH 2 OHOHHOHCO 2NADPH + H +NADP +BLOCK INPENTOSURIANAD +D-XYLULOSEREDUCTASEOO*COC O –C O –H *C OHHO C H NADHNADP + NADPH+ H + NAD + HO C H+ H + H C OHC OHO C HHO C HOH C OHH C OHH C OHHO C HHO C HH C* CH 2 OHCH 2 OHC O –NADH+ H + C OC[2H] C3-Keto-L-gulonateL-GulonateOD-GlucuronateOxalateH 2 OGlycolateL-GulonolactoneO 22BLOCK IN PRIMATESGlycolaldehydeAND GUINEA PIGSBLOCK IN HUMANS2-Keto-L-gulonolactoneD-Xylulose 1-phosphate*CH 2 OHOOHO C H D-XyluloseHO CO COOH C OHHO CO CATPADPMg 2+CH 2 OHD-Xylulose 5-phosphateDietHHO*CCHCH 2 OHL-AscorbateUDPHHO*CCHCH 2 OHOxalateL-DehydroascorbatePentose phosphate pathwayFigure 20–4. Uronic acid pathway. (Asterisk indicates the fate of carbon 1 of glucose; P , ⎯PO 2– 3 .)

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM / 169PHOSPHOHEXOSEISOMERASEGlycogenGlucose 6-phosphateFructose 6-phosphateATPHEXOKINASEGLUCOKINASED-GlucoseGLUCOSE-6-PHOSPHATASEHEXOKINASEATPALDOSEREDUCTASE*D-SorbitolNADPH+ H + NADP +SORBITOLNAD +DEHYDROGENASE NADH+ H +D-FructoseDietFRUCTOSE-1,6-BISPHOSPHATASEATPPHOSPHOFRUCTOKINASEFRUCTOKINASEBLOCK IN ESSENTIALFRUCTOSURIAATPFructose 1,6-bisphosphateFructose 1-phosphateDihydroxyacetone-phosphateBLOCK IN HEREDITARYFRUCTOSE INTOLERANCEALDOLASE BALDOLASE AALDOLASE BPHOSPHO-TRIOSEISOMERASEFatty acidesterificationGlyceraldehyde 3-phosphateATPTRIOKINASED-Glyceraldehyde2-PhosphoglyceratePyruvateFatty acid synthesisFigure 20–5. Metabolism of fructose. Aldolase A is found in all tissues, whereas aldolase B isthe predominant form in liver. (*, not found in liver.)Glucose Is the Precursor of AllAmino Sugars (Hexosamines)Amino sugars are important components of glycoproteins(Chapter 47), of certain glycosphingolipids (eg,gangliosides) (Chapter 14), and of glycosaminoglycans(Chapter 48). The major amino sugars are glucosamine,galactosamine, and mannosamine and thenine-carbon compound sialic acid. The principal sialicacid found in human tissues is N-acetylneuraminic acid(NeuAc). A summary of the metabolic interrelationshipsamong the amino sugars is shown in Figure 20–7.CLINICAL ASPECTSImpairment of the Pentose PhosphatePathway Leads to Erythrocyte HemolysisGenetic deficiency of glucose-6-phosphate dehydrogenase,with consequent impairment of the generation ofNADPH, is common in populations of Mediterraneanand Afro-Caribbean origin. The defect is manifested asred cell hemolysis (hemolytic anemia) when susceptibleindividuals are subjected to oxidants, such as the antimalarialprimaquine, aspirin, or sulfonamides or when

170 / CHAPTER 20AGalactoseGlycogenGLYCOGEN SYNTHASEATPPiPHOSPHORYLASEMg 2+ GALACTOKINASENAD + NAD +ADPGlucose 1-phosphateGalactose1-phosphateBLOCK INGALACTOSEMIAUDPGlcPHOSPHOGLUCOMUTASEGALACTOSE1-PHOSPHATEURIDYL TRANSFERASEGlucose1-phosphateUDPGalURIDINEDIPHOSPHOGALACTOSE4-EPIMERASEGlucose 6-phosphateGLUCOSE-6-PHOSPHATASEGlucoseBATPGlucoseUDPGlcURIDINEDIPHOSPHOGALACTOSE4-EPIMERASEUDPGalADPMg 2+HEXOKINASEUDPGlcPYROPHOSPHORYLASEPHOSPHOGLUCOMUTASEPPiLACTOSESYNTHASEGlucose 6-phosphate Glucose 1-phosphate GlucoseFigure 20–6. Pathway of conversion of (A) galactose to glucose in the liver and (B) glucose to lactose inthe lactating mammary gland.Lactosethey have eaten fava beans (Vicia fava—hence the termfavism). Glutathione peroxidase is dependent upon asupply of NADPH, which in erythrocytes can beformed only via the pentose phosphate pathway. It reducesorganic peroxides and H 2 O 2 as part of the body’sdefense against lipid peroxidation (Figure 14–21).Measurement of erythrocyte transketolase and its activationby thiamin diphosphate is used to assess thiaminnutritional status (Chapter 45).Disruption of the Uronic Acid Pathway IsCaused by Enzyme Defects & Some DrugsIn the rare hereditary disease essential pentosuria, considerablequantities of L-xylulose appear in the urinebecause of absence of the enzyme necessary to reduceL-xylulose to xylitol. Parenteral administration of xylitolmay lead to oxalosis, involving calcium oxalate depositionin brain and kidneys (Figure 20–4). Various drugsmarkedly increase the rate at which glucose enters theuronic acid pathway. For example, administration ofbarbital or of chlorobutanol to rats results in a significantincrease in the conversion of glucose to glucuronate,L-gulonate, and ascorbate.Loading of the Liver With FructoseMay Potentiate Hyperlipidemia& HyperuricemiaIn the liver, fructose increases triacylglycerol synthesisand VLDL secretion, leading to hypertriacylglycerolemia—andincreased LDL cholesterol—which canbe regarded as potentially atherogenic (Chapter 26). Inaddition, acute loading of the liver with fructose, as canoccur with intravenous infusion or following very highfructose intakes, causes sequestration of inorganic phosphatein fructose 1-phosphate and diminished ATPsynthesis. As a result there is less inhibition of de novopurine synthesis by ATP and uric acid formation is in-

THE PENTOSE PHOSPHATE PATHWAY & OTHER PATHWAYS OF HEXOSE METABOLISM / 171GlycogenGlucose 1-phosphateATPADPGlucoseGlucose 6-phosphateFructose 6-phosphateGlutamineATPADPAMIDOTRANSFERASEGlutamateUTPGlucosamineAcetyl-CoAATP ADP–Glucosamine6-phosphateAcetyl-CoAPHOSPHOGLUCO-MUTASEGlucosamine1-phosphateUDPglucosamine*PPiN-AcetylglucosamineN-Acetylglucosamine6-phosphateN-Acetylglucosamine1-phosphateGlycosaminoglycans(eg, heparin)UTPEPIMERASEPhosphoenolpyruvateN-Acetylmannosamine6-phosphatePP iUDP-N-acetylglucosamine*NAD +EPIMERASEGlycosaminoglycans(hyaluronic acid),glycoproteinsN-Acetylneuraminicacid9-phosphateUDP-N-acetylgalactosamine*–InhibitingallostericeffectSialic acid,gangliosides,glycoproteinsGlycosaminoglycans(chondroitins),glycoproteinsFigure 20–7. Summary of the interrelationships in metabolism of amino sugars. (At asterisk: Analogousto UDPGlc.) Other purine or pyrimidine nucleotides may be similarly linked to sugars or amino sugars.Examples are thymidine diphosphate (TDP)-glucosamine and TDP-N-acetylglucosamine.creased, causing hyperuricemia, which is a cause of gout(Chapter 34).Defects in Fructose Metabolism CauseDisease (Figure 20–5)Lack of hepatic fructokinase causes essential fructosuria,and absence of hepatic aldolase B, which cleavesfructose 1-phosphate, leads to hereditary fructose intolerance.Diets low in fructose, sorbitol, and sucroseare beneficial for both conditions. One consequence ofhereditary fructose intolerance and of another conditiondue to fructose-1,6-bisphosphatase deficiency isfructose-induced hypoglycemia despite the presence ofhigh glycogen reserves. The accumulation of fructose1-phosphate and fructose 1,6-bisphosphate allosterically

172 / CHAPTER 20inhibits the activity of liver phosphorylase. The sequestrationof inorganic phosphate also leads to depletion ofATP and hyperuricemia.Fructose & Sorbitol in the Lens AreAssociated With Diabetic CataractBoth fructose and sorbitol are found in the lens of theeye in increased concentrations in diabetes mellitus andmay be involved in the pathogenesis of diabeticcataract. The sorbitol (polyol) pathway (not found inliver) is responsible for fructose formation from glucose(Figure 20–5) and increases in activity as the glucoseconcentration rises in diabetes in those tissues that arenot insulin-sensitive, ie, the lens, peripheral nerves, andrenal glomeruli. Glucose is reduced to sorbitol by aldosereductase, followed by oxidation of sorbitol tofructose in the presence of NAD + and sorbitol dehydrogenase(polyol dehydrogenase). Sorbitol does not diffusethrough cell membranes easily and accumulates,causing osmotic damage. Simultaneously, myoinositollevels fall. Sorbitol accumulation, myoinositol depletion,and diabetic cataract can be prevented by aldosereductase inhibitors in diabetic rats, and promising resultshave been obtained in clinical trials.When sorbitol is administered intravenously, it isconverted to fructose rather than to glucose. It is poorlyabsorbed in the small intestine, and much is fermentedby colonic bacteria to short-chain fatty acids, CO 2 , andH 2 , leading to abdominal pain and diarrhea (sorbitolintolerance).Enzyme Deficiencies in the GalactosePathway Cause GalactosemiaInability to metabolize galactose occurs in the galactosemias,which may be caused by inherited defects ingalactokinase, uridyl transferase, or 4-epimerase (Figure20–6A), though a deficiency in uridyl transferase isthe best known cause. The galactose concentration inthe blood and in the eye is reduced by aldose reductaseto galactitol, which accumulates, causing cataract. Inuridyl transferase deficiency, galactose 1-phosphate accumulatesand depletes the liver of inorganic phosphate.Ultimately, liver failure and mental deteriorationresult. As the epimerase is present in adequate amounts,the galactosemic individual can still form UDPGalfrom glucose, and normal growth and development canoccur regardless of the galactose-free diets used to controlthe symptoms of the disease.SUMMARY• The pentose phosphate pathway, present in the cytosol,can account for the complete oxidation of glucose,producing NADPH and CO 2 but not ATP.• The pathway has an oxidative phase, which is irreversibleand generates NADPH; and a nonoxidativephase, which is reversible and provides ribose precursorsfor nucleotide synthesis. The complete pathwayis present only in those tissues having a requirementfor NADPH for reductive syntheses, eg, lipogenesisor steroidogenesis, whereas the nonoxidative phase ispresent in all cells requiring ribose.• In erythrocytes, the pathway has a major function inpreventing hemolysis by providing NADPH tomaintain glutathione in the reduced state as the substratefor glutathione peroxidase.• The uronic acid pathway is the source of glucuronicacid for conjugation of many endogenous and exogenoussubstances before excretion as glucuronides inurine and bile.• Fructose bypasses the main regulatory step in glycolysis,catalyzed by phosphofructokinase, and stimulatesfatty acid synthesis and hepatic triacylglycerolsecretion.• Galactose is synthesized from glucose in the lactatingmammary gland and in other tissues where it is requiredfor the synthesis of glycolipids, proteoglycans,and glycoproteins.REFERENCESCouet C, Jan P, Debry G: Lactose and cataract in humans: a review.J Am Coll Nutr 1991;10:79.Cox TM: Aldolase B and fructose intolerance. FASEB J 1994;8:62.Cross NCP, Cox TM: Hereditary fructose intolerance. Int JBiochem 1990;22:685.Kador PF: The role of aldose reductase in the development of diabeticcomplications. Med Res Rev 1988;8:325.Kaufman FR, Devgan S: Classical galactosemia: a review. Endocrinologist1995;5:189.Macdonald I, Vrana A (editors): Metabolic Effects of Dietary Carbohydrates.Karger, 1986.Mayes PA: Intermediary metabolism of fructose. Am J Clin Nutr1993(5 Suppl);58:754S.Van den Berghe G: Inborn errors of fructose metabolism. AnnuRev Nutr 1994;14:41.Wood T: Physiological functions of the pentose phosphate pathway.Cell Biol Funct 1986;4:241.

Biosynthesis of Fatty Acids 21Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEFatty acids are synthesized by an extramitochondrialsystem, which is responsible for the complete synthesisof palmitate from acetyl-CoA in the cytosol. In the rat,the pathway is well represented in adipose tissue andliver, whereas in humans adipose tissue may not be animportant site, and liver has only low activity. In birds,lipogenesis is confined to the liver, where it is particularlyimportant in providing lipids for egg formation. Inmost mammals, glucose is the primary substrate forlipogenesis, but in ruminants it is acetate, the main fuelmolecule produced by the diet. Critical diseases of thepathway have not been reported in humans. However,inhibition of lipogenesis occurs in type 1 (insulin-dependent)diabetes mellitus, and variations in its activitymay affect the nature and extent of obesity.THE MAIN PATHWAY FOR DE NOVOSYNTHESIS OF FATTY ACIDS(LIPOGENESIS) OCCURSIN THE CYTOSOLThis system is present in many tissues, including liver,kidney, brain, lung, mammary gland, and adipose tissue.Its cofactor requirements include NADPH, ATP,Mn 2+ , biotin, and HCO − 3 (as a source of CO 2 ). Acetyl-CoA is the immediate substrate, and free palmitate isthe end product.Production of Malonyl-CoA Isthe Initial & Controlling Stepin Fatty Acid SynthesisBicarbonate as a source of CO 2 is required in the initialreaction for the carboxylation of acetyl-CoA to malonyl-CoAin the presence of ATP and acetyl-CoA carboxylase.Acetyl-CoA carboxylase has a requirementfor the vitamin biotin (Figure 21–1). The enzyme is amultienzyme protein containing a variable number ofidentical subunits, each containing biotin, biotin carboxylase,biotin carboxyl carrier protein, and transcarboxylase,as well as a regulatory allosteric site. The reactiontakes place in two steps: (1) carboxylation of biotininvolving ATP and (2) transfer of the carboxyl toacetyl-CoA to form malonyl-CoA.The Fatty Acid Synthase ComplexIs a Polypeptide ContainingSeven Enzyme ActivitiesIn bacteria and plants, the individual enzymes of thefatty acid synthase system are separate, and the acylradicals are found in combination with a protein calledthe acyl carrier protein (ACP). However, in yeast,mammals, and birds, the synthase system is a multienzymepolypeptide complex that incorporates ACP,which takes over the role of CoA. It contains the vitaminpantothenic acid in the form of 4′-phosphopantetheine(Figure 45–18). The use of one multienzymefunctional unit has the advantages of achieving the effectof compartmentalization of the process within thecell without the erection of permeability barriers, andsynthesis of all enzymes in the complex is coordinatedsince it is encoded by a single gene.In mammals, the fatty acid synthase complex is adimer comprising two identical monomers, each containingall seven enzyme activities of fatty acid synthaseon one polypeptide chain (Figure 21–2). Initially, apriming molecule of acetyl-CoA combines with a cysteine⎯SH group catalyzed by acetyl transacylase(Figure 21–3, reaction 1a). Malonyl-CoA combineswith the adjacent ⎯SH on the 4′-phosphopantetheineof ACP of the other monomer, catalyzed by malonyltransacylase (reaction 1b), to form acetyl (acyl)-malonylenzyme. The acetyl group attacks the methylenegroup of the malonyl residue, catalyzed by 3-ketoacylsynthase, and liberates CO 2 , forming 3-ketoacyl enzyme(acetoacetyl enzyme) (reaction 2), freeing the cysteine⎯SH group. Decarboxylation allows the reactionto go to completion, pulling the whole sequence of reactionsin the forward direction. The 3-ketoacyl groupis reduced, dehydrated, and reduced again (reactions 3,4, 5) to form the corresponding saturated acyl-Senzyme.A new malonyl-CoA molecule combines withthe ⎯SH of 4′-phosphopantetheine, displacing the saturatedacyl residue onto the free cysteine ⎯SH group.The sequence of reactions is repeated six more timesuntil a saturated 16-carbon acyl radical (palmityl) hasbeen assembled. It is liberated from the enzyme complexby the activity of a seventh enzyme in the complex,thioesterase (deacylase). The free palmitate must be activatedto acyl-CoA before it can proceed via any other173

174 / CHAPTER 21CH 3 CO S CoAAcetyl-CoACH 2 CoA– OOC CO SEnz biotin COO – Enz biotinMalonyl-CoAADP + PiATP + HCO 3–Figure 21–1.Biosynthesis of malonyl-CoA. (Enz, acetyl-CoA carboxylase.)metabolic pathway. Its usual fate is esterification intoacylglycerols, chain elongation or desaturation, or esterificationto cholesteryl ester. In mammary gland, thereis a separate thioesterase specific for acyl residues of C 8 ,C 10 , or C 12 , which are subsequently found in milklipids.The equation for the overall synthesis of palmitatefrom acetyl-CoA and malonyl-CoA is:+CH2CO⋅S ⋅ CoA + 7HOOC ⋅CH2CO⋅S ⋅ CoA + 14NADPH + 14H+→ CH3( CH2)14COOH + 7CO2 + 6H 2O + 8CoA ⋅ SH + 14NADPThe acetyl-CoA used as a primer forms carbonatoms 15 and 16 of palmitate. The addition of all thesubsequent C 2 units is via malonyl-CoA. Propionyl-CoA acts as primer for the synthesis of long-chain fattyMalonyltransacylaseAcetyltransacylaseHydrataseEnoylreductaseKetoacylreductase1.KetoacylsynthaseACPThioesteraseFunctionaldivisionCys4′-PhosphopantetheineSHSHSubunitdivisionSHSH2.4′-PhosphopantetheineCysThioesteraseACPKetoacylsynthaseKetoacylreductaseEnoylreductaseHydrataseMalonyltransacylaseAcetyltransacylaseFigure 21–2. Fatty acid synthase multienzyme complex. The complex is a dimer of two identical polypeptidemonomers, 1 and 2, each consisting of seven enzyme activities and the acyl carrier protein (ACP). (Cys⎯SH, cysteinethiol.) The ⎯SH of the 4′-phosphopantetheine of one monomer is in close proximity to the ⎯SH of the cysteineresidue of the ketoacyl synthase of the other monomer, suggesting a “head-to-tail” arrangement of the twomonomers. Though each monomer contains all the partial activities of the reaction sequence, the actual functionalunit consists of one-half of one monomer interacting with the complementary half of the other. Thus, twoacyl chains are produced simultaneously. The sequence of the enzymes in each monomer is based on Wakil.

HSHSPanCys12PanFatty acid synthasemultienzyme complex*CO 2Acetyl-CoA*Malonyl-CoAC 2 ACETYL-CoAC 3CARBOXYLASE1aCys SH1b CoAACETYLTRANSACYLASEC n transfer fromSHCoA12C 2MALONYLTRANSACYLASEOCys S C CH 3OPan S C CH 2 *COO –Acyl(acetyl)-malonyl enzyme(C 3 )2 to 1*CO 23-KETOACYLSYNTHASE21CysSHOO2Pan S C CH 2 CCH 3NADPH + H +NADP + Cys SH3-Ketoacyl enzyme(acetoacetyl enzyme)3-KETOACYLREDUCTASE31OOHNADPHGENERATORS2Pan S C CH 2 CHD(–)-3-Hydroxyacyl enzymeCH 3Pentose phosphatepathwayIsocitratedehydrogenaseH 2 OHYDRATASE4Malicenzyme1CysSHO2Pan S C CH CHCH 3NADPH + H +2,3-Unsaturated acyl enzymeENOYL REDUCTASE5NADP + Cys SHPalmitateTHIOESTERASEAfter cycling throughsteps 2 – 5 seven timesH 2 O12OPan S C CH2 CH 2 CH 3 (C n )Acyl enzymeKEY:1 , 2 , individual monomers of fatty acid synthaseFigure 21–3. Biosynthesis of long-chain fatty acids. Details of how addition of a malonyl residuecauses the acyl chain to grow by two carbon atoms. (Cys, cysteine residue; Pan, 4′-phosphopantetheine.)The blocks shown in dark blue contain initially a C 2 unit derived from acetyl-CoA (as illustrated)and subsequently the C n unit formed in reaction 5.175

176 / CHAPTER 21acids having an odd number of carbon atoms, foundparticularly in ruminant fat and milk.The Main Source of NADPHfor Lipogenesis Is the PentosePhosphate PathwayNADPH is involved as donor of reducing equivalentsin both the reduction of the 3-ketoacyl and of the 2,3-unsaturated acyl derivatives (Figure 21–3, reactions 3and 5). The oxidative reactions of the pentose phosphatepathway (see Chapter 20) are the chief source ofthe hydrogen required for the reductive synthesis offatty acids. Significantly, tissues specializing in activelipogenesis—ie, liver, adipose tissue, and the lactatingmammary gland—also possess an active pentose phosphatepathway. Moreover, both metabolic pathways arefound in the cytosol of the cell, so there are no membranesor permeability barriers against the transfer ofNADPH. Other sources of NADPH include the reactionthat converts malate to pyruvate catalyzed by the“malic enzyme” (NADP malate dehydrogenase) (Figure21–4) and the extramitochondrial isocitrate dehydrogenasereaction (probably not a substantial source,except in ruminants).Acetyl-CoA Is the Principal BuildingBlock of Fatty AcidsAcetyl-CoA is formed from glucose via the oxidation ofpyruvate within the mitochondria. However, it doesnot diffuse readily into the extramitochondrial cytosol,GlucosePalmitateGlucose 6-phosphateNADP +H +NADH + H +Citrate Citrate IsocitrateNAD + MalateCOGLYCERALDEHYDE-2ATP ACETYL-3-PHOSPHATECoADEHYDROGENASENADH + H + CARBOXY-OxaloacetateCO 2 LASEPPPNADP +Fructose 6-phosphateOxaloacetateCitrateNADPHMalic + +HNADPH + H+enzymeGlyceraldehydeMALATEDEHYDROGENASE3-phosphateMalonyl-CoAPyruvateAcetyl-CoACYTOSOLCoAATP-CITRATE CoA ATP AcetateLYASE ATPISOCITRATEOutsideDEHYDROGENASETP INNER MITOCHONDRIAL MEMBRANE TPYRUVATEInsideDEHYDROGENASEPyruvateAcetyl-CoAMalateMITOCHONDRIONα-KetoglutarateNAD +MalateCitric acid cycleα-KetoglutarateKFigure 21–4. The provision of acetyl-CoA and NADPH for lipogenesis. (PPP, pentose phosphate pathway;T, tricarboxylate transporter; K, α-ketoglutarate transporter; P, pyruvate transporter.)

BIOSYNTHESIS OF FATTY ACIDS / 177the principal site of fatty acid synthesis. Citrate, formedafter condensation of acetyl-CoA with oxaloacetate inthe citric acid cycle within mitochondria, is translocatedinto the extramitochondrial compartment via thetricarboxylate transporter, where in the presence ofCoA and ATP it undergoes cleavage to acetyl-CoA andoxaloacetate catalyzed by ATP-citrate lyase, which increasesin activity in the well-fed state. The acetyl-CoAis then available for malonyl-CoA formation and synthesisto palmitate (Figure 21–4). The resulting oxaloacetatecan form malate via NADH-linked malatedehydrogenase, followed by the generation of NADPHvia the malic enzyme. The NADPH becomes availablefor lipogenesis, and the pyruvate can be used to regenerateacetyl-CoA after transport into the mitochondrion.This pathway is a means of transferring reducingequivalents from extramitochondrial NADH to NADP.Alternatively, malate itself can be transported into themitochondrion, where it is able to re-form oxaloacetate.Note that the citrate (tricarboxylate) transporter in themitochondrial membrane requires malate to exchangewith citrate (see Figure 12-10). There is little ATPcitratelyase or malic enzyme in ruminants, probablybecause in these species acetate (derived from therumen and activated to acetyl CoA extramitochondrially)is the main source of acetyl-CoA.RCH 2OCSAcyl-CoACoA3-KETOACYL-CoASYNTHASERROOCH 2 C S CoACOOHMalonyl-CoACoA SH + CO 2OCH 2 C CH 2 C S3-Ketoacyl-CoA3-KETOACYL-CoAREDUCTASEOHONADP +CH 2 CH CH 2 C S3-Hydroxyacyl-CoA+CoANADPH + H +CoAElongation of Fatty Acid Chains Occursin the Endoplasmic ReticulumThis pathway (the “microsomal system”) elongates saturatedand unsaturated fatty acyl-CoAs (from C 10 upward)by two carbons, using malonyl-CoA as acetyldonor and NADPH as reductant, and is catalyzed bythe microsomal fatty acid elongase system of enzymes(Figure 21–5). Elongation of stearyl-CoA in brain increasesrapidly during myelination in order to provideC 22 and C 24 fatty acids for sphingolipids.THE NUTRITIONAL STATEREGULATES LIPOGENESISExcess carbohydrate is stored as fat in many animals inanticipation of periods of caloric deficiency such as starvation,hibernation, etc, and to provide energy for usebetween meals in animals, including humans, that taketheir food at spaced intervals. Lipogenesis converts surplusglucose and intermediates such as pyruvate, lactate,and acetyl-CoA to fat, assisting the anabolic phase ofthis feeding cycle. The nutritional state of the organismis the main factor regulating the rate of lipogenesis.Thus, the rate is high in the well-fed animal whose dietcontains a high proportion of carbohydrate. It is depressedunder conditions of restricted caloric intake, on3-HYDROXYACYL-CoADEHYDRASEOR CH 2 CH CH C S2-trans-Enoyl-CoA2-trans-ENOYL-CoAREDUCTASEOR CH 2 CH 2 CH 2 CAcyl-CoAH 2 ONADP +SCoANADPH + H +CoAFigure 21–5. Microsomal elongase system for fattyacid chain elongation. NADH is also used by the reductases,but NADPH is preferred.a fat diet, or when there is a deficiency of insulin, as indiabetes mellitus. These latter conditions are associatedwith increased concentrations of plasma free fatty acids,and an inverse relationship has been demonstrated betweenhepatic lipogenesis and the concentration ofserum-free fatty acids. Lipogenesis is increased when su-

178 / CHAPTER 21crose is fed instead of glucose because fructose bypassesthe phosphofructokinase control point in glycolysis andfloods the lipogenic pathway (Figure 20–5).P iPROTEINPHOSPHATASEH 2 OSHORT- & LONG-TERM MECHANISMSREGULATE LIPOGENESISLong-chain fatty acid synthesis is controlled in theshort term by allosteric and covalent modification ofenzymes and in the long term by changes in gene expressiongoverning rates of synthesis of enzymes.Acetyl-CoAACETYL-CoACARBOXYLASE(active)PACETYL-CoACARBOXYLASE(inactive)Acetyl-CoA Carboxylase Is the MostImportant Enzyme in the Regulationof LipogenesisAcetyl-CoA carboxylase is an allosteric enzyme and isactivated by citrate, which increases in concentration inthe well-fed state and is an indicator of a plentiful supplyof acetyl-CoA. Citrate converts the enzyme from aninactive dimer to an active polymeric form, having amolecular mass of several million. Inactivation is promotedby phosphorylation of the enzyme and by longchainacyl-CoA molecules, an example of negative feedbackinhibition by a product of a reaction. Thus, ifacyl-CoA accumulates because it is not esterifiedquickly enough or because of increased lipolysis or aninflux of free fatty acids into the tissue, it will automaticallyreduce the synthesis of new fatty acid. Acyl-CoAmay also inhibit the mitochondrial tricarboxylatetransporter, thus preventing activation of the enzymeby egress of citrate from the mitochondria into the cytosol.Acetyl-CoA carboxylase is also regulated by hormonessuch as glucagon, epinephrine, and insulin viachanges in its phosphorylation state (details in Figure21–6).Malonyl-CoAAcyl-CoAGlucagonATPH 2 OP icAMP+ +AMPK(active)PAMPK(inactive)+ATPADPAMPKKcAMP-DEPENDENTPROTEIN KINASEFigure 21–6. Regulation of acetyl-CoA carboxylaseby phosphorylation/dephosphorylation. The enzyme isinactivated by phosphorylation by AMP-activated proteinkinase (AMPK), which in turn is phosphorylatedand activated by AMP-activated protein kinase kinase(AMPKK). Glucagon (and epinephrine), after increasingcAMP, activate this latter enzyme via cAMP-dependentprotein kinase. The kinase kinase enzyme is also believedto be activated by acyl-CoA. Insulin activatesacetyl-CoA carboxylase, probably through an “activator”protein and an insulin-stimulated protein kinase.+Pyruvate Dehydrogenase Is AlsoRegulated by Acyl-CoAAcyl-CoA causes an inhibition of pyruvate dehydrogenaseby inhibiting the ATP-ADP exchange transporter ofthe inner mitochondrial membrane, which leads to increasedintramitochondrial [ATP]/[ADP] ratios andtherefore to conversion of active to inactive pyruvatedehydrogenase (see Figure 17–6), thus regulating theavailability of acetyl-CoA for lipogenesis. Furthermore,oxidation of acyl-CoA due to increased levels of freefatty acids may increase the ratios of [acetyl-CoA]/[CoA] and [NADH]/[NAD + ] in mitochondria, inhibitingpyruvate dehydrogenase.Insulin Also Regulates Lipogenesisby Other MechanismsInsulin stimulates lipogenesis by several other mechanismsas well as by increasing acetyl-CoA carboxylaseactivity. It increases the transport of glucose into thecell (eg, in adipose tissue), increasing the availability ofboth pyruvate for fatty acid synthesis and glycerol3-phosphate for esterification of the newly formed fattyacids, and also converts the inactive form of pyruvatedehydrogenase to the active form in adipose tissue butnot in liver. Insulin also—by its ability to depress thelevel of intracellular cAMP—inhibits lipolysis in adiposetissue and thereby reduces the concentration of

BIOSYNTHESIS OF FATTY ACIDS / 179plasma free fatty acids and therefore long-chain acyl-CoA, an inhibitor of lipogenesis.The Fatty Acid Synthase Complex& Acetyl-CoA Carboxylase AreAdaptive EnzymesThese enzymes adapt to the body’s physiologic needsby increasing in total amount in the fed state and bydecreasing in starvation, feeding of fat, and in diabetes.Insulin is an important hormone causing gene expressionand induction of enzyme biosynthesis, andglucagon (via cAMP) antagonizes this effect. Feedingfats containing polyunsaturated fatty acids coordinatelyregulates the inhibition of expression of key enzymes ofglycolysis and lipogenesis. These mechanisms forlonger-term regulation of lipogenesis take several daysto become fully manifested and augment the direct andimmediate effect of free fatty acids and hormones suchas insulin and glucagon.SUMMARY• The synthesis of long-chain fatty acids (lipogenesis) iscarried out by two enzyme systems: acetyl-CoA carboxylaseand fatty acid synthase.• The pathway converts acetyl-CoA to palmitate andrequires NADPH, ATP, Mn 2+ , biotin, pantothenicacid, and HCO − 3 as cofactors.• Acetyl-CoA carboxylase is required to convert acetyl-CoA to malonyl-CoA. In turn, fatty acid synthase, amultienzyme complex of one polypeptide chain withseven separate enzymatic activities, catalyzes the assemblyof palmitate from one acetyl-CoA and sevenmalonyl-CoA molecules.• Lipogenesis is regulated at the acetyl-CoA carboxylasestep by allosteric modifiers, phosphorylation/dephosphorylation,and induction and repression of enzymesynthesis. Citrate activates the enzyme, andlong-chain acyl-CoA inhibits its activity. Insulin activatesacetyl-CoA carboxylase whereas glucagon andepinephrine have opposite actions.REFERENCESHudgins LC et al: Human fatty acid synthesis is stimulated by aeucaloric low fat, high carbohydrate diet. J Clin Invest1996;97:2081.Jump DB et al: Coordinate regulation of glycolytic and lipogenicgene expression by polyunsaturated fatty acids. J Lipid Res1994;35:1076.Kim KH: Regulation of mammalian acetyl-coenzyme A carboxylase.Annu Rev Nutr 1997;17:77.Salati LM, Goodridge AG: Fatty acid synthesis in eukaryotes. In:Biochemistry of Lipids, Lipoproteins and Membranes. VanceDE, Vance JE (editors). Elsevier, 1996.Wakil SJ: Fatty acid synthase, a proficient multifunctional enzyme.Biochemistry 1989;28:4523.

Oxidation of Fatty Acids:Ketogenesis 22Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEAlthough fatty acids are both oxidized to acetyl-CoAand synthesized from acetyl-CoA, fatty acid oxidation isnot the simple reverse of fatty acid biosynthesis but anentirely different process taking place in a separate compartmentof the cell. The separation of fatty acid oxidationin mitochondria from biosynthesis in the cytosolallows each process to be individually controlled andintegrated with tissue requirements. Each step in fattyacid oxidation involves acyl-CoA derivatives catalyzedby separate enzymes, utilizes NAD + and FAD as coenzymes,and generates ATP. It is an aerobic process, requiringthe presence of oxygen.Increased fatty acid oxidation is a characteristic ofstarvation and of diabetes mellitus, leading to ketonebody production by the liver (ketosis). Ketone bodiesare acidic and when produced in excess over long periods,as in diabetes, cause ketoacidosis, which is ultimatelyfatal. Because gluconeogenesis is dependentupon fatty acid oxidation, any impairment in fatty acidoxidation leads to hypoglycemia. This occurs in variousstates of carnitine deficiency or deficiency of essentialenzymes in fatty acid oxidation, eg, carnitinepalmitoyltransferase, or inhibition of fatty acid oxidationby poisons, eg, hypoglycin.OXIDATION OF FATTY ACIDS OCCURSIN MITOCHONDRIAFatty Acids Are Transported in theBlood as Free Fatty Acids (FFA)Free fatty acids—also called unesterified (UFA) or nonesterified(NEFA) fatty acids—are fatty acids that are inthe unesterified state. In plasma, longer-chain FFA arecombined with albumin, and in the cell they are attachedto a fatty acid-binding protein, so that in factthey are never really “free.” Shorter-chain fatty acids are180more water-soluble and exist as the un-ionized acid oras a fatty acid anion.Fatty Acids Are Activated BeforeBeing CatabolizedFatty acids must first be converted to an active intermediatebefore they can be catabolized. This is the onlystep in the complete degradation of a fatty acid that requiresenergy from ATP. In the presence of ATP andcoenzyme A, the enzyme acyl-CoA synthetase (thiokinase)catalyzes the conversion of a fatty acid (or freefatty acid) to an “active fatty acid” or acyl-CoA, whichuses one high-energy phosphate with the formation ofAMP and PP i (Figure 22–1). The PP i is hydrolyzed byinorganic pyrophosphatase with the loss of a furtherhigh-energy phosphate, ensuring that the overall reactiongoes to completion. Acyl-CoA synthetases arefound in the endoplasmic reticulum, peroxisomes, andinside and on the outer membrane of mitochondria.Long-Chain Fatty Acids Penetrate theInner Mitochondrial Membrane asCarnitine DerivativesCarnitine (β-hydroxy-γ-trimethylammonium butyrate),(CH 3 ) 3 N + ⎯CH 2 ⎯CH(OH)⎯CH 2 ⎯COO − , iswidely distributed and is particularly abundant in muscle.Long-chain acyl-CoA (or FFA) will not penetratethe inner membrane of mitochondria. However, carnitinepalmitoyltransferase-I, present in the outermitochondrial membrane, converts long-chain acyl-CoA to acylcarnitine, which is able to penetrate theinner membrane and gain access to the β-oxidationsystem of enzymes (Figure 22–1). Carnitine-acylcarnitinetranslocase acts as an inner membrane exchangetransporter. Acylcarnitine is transported in,coupled with the transport out of one molecule of carnitine.The acylcarnitine then reacts with CoA, cat-

OXIDATION OF FATTY ACIDS: KETOGENESIS / 181ATP+CoAFFAAMP + PPiAcyl-CoAH 3 CPalmitoyl-CoACoA SHαβ CO S CoAAcyl-CoASYNTHETASEAcyl-CoACarnitineCARNITINEPALMITOYL- OUTERTRANSFERASE MITOCHONDRIALIMEMBRANECoAAcylcarnitineH 3 Cαβ COCH 3S+COAcetyl-CoASuccessive removal of acetyl-CoA (C 2 ) unitsCoAS CoACARNITINEPALMITOYL-TRANSFERASEIICARNITINE INNERACYLCAR-MITOCHONDRIALNITINETRANSLOCASEMEMBRANE8 CH 3 CO S CoAAcetyl-CoAFigure 22–2. Overview of β-oxidation of fatty acids.CoAAcylcarnitineCarnitineAcyl-CoAβ-OxidationAcylcarnitineFigure 22–1. Role of carnitine in the transport oflong-chain fatty acids through the inner mitochondrialmembrane. Long-chain acyl-CoA cannot pass throughthe inner mitochondrial membrane, but its metabolicproduct, acylcarnitine, can.alyzed by carnitine palmitoyltransferase-II, locatedon the inside of the inner membrane. Acyl-CoA is reformedin the mitochondrial matrix, and carnitine isliberated.-OXIDATION OF FATTY ACIDSINVOLVES SUCCESSIVE CLEAVAGEWITH RELEASE OF ACETYL-CoAIn β-oxidation (Figure 22–2), two carbons at a time arecleaved from acyl-CoA molecules, starting at the carboxylend. The chain is broken between the α(2)- andβ(3)-carbon atoms—hence the name β-oxidation. Thetwo-carbon units formed are acetyl-CoA; thus, palmitoyl-CoAforms eight acetyl-CoA molecules.The Cyclic Reaction Sequence GeneratesFADH 2 & NADHSeveral enzymes, known collectively as “fatty acid oxidase,”are found in the mitochondrial matrix or innermembrane adjacent to the respiratory chain. These catalyzethe oxidation of acyl-CoA to acetyl-CoA, the systembeing coupled with the phosphorylation of ADP toATP (Figure 22–3).The first step is the removal of two hydrogen atomsfrom the 2(α)- and 3(β)-carbon atoms, catalyzed byacyl-CoA dehydrogenase and requiring FAD. This resultsin the formation of ∆ 2 -trans-enoyl-CoA andFADH 2 . The reoxidation of FADH 2 by the respiratorychain requires the mediation of another flavoprotein,termed electron-transferring flavoprotein (Chapter11). Water is added to saturate the double bond andform 3-hydroxyacyl-CoA, catalyzed by 2 -enoyl-CoAhydratase. The 3-hydroxy derivative undergoes furtherdehydrogenation on the 3-carbon catalyzed by L(+)-3-hydroxyacyl-CoA dehydrogenase to form the corresponding3-ketoacyl-CoA compound. In this case,NAD + is the coenzyme involved. Finally, 3-ketoacyl-CoA is split at the 2,3- position by thiolase (3-ketoacyl-CoA-thiolase),forming acetyl-CoA and a new acyl-CoA two carbons shorter than the original acyl-CoAmolecule. The acyl-CoA formed in the cleavage reactionreenters the oxidative pathway at reaction 2 (Figure22–3). In this way, a long-chain fatty acid may bedegraded completely to acetyl-CoA (C 2 units). Sinceacetyl-CoA can be oxidized to CO 2 and water via the

182 / CHAPTER 221CoAO3 2R CH2 CH2 CSHACYL-CoASYNTHETASEFatty acidMg 2+O3 2R CH2 CH2 CAcyl-CoAATPAMP + PPiSO – CoAFigure 22–3. β-Oxidation of fatty acids. Long-chainacyl-CoA is cycled through reactions 2–5, acetyl-CoAbeing split off, each cycle, by thiolase (reaction 5).When the acyl radical is only four carbon atoms inlength, two acetyl-CoA molecules are formed in reaction5.citric acid cycle (which is also found within the mitochondria),the complete oxidation of fatty acids isachieved.INNER MITOCHONDRIAL MEMBRANE2345O3 2R CH2 CH2 CACYL-CoADEHYDROGENASEAcyl-CoASFADCoA(outside)C sideM side(inside)FADH 2H 2 OORespiratorychain3 2 R CH CH C S CoA∆ 2 -trans-Enoyl-CoA∆ 2 -ENOYL-CoAHYDRATASEOHO3 2 R CH CH2 C SL(+)-3-Hydroxyacyl-CoAL(+)-3-HYDROXYACYL-CoA DEHYDROGENASEROCAcyl-CoAOTHIOLASECO3 2 R C CH2 CS CoA + CH 3CARNITINE TRANSPORTERH 2 ONAD +CoANADH + H +S3-Ketoacyl-CoACoAOCAcetyl-CoACitricacidcycleCoASSH2RespiratorychainCoAP3 PH 2 OOxidation of a Fatty Acid With an OddNumber of Carbon Atoms Yields Acetyl-CoA Plus a Molecule of Propionyl-CoAFatty acids with an odd number of carbon atoms are oxidizedby the pathway of β-oxidation, producing acetyl-CoA, until a three-carbon (propionyl-CoA) residue remains.This compound is converted to succinyl-CoA, aconstituent of the citric acid cycle (Figure 19–2). Hence,the propionyl residue from an odd-chain fatty acid isthe only part of a fatty acid that is glucogenic.Oxidation of Fatty Acids Producesa Large Quantity of ATPTransport in the respiratory chain of electrons fromFADH 2 and NADH will lead to the synthesis of fivehigh-energy phosphates (Chapter 12) for each of thefirst seven acetyl-CoA molecules formed by β-oxidationof palmitate (7 × 5 = 35). A total of 8 mol of acetyl-CoA is formed, and each will give rise to 12 mol ofATP on oxidation in the citric acid cycle, making 8 ×12 = 96 mol. Two must be subtracted for the initial activationof the fatty acid, yielding a net gain of 129 molof ATP per mole of palmitate, or 129 × 51.6* = 6656kJ. This represents 68% of the free energy of combustionof palmitic acid.Peroxisomes Oxidize Very LongChain Fatty AcidsA modified form of β-oxidation is found in peroxisomesand leads to the formation of acetyl-CoA andH 2 O 2 (from the flavoprotein-linked dehydrogenasestep), which is broken down by catalase. Thus, this dehydrogenationin peroxisomes is not linked directly tophosphorylation and the generation of ATP. The systemfacilitates the oxidation of very long chain fattyacids (eg, C 20 , C 22 ). These enzymes are induced by2CO 2* ∆G for the ATP reaction, as explained in Chapter 17.

OXIDATION OF FATTY ACIDS: KETOGENESIS / 183Figure 22–4. Sequence of reactions in the oxidationof unsaturated fatty acids, eg, linoleic acid. ∆ 4 -cis-fattyacids or fatty acids forming ∆ 4 -cis-enoyl-CoA enter thepathway at the position shown. NADPH for the dienoyl-CoA reductase step is supplied by intramitochondrialsources such as glutamate dehydrogenase, isocitratedehydrogenase, and NAD(P)H transhydrogenase.high-fat diets and in some species by hypolipidemicdrugs such as clofibrate.The enzymes in peroxisomes do not attack shorterchainfatty acids; the β-oxidation sequence ends at octanoyl-CoA.Octanoyl and acetyl groups are both furtheroxidized in mitochondria. Another role of peroxisomalβ-oxidation is to shorten the side chain of cholesterol inbile acid formation (Chapter 26). Peroxisomes also takepart in the synthesis of ether glycerolipids (Chapter 24),cholesterol, and dolichol (Figure 26–2).OXIDATION OF UNSATURATED FATTYACIDS OCCURS BY A MODIFIED-OXIDATION PATHWAYThe CoA esters of these acids are degraded by the enzymesnormally responsible for β-oxidation until eithera ∆ 3 -cis-acyl-CoA compound or a ∆ 4 -cis-acyl-CoA compoundis formed, depending upon the position of thedouble bonds (Figure 22–4). The former compound isisomerized ( 3 cis v 2 -trans-enoyl-CoA isomerase)to the corresponding ∆ 2 -trans-CoA stage of β-oxidationfor subsequent hydration and oxidation. Any ∆ 4 -cis-acyl-CoA either remaining, as in the case of linoleic acid, orentering the pathway at this point after conversion byacyl-CoA dehydrogenase to ∆ 2 -trans-∆ 4 -cis-dienoyl-CoA, is then metabolized as indicated in Figure 22–4.KETOGENESIS OCCURS WHEN THEREIS A HIGH RATE OF FATTY ACIDOXIDATION IN THE LIVERUnder metabolic conditions associated with a high rateof fatty acid oxidation, the liver produces considerablequantities of acetoacetate and D()-3-hydroxybutyrate(β-hydroxybutyrate). Acetoacetate continually undergoesspontaneous decarboxylation to yield acetone.These three substances are collectively known as the ketonebodies (also called acetone bodies or [incorrectly*]“ketones”) (Figure 22–5). Acetoacetate and 3-hydroxybu-* The term “ketones” should not be used because 3-hydroxybutyrateis not a ketone and there are ketones in blood that are notketone bodies, eg, pyruvate, fructose.cis 12cis3 Cycles ofβ-oxidationH + + NADPHNADP +Linoleyl-CoAcis 6ciscis6 2CtransOO9 C S CoASOCoA3 Acetyl-CoA3 C S CoA∆ 3 -cis-∆ 6 -cis-Dienoyl-CoAOC∆ 3 -cis (or trans) → ∆ 2 -trans-ENOYL-CoAISOMERASESCoA∆ 2 -trans-∆ 6 -cis-Dienoyl-CoA(∆ 2 -trans-Enoyl-CoA stage of β-oxidation)1 Cycle ofβ-oxidationcis4 2trans∆ 2 -trans-∆ 4 -cis-Dienoyl-CoAtrans3CS∆ 3 -trans-Enoyl-CoAtransC2OOS∆ 2 -trans-Enoyl-CoA4 Cycles ofβ-oxidation5 Acetyl-CoACoAACYL-CoADEHYDROGENASE∆ 4 -cis-Enoyl-CoA∆ 2 -trans-∆ 4 -cis-DIENOYL-CoAREDUCTASE∆ 3 -cis (or trans) → ∆ 2 -trans-ENOYL-CoAISOMERASECoAAcetyl-CoA

184 / CHAPTER 22CO 2OCH 3 C CH 2 COO –AcetoacetateNADH + H +NAD +SpontaneousOCH 3 C CH 3AcetoneD(–)-3-HYDROXYBUTYRATEDEHYDROGENASEOHCH 3 CH CH 2COO –D(–)-3-HydroxybutyrateFigure 22–5. Interrelationships of the ketone bodies.D(−)-3-hydroxybutyrate dehydrogenase is a mitochondrialenzyme.tyrate are interconverted by the mitochondrial enzymeD()-3-hydroxybutyrate dehydrogenase; the equilibriumis controlled by the mitochondrial [NAD + ]/[NADH] ratio, ie, the redox state. The concentrationof total ketone bodies in the blood of well-fed mammalsdoes not normally exceed 0.2 mmol/L except inruminants, where 3-hydroxybutyrate is formed continuouslyfrom butyric acid (a product of ruminal fermentation)in the rumen wall. In vivo, the liver appears tobe the only organ in nonruminants to add significantquantities of ketone bodies to the blood. Extrahepatictissues utilize them as respiratory substrates. The netflow of ketone bodies from the liver to the extrahepatictissues results from active hepatic synthesis coupledwith very low utilization. The reverse situation occursin extrahepatic tissues (Figure 22–6).3-Hydroxy-3-Methylglutaryl-CoA(HMG-CoA) Is an Intermediatein the Pathway of KetogenesisEnzymes responsible for ketone body formation are associatedmainly with the mitochondria. Two acetyl-CoA molecules formed in β-oxidation condense withone another to form acetoacetyl-CoA by a reversal ofthe thiolase reaction. Acetoacetyl-CoA, which is theLIVERBLOODEXTRAHEPATICTISSUESAcyl-CoAFFAGlucoseGlucoseAcyl-CoAAcetyl-CoAURINEAcetyl-CoACitricacidcycleKetonebodiesKetone bodiesAcetoneLUNGSKetonebodiesCitricacidcycle2CO 22CO 2Figure 22–6. Formation, utilization, and excretion of ketone bodies. (The mainpathway is indicated by the solid arrows.)

OXIDATION OF FATTY ACIDS: KETOGENESIS / 185starting material for ketogenesis, also arises directlyfrom the terminal four carbons of a fatty acid duringβ-oxidation (Figure 22–7). Condensation of acetoacetyl-CoAwith another molecule of acetyl-CoAby 3-hydroxy-3-methylglutaryl-CoA synthase formsHMG-CoA. 3-Hydroxy-3-methylglutaryl-CoA lyasethen causes acetyl-CoA to split off from the HMG-CoA, leaving free acetoacetate. The carbon atoms splitoff in the acetyl-CoA molecule are derived from theoriginal acetoacetyl-CoA molecule. Both enzymesmust be present in mitochondria for ketogenesis totake place. This occurs solely in liver and rumen epithelium.D(−)-3-Hydroxybutyrate is quantitatively thepredominant ketone body present in the blood andurine in ketosis.Ketone Bodies Serve as a Fuelfor Extrahepatic TissuesWhile an active enzymatic mechanism produces acetoacetatefrom acetoacetyl-CoA in the liver, acetoacetateonce formed cannot be reactivated directly exceptin the cytosol, where it is used in a much less activepathway as a precursor in cholesterol synthesis. This accountsfor the net production of ketone bodies by theliver.ATPCoAFFAACYL-CoASYNTHETASEAcyl-CoAEsterificationTriacylglycerolPhospholipid(Acetyl-CoA) nβ-OxidationOOCH 3C CH 2 C SAcetoacetyl-CoACoAHMG-CoASYNTHASECoA SHTHIOLASEOH OH 2 OCH 3 C CH 2 C S CoAO* CH2 * COO–* CH3 * C S CoACoA SH3-Hydroxy-3-methylglutaryl-CoA(HMG-CoA)Acetyl-CoAHMG-CoACH 3 CO S CoALYASEAcetyl-CoACitricOacidcycleCH 3 C* CH2 * COO–Acetoacetate2CO 2NADH + H +D(–)-3-HYDROXYBUTYRATEDEHYDROGENASENAD +OHCH 3* CH2 * COO–CHD(–)-3-HydroxybutyrateFigure 22–7. Pathways of ketogenesis in the liver. (FFA, free fatty acids; HMG, 3-hydroxy-3-methylglutaryl.)

186 / CHAPTER 22In extrahepatic tissues, acetoacetate is activated toacetoacetyl-CoA by succinyl-CoA-acetoacetate CoAtransferase. CoA is transferred from succinyl-CoA toform acetoacetyl-CoA (Figure 22–8). The acetoacetyl-CoA is split to acetyl-CoA by thiolase and oxidized inthe citric acid cycle. If the blood level is raised, oxidationof ketone bodies increases until, at a concentrationof approximately 12 mmol/L, they saturate the oxidativemachinery. When this occurs, a large proportion ofthe oxygen consumption may be accounted for by theoxidation of ketone bodies.In most cases, ketonemia is due to increased productionof ketone bodies by the liver rather than to adeficiency in their utilization by extrahepatic tissues.While acetoacetate and D(−)-3-hydroxybutyrate arereadily oxidized by extrahepatic tissues, acetone is difficultto oxidize in vivo and to a large extent is volatilizedin the lungs.In moderate ketonemia, the loss of ketone bodies viathe urine is only a few percent of the total ketone bodyproduction and utilization. Since there are renal threshold-likeeffects (there is not a true threshold) that varybetween species and individuals, measurement of theketonemia, not the ketonuria, is the preferred methodof assessing the severity of ketosis.KETOGENESIS IS REGULATEDAT THREE CRUCIAL STEPS(1) Ketosis does not occur in vivo unless there is anincrease in the level of circulating free fatty acids thatarise from lipolysis of triacylglycerol in adipose tissue.Free fatty acids are the precursors of ketone bodiesin the liver. The liver, both in fed and in fasting conditions,extracts about 30% of the free fatty acids passingthrough it, so that at high concentrations the flux passinginto the liver is substantial. Therefore, the factorsregulating mobilization of free fatty acids from adiposetissue are important in controlling ketogenesis(Figures 22–9 and 25–8).(2) After uptake by the liver, free fatty acids are either-oxidized to CO 2 or ketone bodies or esterifiedto triacylglycerol and phospholipid. There is regulationof entry of fatty acids into the oxidative pathway by carnitinepalmitoyltransferase-I (CPT-I), and the remainderof the fatty acid uptake is esterified. CPT-I activity isEXTRAHEPATIC TISSUESeg, MUSCLEFFAAcyl-CoAβ-OxidationAcetyl-CoAHMG-CoAAcetoacetateNADH + H +3-HydroxybutyrateNAD +LIVERAcetyl-CoATHIOLASEAcetoacetyl-CoASuccinateCoATRANSFERASECitric acid cycleSuccinyl- CitrateCoAAcetoacetate2CO 2NADH + H +NAD +3-HydroxybutyrateOAATransport of ketone bodies from the liver and pathways of utilization and oxidation in extrahe-Figure 22–8.patic tissues.

OXIDATION OF FATTY ACIDS: KETOGENESIS / 187ADIPOSE TISSUEBLOODLIVERCPT-Igatewayβ-OxidationKetogenesisTriacylglycerolFFAFFA1Acyl-CoA2Acetyl-CoA3Ketone bodiesLipolysisEsterificationAcylglycerolsCitric acidcycleCO 2Figure 22–9. Regulation of ketogenesis. 1 –3 showthree crucial steps in the pathway of metabolism of freefatty acids (FFA) that determine the magnitude of ketogenesis.(CPT-I, carnitine palmitoyltransferase-I.)low in the fed state, leading to depression of fatty acidoxidation, and high in starvation, allowing fatty acid oxidationto increase. Malonyl-CoA, the initial intermediatein fatty acid biosynthesis (Figure 21–1), formed byacetyl-CoA carboxylase in the fed state, is a potent inhibitorof CPT-I (Figure 22–10). Under these conditions,free fatty acids enter the liver cell in low concentrationsand are nearly all esterified to acylglycerols andtransported out of the liver in very low density lipoproteins(VLDL). However, as the concentration of freefatty acids increases with the onset of starvation, acetyl-CoA carboxylase is inhibited directly by acyl-CoA, and[malonyl-CoA] decreases, releasing the inhibition ofCPT-I and allowing more acyl-CoA to be β-oxidized.These events are reinforced in starvation by decrease inthe [insulin]/[glucagon] ratio. Thus, β-oxidation fromfree fatty acids is controlled by the CPT-I gateway intothe mitochondria, and the balance of the free fatty aciduptake not oxidized is esterified.(3) In turn, the acetyl-CoA formed in β-oxidation isoxidized in the citric acid cycle, or it enters the pathwayof ketogenesis to form ketone bodies. As the level ofserum free fatty acids is raised, proportionately morefree fatty acid is converted to ketone bodies and less isoxidized via the citric acid cycle to CO 2 . The partitionof acetyl-CoA between the ketogenic pathway and thepathway of oxidation to CO 2 is so regulated that thetotal free energy captured in ATP which results fromthe oxidation of free fatty acids remains constant. Thismay be appreciated when it is realized that completeoxidation of 1 mol of palmitate involves a net productionof 129 mol of ATP via β-oxidation and CO 2 productionin the citric acid cycle (see above), whereas only33 mol of ATP are produced when acetoacetate is theend product and only 21 mol when 3-hydroxybutyrateis the end product. Thus, ketogenesis may be regardedas a mechanism that allows the liver to oxidize increasingquantities of fatty acids within the constraints of atightly coupled system of oxidative phosphorylation—without increasing its total energy expenditure.Theoretically, a fall in concentration of oxaloacetate,particularly within the mitochondria, could impair theability of the citric acid cycle to metabolize acetyl-CoAand divert fatty acid oxidation toward ketogenesis.Such a fall may occur because of an increase in the[NADH]/[NAD + ] ratio caused by increased β-oxidationaffecting the equilibrium between oxaloacetate andmalate and decreasing the concentration of oxaloacetate.However, pyruvate carboxylase, which catalyzesthe conversion of pyruvate to oxaloacetate, is activatedby acetyl-CoA. Consequently, when there are significantamounts of acetyl-CoA, there should be sufficientoxaloacetate to initiate the condensing reaction of thecitric acid cycle.CLINICAL ASPECTSImpaired Oxidation of Fatty AcidsGives Rise to Diseases OftenAssociated With HypoglycemiaCarnitine deficiency can occur particularly in the newborn—andespecially in preterm infants—owing to inadequatebiosynthesis or renal leakage. Losses can alsooccur in hemodialysis. This suggests a vitamin-like dietaryrequirement for carnitine in some individuals.Symptoms of deficiency include hypoglycemia, whichis a consequence of impaired fatty acid oxidation andlipid accumulation with muscular weakness. Treatmentis by oral supplementation with carnitine.Inherited CPT-I deficiency affects only the liver,resulting in reduced fatty acid oxidation and ketogenesis,with hypoglycemia. CPT-II deficiency affects pri-

188 / CHAPTER 22GlucoseBLOODFFAVLDLAcetyl-CoALIVERAcylglycerolsLipogenesisInsulin+ −ACETYL-CoACARBOXYLASE−GlucagonAcyl-CoAEsterificationCytosolMalonyl-CoAPalmitate−MitochondrionKetogenesisKetone bodiesCARNITINEPALMITOYL-TRANSFERASE IAcyl-CoAAcetyl-CoAβ-OxidationCO2MitochondrialmembraneFigure 22–10. Regulation oflong-chain fatty acid oxidation in theliver. (FFA, free fatty acids; VLDL,very low density lipoprotein.) Positive(+ ) and negative (− ) regulatoryeffects are represented by brokenarrows and substrate flow by solidarrows.marily skeletal muscle and, when severe, the liver. Thesulfonylurea drugs (glyburide [glibenclamide] andtolbutamide), used in the treatment of type 2 diabetesmellitus, reduce fatty acid oxidation and, therefore, hyperglycemiaby inhibiting CPT-I.Inherited defects in the enzymes of β-oxidation andketogenesis also lead to nonketotic hypoglycemia, coma,and fatty liver. Defects are known in long- and shortchain3-hydroxyacyl-CoA dehydrogenase (deficiency ofthe long-chain enzyme may be a cause of acute fattyliver of pregnancy). 3-Ketoacyl-CoA thiolase andHMG-CoA lyase deficiency also affect the degradationof leucine, a ketogenic amino acid (Chapter 30).Jamaican vomiting sickness is caused by eating theunripe fruit of the akee tree, which contains a toxin,hypoglycin, that inactivates medium- and short-chainacyl-CoA dehydrogenase, inhibiting β-oxidation andcausing hypoglycemia. Dicarboxylic aciduria is characterizedby the excretion of C 6 –C 10 ω-dicarboxylicacids and by nonketotic hypoglycemia. It is caused by alack of mitochondrial medium-chain acyl-CoA dehydrogenase.Refsum’s disease is a rare neurologic disorderdue to a defect that causes the accumulation of phytanicacid, which is found in plant foodstuffs andblocks β-oxidation. Zellweger’s (cerebrohepatorenal)syndrome occurs in individuals with a rare inheritedabsence of peroxisomes in all tissues. They accumulateC 26 –C 38 polyenoic acids in brain tissue and also exhibita generalized loss of peroxisomal functions, eg, impairedbile acid and ether lipid synthesis.Ketoacidosis Results FromProlonged KetosisHigher than normal quantities of ketone bodies presentin the blood or urine constitute ketonemia (hyperketonemia)or ketonuria, respectively. The overall conditionis called ketosis. Acetoacetic and 3-hydroxybutyricacids are both moderately strong acids and are bufferedwhen present in blood or other tissues. However, theircontinual excretion in quantity progressively depletesthe alkali reserve, causing ketoacidosis. This may befatal in uncontrolled diabetes mellitus.The basic form of ketosis occurs in starvation andinvolves depletion of available carbohydrate coupledwith mobilization of free fatty acids. This general patternof metabolism is exaggerated to produce the pathologicstates found in diabetes mellitus, twin lamb disease,and ketosis in lactating cattle. Nonpathologicforms of ketosis are found under conditions of high-fat

OXIDATION OF FATTY ACIDS: KETOGENESIS / 189feeding and after severe exercise in the postabsorptivestate.SUMMARY• Fatty acid oxidation in mitochondria leads to the generationof large quantities of ATP by a process calledβ-oxidation that cleaves acetyl-CoA units sequentiallyfrom fatty acyl chains. The acetyl-CoA is oxidized inthe citric acid cycle, generating further ATP.• The ketone bodies (acetoacetate, 3-hydroxybutyrate,and acetone) are formed in hepatic mitochondriawhen there is a high rate of fatty acid oxidation. Thepathway of ketogenesis involves synthesis and breakdownof 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by two key enzymes, HMG-CoA synthase andHMG-CoA lyase.• Ketone bodies are important fuels in extrahepatic tissues.• Ketogenesis is regulated at three crucial steps: (1)control of free fatty acid mobilization from adiposetissue; (2) the activity of carnitine palmitoyltransferase-Iin liver, which determines the proportion ofthe fatty acid flux that is oxidized rather than esterified;and (3) partition of acetyl-CoA between thepathway of ketogenesis and the citric acid cycle.• Diseases associated with impairment of fatty acid oxidationlead to hypoglycemia, fatty infiltration of organs,and hypoketonemia.• Ketosis is mild in starvation but severe in diabetesmellitus and ruminant ketosis.REFERENCESEaton S, Bartlett K, Pourfarzam M: Mammalian mitochondrial β-oxidation. Biochem J 1996;320:345.Mayes PA, Laker ME: Regulation of ketogenesis in the liver.Biochem Soc Trans 1981;9:339.McGarry JD, Foster DW: Regulation of hepatic fatty acid oxidationand ketone body production. Annu Rev Biochem1980;49:395.Osmundsen H, Hovik R: β-Oxidation of polyunsaturated fattyacids. Biochem Soc Trans 1988;16:420.Reddy JK, Mannaerts GP: Peroxisomal lipid metabolism. AnnuRev Nutr 1994;14:343.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Treem WR et al: Acute fatty liver of pregnancy and long-chain 3-hydroxyacyl-coenzyme A dehydrogenase deficiency. Hepatology1994;19:339.Wood PA: Defects in mitochondrial beta-oxidation of fatty acids.Curr Opin Lipidol 1999;10:107.

Metabolism of Unsaturated FattyAcids & Eicosanoids 23Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEUnsaturated fatty acids in phospholipids of the cellmembrane are important in maintaining membranefluidity. A high ratio of polyunsaturated fatty acids tosaturated fatty acids (P:S ratio) in the diet is a majorfactor in lowering plasma cholesterol concentrationsand is considered to be beneficial in preventing coronaryheart disease. Animal tissues have limited capacityfor desaturating fatty acids, and that process requirescertain dietary polyunsaturated fatty acids derivedfrom plants. These essential fatty acids are used toform eicosanoic (C 20 ) fatty acids, which in turn giverise to the prostaglandins and thromboxanes and toleukotrienes and lipoxins—known collectively aseicosanoids. The prostaglandins and thromboxanes arelocal hormones that are synthesized rapidly when required.Prostaglandins mediate inflammation, producepain, and induce sleep as well as being involved in theregulation of blood coagulation and reproduction.Nonsteroidal anti-inflammatory drugs such as aspirinact by inhibiting prostaglandin synthesis. Leukotrieneshave muscle contractant and chemotactic propertiesand are important in allergic reactions and inflammation.190SOME POLYUNSATURATED FATTYACIDS CANNOT BE SYNTHESIZEDBY MAMMALS & ARENUTRITIONALLY ESSENTIALCertain long-chain unsaturated fatty acids of metabolicsignificance in mammals are shown in Figure 23–1.Other C 20 , C 22 , and C 24 polyenoic fatty acids may bederived from oleic, linoleic, and α-linolenic acids bychain elongation. Palmitoleic and oleic acids are not essentialin the diet because the tissues can introduce adouble bond at the ∆ 9 position of a saturated fatty acid.Linoleic and -linolenic acids are the only fatty acidsknown to be essential for the complete nutrition ofmany species of animals, including humans, and areknown as the nutritionally essential fatty acids. Inmost mammals, arachidonic acid can be formed fromlinoleic acid (Figure 23–4). Double bonds can be introducedat the ∆ 4 , ∆ 5 , ∆ 6 , and ∆ 9 positions (see Chapter14) in most animals, but never beyond the ∆ 9 position.In contrast, plants are able to synthesize the nutritionallyessential fatty acids by introducing double bonds atthe ∆ 12 and ∆ 15 positions.16 9COOH1820Palmitoleic acid (ω7, 16:1, ∆ 9 )18 9Oleic acid (ω9, 18:1, ∆ 9 )12*Linoleic acid (ω6, 18:2, ∆ 9,12 )14911 8 5COOH*α-Linolenic acid (ω3, 18:3, ∆ 9,12,15 )COOH18 15 12 9 COOH*Arachidonic acid (ω6, 20:4, ∆ 5,8,11,14 )Eicosapentaenoic acid (ω3, 20:5, ∆ 5,8,11,14,17 )COOH20 17 14 11 8 5 COOHFigure 23–1. Structure of some unsaturated fattyacids. Although the carbon atoms in the molecules areconventionally numbered—ie, numbered from the carboxylterminal—the ω numbers (eg, ω7 in palmitoleicacid) are calculated from the reverse end (the methylterminal) of the molecules. The information in parenthesesshows, for instance, that α-linolenic acid containsdouble bonds starting at the third carbon fromthe methyl terminal, has 18 carbons and 3 doublebonds, and has these double bonds at the 9th, 12th,and 15th carbons from the carboxyl terminal. (Asterisks:Classified as “essential fatty acids.”)

METABOLISM OF UNSATURATED FATTY ACIDS & EICOSANOIDS / 191Figure 23–2.Stearoyl∆ 9 DESATURASEOleoylCoACyt b 5O 2 + NADH + H +CoANAD + + 2H 2 OMicrosomal ∆ 9 desaturase.MONOUNSATURATED FATTYACIDS ARE SYNTHESIZED BYA 9 DESATURASE SYSTEMSeveral tissues including the liver are considered to be responsiblefor the formation of nonessential monounsaturatedfatty acids from saturated fatty acids. The first doublebond introduced into a saturated fatty acid is nearlyalways in the ∆ 9 position. An enzyme system— 9 desaturase(Figure 23–2)—in the endoplasmic reticulum willcatalyze the conversion of palmitoyl-CoA or stearoyl-CoAto palmitoleoyl-CoA or oleoyl-CoA, respectively. Oxygenand either NADH or NADPH are necessary for the reaction.The enzymes appear to be similar to a monooxygenasesystem involving cytochrome b 5 (Chapter 11).SYNTHESIS OF POLYUNSATURATEDFATTY ACIDS INVOLVES DESATURASE& ELONGASE ENZYME SYSTEMSAdditional double bonds introduced into existing monounsaturatedfatty acids are always separated from eachother by a methylene group (methylene interrupted) exceptin bacteria. Since animals have a ∆ 9 desaturase, theyare able to synthesize the ω9 (oleic acid) family of unsaturatedfatty acids completely by a combination of chainelongation and desaturation (Figure 23–3). However, asindicated above, linoleic (ω6) or α-linolenic (ω3) acidsrequired for the synthesis of the other members of theω6 or ω3 families must be supplied in the diet.Linoleate may be converted to arachidonate via -linolenate by the pathway shown in Figure 23–4. Thenutritional requirement for arachidonate may thus bedispensed with if there is adequate linoleate in the diet.The desaturation and chain elongation system is greatlydiminished in the starving state, in response to glucagonand epinephrine administration, and in the absence ofinsulin as in type 1 diabetes mellitus.DEFICIENCY SYMPTOMS ARE PRODUCEDWHEN THE ESSENTIAL FATTY ACIDS(EFA) ARE ABSENT FROM THE DIETRats fed a purified nonlipid diet containing vitamins Aand D exhibit a reduced growth rate and reproductivedeficiency which may be cured by the addition oflinoleic, -linolenic, and arachidonic acids to the diet.These fatty acids are found in high concentrations invegetable oils (Table 14–2) and in small amounts in animalcarcasses. These essential fatty acids are required forprostaglandin, thromboxane, leukotriene, and lipoxinformation (see below), and they also have various otherfunctions which are less well defined. Essential fatty acidsare found in the structural lipids of the cell, often in the2 position of phospholipids, and are concerned with thestructural integrity of the mitochondrial membrane.Arachidonic acid is present in membranes and accountsfor 5–15% of the fatty acids in phospholipids.Docosahexaenoic acid (DHA; ω3, 22:6), which is syn-24:1ω9Family1 122:1ω6Familyω3FamilyOleic acid18:1120:1Linoleic acid18:2120:2α-Linolenicacid18:32 1 3 1 418:2 20:2 20:3 22:3 22:4—2 1 3 1 418:3 20:3 20:4 22:4 22:5—Accumulates in essentialfatty acid deficiency2 1 3 1 418:3 20:3 20:4 22:4 22:5Figure 23–3. Biosynthesis of the ω9, ω6, and ω3 families of polyunsaturated fattyacids. Each step is catalyzed by the microsomal chain elongation or desaturase system:1, elongase; 2, ∆ 6 desaturase; 3, ∆ 5 desaturase; 4, ∆ 4 desaturase. (—, Inhibition.)

192 / CHAPTER 2318O12 9C S CoALinoleoyl-CoA (∆ 9,12 -octadecadienoyl-CoA)fatty acids in phospholipids, other complex lipids, andmembranes, particularly with ∆ 5,8,11 -eicosatrienoic acid(ω9 20:3) (Figure 23–3). The triene:tetraene ratio inplasma lipids can be used to diagnose the extent of essentialfatty acid deficiency.182020∆ 6DESATURASE2H 2 O + NAD +12 9 6C S CoAOγ-Linolenoyl-CoA (∆ 6,9,12 -octadecatrienoyl-CoA)C 2(Malonyl-CoA,MICROSOMAL CHAINNADPH) ELONGATION SYSTEM(ELONGASE)14 11 8C S CoAODihomo-γ-linolenoyl-CoA (∆ 8,11,14 -eicosatrienoyl-CoA)O 2 + NADH + H +O 2 + NADH + H + ∆ 52H 2 O + NAD +1411DESATURASEArachidonoyl-CoA (∆ 5,8,11,14 -eicosatetraenoyl-CoA)thesized from α-linolenic acid or obtained directly fromfish oils, is present in high concentrations in retina,cerebral cortex, testis, and sperm. DHA is particularlyneeded for development of the brain and retina and issupplied via the placenta and milk. Patients with retinitispigmentosa are reported to have low blood levels ofDHA. In essential fatty acid deficiency, nonessentialpolyenoic acids of the ω9 family replace the essential85OCSCoAFigure 23–4. Conversion of linoleate to arachidonate.Cats cannot carry out this conversion owing to absenceof ∆ 6 desaturase and must obtain arachidonate intheir diet.Trans Fatty Acids Are Implicatedin Various DisordersSmall amounts of trans-unsaturated fatty acids are foundin ruminant fat (eg, butter fat has 2–7%), where theyarise from the action of microorganisms in the rumen,but the main source in the human diet is from partiallyhydrogenated vegetable oils (eg, margarine). Trans fattyacids compete with essential fatty acids and may exacerbateessential fatty acid deficiency. Moreover, they arestructurally similar to saturated fatty acids (Chapter 14)and have comparable effects in the promotion of hypercholesterolemiaand atherosclerosis (Chapter 26).EICOSANOIDS ARE FORMED FROM C 20POLYUNSATURATED FATTY ACIDSArachidonate and some other C 20 polyunsaturated fattyacids give rise to eicosanoids, physiologically and pharmacologicallyactive compounds known as prostaglandins(PG), thromboxanes (TX), leukotrienes(LT), and lipoxins (LX) (Chapter 14). Physiologically,they are considered to act as local hormones functioningthrough G-protein-linked receptors to elicit theirbiochemical effects.There are three groups of eicosanoids that are synthesizedfrom C 20 eicosanoic acids derived from the essentialfatty acids linoleate and -linolenate, or directlyfrom dietary arachidonate and eicosapentaenoate(Figure 23–5). Arachidonate, usually derived from the2 position of phospholipids in the plasma membrane bythe action of phospholipase A 2 (Figure 24–6)—but alsofrom the diet—is the substrate for the synthesis of thePG 2 , TX 2 series (prostanoids) by the cyclooxygenasepathway, or the LT 4 and LX 4 series by the lipoxygenasepathway, with the two pathways competing forthe arachidonate substrate (Figure 23–5).THE CYCLOOXYGENASEPATHWAY IS RESPONSIBLE FORPROSTANOID SYNTHESISProstanoid synthesis (Figure 23–6) involves the consumptionof two molecules of O 2 catalyzed byprostaglandin H synthase (PGHS), which consists oftwo enzymes, cyclooxygenase and peroxidase. PGHSis present as two isoenzymes, PGHS-1 and PGHS-2.The product, an endoperoxide (PGH), is converted toprostaglandins D, E, and F as well as to a thromboxane

METABOLISM OF UNSATURATED FATTY ACIDS & EICOSANOIDS / 193DietMembrane phospholipidLinoleate–2HPHOSPHOLIPASEA 2+Angiotensin IIBradykininEpinephrineThrombinγ-Linolenate+2C8,11,14-Eicosatrienoate(dihomo γ-linolenate)COOH12GROUP 1ProstanoidsPGE 1PGF 1TXA 1LeukotrienesLTA 3LTC 3LTD 3–2HDiet GROUP 2ProstanoidsPGD 21PGE 2PGFCOOH2PGI 2TXA 25,8,11,14-EicosatetraenoateArachidonate2LeukotrienesLTA 4LTB 4LTC 4LTD 4LTE 4LipoxinsLXA 4LXB 4LXC 4LXD 4LXE 4–2HEicosatetraenoate+2COctadecatetraenoate5,8,11,14,17-EicosapentaenoateCOOH12GROUP 3ProstanoidsPGD 3PGE 3PGF 3PGI 3TXA 3LeukotrienesLTA 5LTB 5LTC 5–2HDietα-LinolenateDietFigure 23–5. The three groups of eicosanoids and their biosynthetic origins. (PG, prostaglandin; PGI, prostacyclin;TX, thromboxane; LT, leukotriene; LX, lipoxin; 1 , cyclooxygenase pathway; 2 , lipoxygenase pathway.)The subscript denotes the total number of double bonds in the molecule and the series to which the compoundbelongs.(TXA 2 ) and prostacyclin (PGI 2 ). Each cell type producesonly one type of prostanoid. Aspirin, a nonsteroidalanti-inflammatory drug (NSAID), inhibits cyclooxygenaseof both PGHS-1 and PGHS-2 byacetylation. Most other NSAIDs, such as indomethacinand ibuprofen, inhibit cyclooxygenases by competingwith arachidonate. Transcription of PGHS-2—but notof PGHS-1—is completely inhibited by anti-inflammatorycorticosteroids.Essential Fatty Acids Do Not ExertAll Their Physiologic Effects ViaProstaglandin SynthesisThe role of essential fatty acids in membrane formationis unrelated to prostaglandin formation. Prostaglandinsdo not relieve symptoms of essential fatty acid deficiency,and an essential fatty acid deficiency is notcaused by inhibition of prostaglandin synthesis.

194 / CHAPTER 23COOHO O Arachidonate2O 2 CYCLOOXYGENASEAspirin– IndomethacinOIbuprofenCOOHCOOHOPGI2OOHPROSTACYCLIN PGG 2*OSYNTHASEPEROXIDASE OOC HCOOH+OC HOH OHOHOOHPGH2Malondialdehyde + HHTCOOHISOMERASETHROMBOXANEImidazoleOSYNTHASE –COOHOCOOHOOH OHOH OHOH TXA 26-Keto PGF 1αPGE2ISOMERASEOHREDUCTASEOH OH*COOHCOOHCOOHCOOHOHOHOOHHOOOHPGF 2 αPGD 2TXB 2Figure 23–6. Conversion of arachidonic acid to prostaglandins and thromboxanes of series 2. (PG,prostaglandin; TX, thromboxane; PGI, prostacyclin; HHT, hydroxyheptadecatrienoate.) (Asterisk: Both of thesestarred activities are attributed to one enzyme: prostaglandin H synthase. Similar conversions occur inprostaglandins and thromboxanes of series 1 and 3.)Cyclooxygenase Is a “Suicide Enzyme”“Switching off” of prostaglandin activity is partly achievedby a remarkable property of cyclooxygenase—that ofself-catalyzed destruction; ie, it is a “suicide enzyme.”Furthermore, the inactivation of prostaglandins by 15-hydroxyprostaglandin dehydrogenase is rapid. Blockingthe action of this enzyme with sulfasalazine or indomethacincan prolong the half-life of prostaglandinsin the body.LEUKOTRIENES & LIPOXINSARE FORMED BY THELIPOXYGENASE PATHWAYThe leukotrienes are a family of conjugated trienesformed from eicosanoic acids in leukocytes, mastocytomacells, platelets, and macrophages by the lipoxygenasepathway in response to both immunologic andnonimmunologic stimuli. Three different lipoxygenases(dioxygenases) insert oxygen into the 5, 12, and 15 positionsof arachidonic acid, giving rise to hydroperoxides(HPETE). Only 5-lipoxygenase forms leukotrienes(details in Figure 23–7). Lipoxins are a family ofconjugated tetraenes also arising in leukocytes. They areformed by the combined action of more than onelipoxygenase (Figure 23–7).CLINICAL ASPECTSSymptoms of Essential Fatty AcidDeficiency in Humans Include SkinLesions & Impairment of Lipid TransportIn adults subsisting on ordinary diets, no signs of essentialfatty acid deficiencies have been reported. How-

METABOLISM OF UNSATURATED FATTY ACIDS & EICOSANOIDS / 195COOH12-LIPOXYGENASECOOHArachidonate15-LIPOXYGENASECOOHHOO12-HPETE1O 25-LIPOXYGENASEOOH15-HPETE1COOHHOCOOHOH15-HETE12-HETEOOH5-HPETECOOH1OH5-HETECOOH5-LIPOXYGENASEOHCOOHH 2 OOH 2 OCOOHOHOHCOOHOH215-LIPOXYGENASEOHLeukotriene B 4Leukotriene A 4Lipoxins, eg, LXA 43GlutathioneGlutamic acidHOOGlycineO NHNHO SNH 2OHOCysteine Glutamic acidHOGlycineO NH 2NHO SCysteineGlycineHOONH 2SCysteineOHCOOH4OHCOOH5OHCOOHLeukotriene C 4Leukotriene D 4Leukotriene E 4Figure 23–7. Conversion of arachidonic acid to leukotrienes and lipoxins of series 4 via the lipoxygenase pathway.Some similar conversions occur in series 3 and 5 leukotrienes. (HPETE, hydroperoxyeicosatetraenoate; HETE,hydroxyeicosatetraenoate; 1 , peroxidase; 2 , leukotriene A 4 epoxide hydrolase; 3 , glutathione S-transferase;4 , γ-glutamyltranspeptidase; 5 , cysteinyl-glycine dipeptidase.)ever, infants receiving formula diets low in fat and patientsmaintained for long periods exclusively by intravenousnutrition low in essential fatty acids show deficiencysymptoms that can be prevented by an essentialfatty acid intake of 1–2% of the total caloric requirement.Abnormal Metabolism of Essential FattyAcids Occurs in Several DiseasesAbnormal metabolism of essential fatty acids, whichmay be connected with dietary insufficiency, has beennoted in cystic fibrosis, acrodermatitis enteropathica,

196 / CHAPTER 23hepatorenal syndrome, Sjögren-Larsson syndrome,multisystem neuronal degeneration, Crohn’s disease,cirrhosis and alcoholism, and Reye’s syndrome. Elevatedlevels of very long chain polyenoic acids havebeen found in the brains of patients with Zellweger’ssyndrome (Chapter 22). Diets with a high P:S (polyunsaturated:saturatedfatty acid) ratio reduce serum cholesterollevels and are considered to be beneficial interms of the risk of development of coronary heart disease.Prostanoids Are Potent BiologicallyActive SubstancesThromboxanes are synthesized in platelets and uponrelease cause vasoconstriction and platelet aggregation.Their synthesis is specifically inhibited by low-dose aspirin.Prostacyclins (PGI 2 ) are produced by blood vesselwalls and are potent inhibitors of platelet aggregation.Thus, thromboxanes and prostacyclins areantagonistic. PG 3 and TX 3 , formed from eicosapentaenoicacid (EPA) in fish oils, inhibit the release ofarachidonate from phospholipids and the formationof PG 2 and TX 2 . PGI 3 is as potent an antiaggregator ofplatelets as PGI 2 , but TXA 3 is a weaker aggregator thanTXA 2 , changing the balance of activity and favoringlonger clotting times. As little as 1 ng/mL of plasmaprostaglandins causes contraction of smooth muscle inanimals. Potential therapeutic uses include preventionof conception, induction of labor at term, terminationof pregnancy, prevention or alleviation of gastric ulcers,control of inflammation and of blood pressure, and reliefof asthma and nasal congestion. In addition, PGD 2is a potent sleep-promoting substance. Prostaglandinsincrease cAMP in platelets, thyroid, corpus luteum,fetal bone, adenohypophysis, and lung but reducecAMP in renal tubule cells and adipose tissue (Chapter25).Leukotrienes & Lipoxins Are PotentRegulators of Many Disease ProcessesSlow-reacting substance of anaphylaxis (SRS-A) is amixture of leukotrienes C 4 , D 4 , and E 4 . This mixture ofleukotrienes is a potent constrictor of the bronchial airwaymusculature. These leukotrienes together withleukotriene B 4 also cause vascular permeability and attractionand activation of leukocytes and are importantregulators in many diseases involving inflammatory orimmediate hypersensitivity reactions, such as asthma.Leukotrienes are vasoactive, and 5-lipoxygenase hasbeen found in arterial walls. Evidence supports a rolefor lipoxins in vasoactive and immunoregulatory function,eg, as counterregulatory compounds (chalones) ofthe immune response.SUMMARY• Biosynthesis of unsaturated long-chain fatty acids isachieved by desaturase and elongase enzymes, whichintroduce double bonds and lengthen existing acylchains, respectively.• Higher animals have ∆ 4 , ∆ 5 , ∆ 6 , and ∆ 9 desaturasesbut cannot insert new double bonds beyond the 9position of fatty acids. Thus, the essential fatty acidslinoleic (ω6) and α-linolenic (ω3) must be obtainedfrom the diet.• Eicosanoids are derived from C 20 (eicosanoic) fattyacids synthesized from the essential fatty acids andcomprise important groups of physiologically andpharmacologically active compounds, including theprostaglandins, thromboxanes, leukotrienes, andlipoxins.REFERENCESConnor WE: The beneficial effects of omega-3 fatty acids: cardiovasculardisease and neurodevelopment. Curr Opin Lipidol1997;8:1.Fischer S: Dietary polyunsaturated fatty acids and eicosanoid formationin humans. Adv Lipid Res 1989;23:169.Lagarde M, Gualde N, Rigaud M: Metabolic interactions betweeneicosanoids in blood and vascular cells. Biochem J 1989;257:313.Neuringer M, Anderson GJ, Connor WE: The essentiality of n-3fatty acids for the development and function of the retina andbrain. Annu Rev Nutr 1988;8:517.Serhan CN: Lipoxin biosynthesis and its impact in inflammatoryand vascular events. Biochim Biophys Acta 1994;1212:1.Smith WL, Fitzpatrick FA: The eicosanoids: Cyclooxygenase,lipoxygenase, and epoxygenase pathways. In: Biochemistry ofLipids, Lipoproteins and Membranes. Vance DE, Vance JE(editors). Elsevier, 1996.Tocher DR, Leaver MJ, Hodgson PA: Recent advances in the biochemistryand molecular biology of fatty acyl desaturases.Prog Lipid Res 1998;37:73.Valenzuela A, Morgado N: Trans fatty acid isomers in humanhealth and the food industry. Biol Res 1999;32:273.

Metabolism of Acylglycerols& Sphingolipids 24Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEAcylglycerols constitute the majority of lipids in thebody. Triacylglycerols are the major lipids in fat depositsand in food, and their roles in lipid transport andstorage and in various diseases such as obesity, diabetes,and hyperlipoproteinemia will be described in subsequentchapters. The amphipathic nature of phospholipidsand sphingolipids makes them ideally suitable asthe main lipid component of cell membranes. Phospholipidsalso take part in the metabolism of manyother lipids. Some phospholipids have specialized functions;eg, dipalmitoyl lecithin is a major component oflung surfactant, which is lacking in respiratory distresssyndrome of the newborn. Inositol phospholipids in thecell membrane act as precursors of hormone secondmessengers, and platelet-activating factor is an alkylphospholipid.Glycosphingolipids, containing sphingosineand sugar residues as well as fatty acid and found inthe outer leaflet of the plasma membrane with theiroligosaccharide chains facing outward, form part of theglycocalyx of the cell surface and are important (1) incell adhesion and cell recognition; (2) as receptors forbacterial toxins (eg, the toxin that causes cholera); and(3) as ABO blood group substances. A dozen or so glycolipidstorage diseases have been described (eg,Gaucher’s disease, Tay-Sachs disease), each due to a geneticdefect in the pathway for glycolipid degradationin the lysosomes.HYDROLYSIS INITIATES CATABOLISMOF TRIACYLGLYCEROLSTriacylglycerols must be hydrolyzed by a lipase to theirconstituent fatty acids and glycerol before further catabolismcan proceed. Much of this hydrolysis (lipolysis)occurs in adipose tissue with release of free fatty acidsinto the plasma, where they are found combined withserum albumin. This is followed by free fatty acid uptakeinto tissues (including liver, heart, kidney, muscle,lung, testis, and adipose tissue, but not readily bybrain), where they are oxidized or reesterified. The utilizationof glycerol depends upon whether such tissuespossess glycerol kinase, found in significant amountsin liver, kidney, intestine, brown adipose tissue, andlactating mammary gland.TRIACYLGLYCEROLS &PHOSPHOGLYCEROLS ARE FORMED BYACYLATION OF TRIOSE PHOSPHATESThe major pathways of triacylglycerol and phosphoglycerolbiosynthesis are outlined in Figure 24–1. Importantsubstances such as triacylglycerols, phosphatidylcholine,phosphatidylethanolamine, phosphatidylinositol,and cardiolipin, a constituent of mitochondrial membranes,are formed from glycerol-3-phosphate. Significantbranch points in the pathway occur at the phosphatidateand diacylglycerol steps. From dihydroxyacetonephosphate are derived phosphoglycerols containing anether link (⎯C⎯O⎯C⎯), the best-known of whichare plasmalogens and platelet-activating factor (PAF).Glycerol 3-phosphate and dihydroxyacetone phosphateare intermediates in glycolysis, making a very importantconnection between carbohydrate and lipid metabolism.Glycerol 3-phosphatePhosphatidateDiacylglycerolPhosphatidylcholinePhosphatidylethanolamineCardiolipinTriacylglycerolDihydroxyacetone phosphatePlasmalogensPAFPhosphatidylinositolPhosphatidylinositol4,5-bisphosphateFigure 24–1. Overview of acylglycerol biosynthesis.(PAF, platelet-activating factor.)197

ATPADPNAD + NADH + H +H 2 COHH 2 COHH 2 COHHOCHHOCHCOGlycolysisH 2 COHGlycerolGLYCEROL KINASEH 2 C O Psn-Glycerol3-phosphateGLYCEROL-3-PHOSPHATEDEHYDROGENASEH 2 C O PDihydroxyacetonephosphateAcyl-CoA (mainly saturated)2GLYCEROL-3-PHOSPHATEACYLTRANSFERASEH 2 COCoAOC R 1R 2H 2 C OHC O C HO H 2 C OH2-MonoacylglycerolHO CHH 2 C OP1-Acylglycerol-3-phosphate(lysophosphatidate)Acyl-CoA (usually unsaturated)Acyl-CoAMONOACYLGLYCEROLACYLTRANSFERASE(INTESTINE)CoA1R 2COH 2 CCCoAOO C R 1H1-ACYLGLYCEROL-3-PHOSPHATEACYLTRANSFERASEOH 2 CO1,2-Diacylglycerolphosphate(phosphatidate)PCholineH 2 OCTPATPCHOLINEKINASEPHOSPHATIDATEPHOSPHOHYDROLASECDP-DGSYNTHASEP 1PP 1ADPPhosphocholineCTPCTP:PHOSPHOCHOLINECYTIDYLTRANSFERASER 2COH 2 COO C HH 2 COH1,2-DiacylglycerolOC R 1R 2COH 2 CO CH 2 COHOC R 1O P PCytidineCDP-diacylglycerolCardiolipinPP 1H 2 CCDP-cholineAcyl-CoAInositolCDP-CHOLINE:DIACYLGLYCEROLPHOSPHOCHOLINETRANSFERASEDIACYLGLYCEROLACYLTRANSFERASEPHOSPHATIDYL-INOSITOL SYNTHASECMPOCoAOCMPATPOKINASEADPOOC R 1H 2 COC R 1H 2 COC R 1H 2 COC R 1R 2 C O C HO H2C O PCholineR 2COOCHH2C O C R3TriacyglycerolPhosphatidylcholinePHOSPHATIDYLETHANOLAMINEN-METHYLTRANSFERASEPhosphatidylethanolamineCO2PhosphatidylserineO(–CH 3 ) 3SerineR 2EthanolamineCOOCHH2CO PInositolPhosphatidylinositolFigure 24–2 . Biosynthesis of triacylglycerol and phospholipids.(1 , Monoacylglycerol pathway; 2 , glycerol phosphate pathway.)Phosphatidylethanolamine may be formed from ethanolamine by apathway similar to that shown for the formation of phosphatidylcholinefrom choline.R 2 C O C HO H2C O P Inositol PPhosphatidylinositol 4-phosphateATPADPKINASEH 2 COC R 1R 2 C O C HO H2C O P Inositol PPPhosphatidylinositol 4,5-bisphosphateO

METABOLISM OF ACYLGLYCEROLS & SPHINGOLIPIDS / 199Phosphatidate Is the Common Precursorin the Biosynthesis of Triacylglycerols,Many Phosphoglycerols, & CardiolipinBoth glycerol and fatty acids must be activated by ATPbefore they can be incorporated into acylglycerols.Glycerol kinase catalyzes the activation of glycerol tosn-glycerol 3-phosphate. If the activity of this enzyme isabsent or low, as in muscle or adipose tissue, most ofthe glycerol 3-phosphate is formed from dihydroxyacetonephosphate by glycerol-3-phosphate dehydrogenase(Figure 24–2).A. BIOSYNTHESIS OF TRIACYLGLYCEROLSTwo molecules of acyl-CoA, formed by the activationof fatty acids by acyl-CoA synthetase (Chapter 22),combine with glycerol 3-phosphate to form phosphatidate(1,2-diacylglycerol phosphate). This takes place intwo stages, catalyzed by glycerol-3-phosphate acyltransferaseand 1-acylglycerol-3-phosphate acyltransferase.Phosphatidate is converted by phosphatidatephosphohydrolase and diacylglycerol acyltransferaseto 1,2-diacylglycerol and then triacylglycerol. In intestinalmucosa, monoacylglycerol acyltransferase convertsmonoacylglycerol to 1,2-diacylglycerol in themonoacylglycerol pathway. Most of the activity ofthese enzymes resides in the endoplasmic reticulum ofthe cell, but some is found in mitochondria. Phosphatidatephosphohydrolase is found mainly in the cytosol,but the active form of the enzyme is membrane-bound.In the biosynthesis of phosphatidylcholine andphosphatidylethanolamine (Figure 24–2), choline orethanolamine must first be activated by phosphorylationby ATP followed by linkage to CTP. The resultingCDP-choline or CDP-ethanolamine reacts with 1,2-diacylglycerolto form either phosphatidylcholine orphosphatidylethanolamine, respectively. Phosphatidylserineis formed from phosphatidylethanolamine directlyby reaction with serine (Figure 24–2). Phosphatidylserinemay re-form phosphatidylethanolamineby decarboxylation. An alternative pathway in liver enablesphosphatidylethanolamine to give rise directly tophosphatidylcholine by progressive methylation of theethanolamine residue. In spite of these sources ofcholine, it is considered to be an essential nutrient inmany mammalian species, but this has not been establishedin humans.The regulation of triacylglycerol, phosphatidylcholine,and phosphatidylethanolamine biosynthesis isdriven by the availability of free fatty acids. Those thatescape oxidation are preferentially converted to phospholipids,and when this requirement is satisfied theyare used for triacylglycerol synthesis.A phospholipid present in mitochondria is cardiolipin(diphosphatidylglycerol; Figure 14–8). It is formedfrom phosphatidylglycerol, which in turn is synthesizedfrom CDP-diacylglycerol (Figure 24–2) and glycerol3-phosphate according to the scheme shown in Figure24–3. Cardiolipin, found in the inner membrane ofmitochondria, is specifically required for the functioningof the phosphate transporter and for cytochromeoxidase activity.B. BIOSYNTHESIS OF GLYCEROL ETHER PHOSPHOLIPIDSThis pathway is located in peroxisomes. Dihydroxyacetonephosphate is the precursor of the glycerol moietyof glycerol ether phospholipids (Figure 24–4). Thiscompound combines with acyl-CoA to give 1-acyldihydroxyacetonephosphate. The ether link is formed inthe next reaction, producing 1-alkyldihydroxyacetonephosphate, which is then converted to 1-alkylglycerol3-phosphate. After further acylation in the 2 position,the resulting 1-alkyl-2-acylglycerol 3-phosphate (analogousto phosphatidate in Figure 24–2) is hydrolyzed togive the free glycerol derivative. Plasmalogens, whichcomprise much of the phospholipid in mitochondria,are formed by desaturation of the analogous 3-phosphoethanolaminederivative (Figure 24–4). Plateletactivatingfactor (PAF) (1-alkyl-2-acetyl-sn-glycerol-3-phosphocholine) is synthesized from the corresponding3-phosphocholine derivative. It is formed by manyblood cells and other tissues and aggregates platelets atconcentrations as low as 10 −11 mol/L. It also has hypotensiveand ulcerogenic properties and is involved ina variety of biologic responses, including inflammation,chemotaxis, and protein phosphorylation.CDP-DiacylglycerolFigure 24–3.CMPPhosphatidylglycerol phosphateCMPPhosphatidylglycerolCardiolipin(diphosphatidylglycerol)sn-Glycerol3-phosphateH 2 OP iBiosynthesis of cardiolipin.

200 / CHAPTER 24H 2 COHO CH 2 C O PAcyl-CoAACYL-TRANSFERASEOH 2 CO CH 2 C O POHNADPH+ H + NADP +H 2 C O (CH 2 ) 2 R 2H 2 C O (CH 2 ) 2 R 2O CHO C HSYNTHASE H 2 C O PREDUCTASE H 2 C O PO C R 1R 2HOOC R 1Dihydroxyacetonephosphate1-Acyldihydroxyacetonephosphate1-Alkyldihydroxyacetonephosphate1-Alkylglycerol 3-phosphateAcyl-CoA(CH 2 ) 2 R 2(CH 2 ) 2 R 2ACYL-TRANSFERASECDP-CMP EthanolamineP i H 2 OO H 2 C OO H 2 C OO H 2 C O (CH 2 ) 2 R 2R 3 C O C HR 3 C O C HR 3 C O C HH 2 C O P CH 2 CH 2 NH 2CDP-ETHANOLAMINE:ALKYLACYLGLYCEROLH 2 C OH PHOSPHOHYDROLASEH 2 C O P1-Alkyl-2-acylglycerolPHOSPHOETHANOLAMINETRANSFERASE3-phosphoethanolamine1-Alkyl-2-acylglycerol1-Alkyl-2-acylglycerol 3-phosphateCDP-cholineNADPH, O 2,DESATURASECDP-CHOLINE:Cyt b ALKYLACYLGLYCEROL5PHOSPHOCHOLINEAlkyl, diacyl glycerolsTRANSFERASER 3OCMPH 2 C O CH CH R 2O H 2 C O (CH 2 ) 2 R 2C O C HH 2 O R 3 COOHR 3 C O C HH 2 C O (CH 2 ) 2 R 2H 2 C O P (CH 2 ) 2 NH 2H 2 C O PHO C HPHOSPHOLIPASE A1-Alkenyl-2-acylglycerol2CholineH 2 C O P3-phosphoethanolamineplasmalogen1-Alkyl-2-acylglycerolCholine3-phosphocholine1-Alkyl-2-lysoglycerolAcetyl-CoA3-phosphocholineACETYLTRANSFERASE*OH 2 CO(CH 2 ) 2 R 2H 3 CCOCHH 2 COPCholine1-Alkyl-2-acetylglycerol 3-phosphocholinePAFFigure 24–4. Biosynthesis of ether lipids, including plasmalogens, and platelet-activating factor (PAF). In thede novo pathway for PAF synthesis, acetyl-CoA is incorporated at stage *, avoiding the last two steps in the pathwayshown here.Phospholipases Allow Degradation& Remodeling of PhosphoglycerolsAlthough phospholipids are actively degraded, eachportion of the molecule turns over at a different rate—eg, the turnover time of the phosphate group is differentfrom that of the 1-acyl group. This is due to thepresence of enzymes that allow partial degradation followedby resynthesis (Figure 24–5). Phospholipase A 2catalyzes the hydrolysis of glycerophospholipids to forma free fatty acid and lysophospholipid, which in turnmay be reacylated by acyl-CoA in the presence of anacyltransferase. Alternatively, lysophospholipid (eg, lysolecithin)is attacked by lysophospholipase, formingthe corresponding glyceryl phosphoryl base, which inturn may be split by a hydrolase liberating glycerol3-phosphate plus base. Phospholipases A 1 , A 2 , B, C,and D attack the bonds indicated in Figure 24–6.Phospholipase A 2 is found in pancreatic fluid andsnake venom as well as in many types of cells; phospholipaseC is one of the major toxins secreted by bacteria;and phospholipase D is known to be involved inmammalian signal transduction.Lysolecithin (lysophosphatidylcholine) may beformed by an alternative route that involves lecithin:cholesterol acyltransferase (LCAT). This enzyme,

METABOLISM OF ACYLGLYCEROLS & SPHINGOLIPIDS / 201ACYLTRANSFERASEAcyl-CoAFigure 24–5.(lecithin).OR 2 C O C HH 2 C OOH 2 C O C R 1PPhosphatidylcholineH 2 OCholinePHOSPHOLIPASE A 2R 2H 2 CH 2 CCOOHOH 2 C O C R 1HO C HH 2 C O P CholineLysophosphatidylcholine (lysolecithin)H 2 OLYSOPHOSPHOLIPASER 1 COOHH 2 C OHHO C HO P CholineGlycerylphosphocholineH 2 OGLYCERYLPHOSPHO-CHOLINE HYDROLASEH 2 C OHHO C H + CholineO Psn-Glycerol 3-phosphateMetabolism of phosphatidylcholinePHOSPHOLIPASE BOR 2 C O C HH 2 C OPHOSPHOLIPASE A 2H 2 C O C R 1PHOSPHOLIPASE A 1OOPO –OPHOSPHOLIPASE CPHOSPHOLIPASE DN-BASEFigure 24–6. Sites of the hydrolytic activity of phospholipaseson a phospholipid substrate.found in plasma, catalyzes the transfer of a fatty acidresidue from the 2 position of lecithin to cholesterol toform cholesteryl ester and lysolecithin and is consideredto be responsible for much of the cholesteryl ester inplasma lipoproteins. Long-chain saturated fatty acidsare found predominantly in the 1 position of phospholipids,whereas the polyunsaturated acids (eg, the precursorsof prostaglandins) are incorporated more intothe 2 position. The incorporation of fatty acids intolecithin occurs by complete synthesis of the phospholipid,by transacylation between cholesteryl ester andlysolecithin, and by direct acylation of lysolecithin byacyl-CoA. Thus, a continuous exchange of the fattyacids is possible, particularly with regard to introducingessential fatty acids into phospholipid molecules.ALL SPHINGOLIPIDS ARE FORMEDFROM CERAMIDECeramide is synthesized in the endoplasmic reticulumfrom the amino acid serine according to Figure 24–7.Ceramide is an important signaling molecule (secondmessenger) regulating pathways including apoptosis(processes leading to cell death), cell senescence, anddifferentiation, and opposes some of the actions of diacylglycerol.Sphingomyelins (Figure 14–11) are phospholipidsand are formed when ceramide reacts with phosphatidylcholineto form sphingomyelin plus diacylglycerol(Figure 24–8A). This occurs mainly in the Golgiapparatus and to a lesser extent in the plasma membrane.Glycosphingolipids Are a Combinationof Ceramide With One or MoreSugar ResiduesThe simplest glycosphingolipids (cerebrosides) aregalactosylceramide (GalCer) and glucosylceramide(GlcCer). GalCer is a major lipid of myelin, whereasGlcCer is the major glycosphingolipid of extraneuraltissues and a precursor of most of the more complexglycosphingolipids. Galactosylceramide (Figure 24–8B)is formed in a reaction between ceramide and UDPGal(formed by epimerization from UDPGlc—Figure20–6). Sulfogalactosylceramide and other sulfolipidssuch as the sulfo(galacto)-glycerolipids and thesteroid sulfates are formed after further reactions involving3′-phosphoadenosine-5′-phosphosulfate (PAPS;“active sulfate”). Gangliosides are synthesized fromceramide by the stepwise addition of activated sugars (eg,UDPGlc and UDPGal) and a sialic acid, usually N-acetylneuraminic acid (Figure 24–9). A large numberof gangliosides of increasing molecular weight may beformed. Most of the enzymes transferring sugars from

202 / CHAPTER 24CH 3O(CH 2 ) 14 C SPalmitoyl-CoACoAPyridoxal phosphate, Mn 2+CO 2OCH 3 (CH 2 ) 12 CH 2 CH 2 C CH CH 2 OH+NH 33-Ketosphinganine3-KETOSPHINGANINEREDUCTASENADPH + H +NADP +CH 3 (CH 2 ) 12 CH 2 CH 2 CH CH CH 2 OHOH+NH 3Dihydrosphingosine (sphinganine)RCoACOSHSAcyl-CoACoASHCoA+ NH3− OOC CH CH2 OHSerineSERINEPALMITOYLTRANSFERASEDIHYDROSPHINGOSINEN-ACYLTRANSFERASECH 3 (CH 2 ) 12 CH 2 CH 2 CH CH CH 2 OHOH NH CO RDihydroceramideCH 3Figure 24–7.(CH 2 ) 122HCHCHDIHYDROCERAMIDEDESATURASECHOHCeramideCHNHBiosynthesis of ceramide.CH 2COOHRnucleotide sugars (glycosyl transferases) are found inthe Golgi apparatus.Glycosphingolipids are constituents of the outerleaflet of plasma membranes and are important in celladhesion and cell recognition. Some are antigens, eg,ABO blood group substances. Certain gangliosidesfunction as receptors for bacterial toxins (eg, for choleratoxin, which subsequently activates adenylyl cyclase).CLINICAL ASPECTSDeficiency of Lung Surfactant CausesRespiratory Distress SyndromeLung surfactant is composed mainly of lipid withsome proteins and carbohydrate and prevents the alveolifrom collapsing. Surfactant activity is largely attributedto dipalmitoylphosphatidylcholine, which issynthesized shortly before parturition in full-term infants.Deficiency of lung surfactant in the lungs ofmany preterm newborns gives rise to respiratory distresssyndrome. Administration of either natural or artificialsurfactant has been of therapeutic benefit.Phospholipids & SphingolipidsAre Involved in Multiple Sclerosisand LipidosesCertain diseases are characterized by abnormal quantitiesof these lipids in the tissues, often in the nervoussystem. They may be classified into two groups: (1) truedemyelinating diseases and (2) sphingolipidoses.In multiple sclerosis, which is a demyelinating disease,there is loss of both phospholipids (particularlyethanolamine plasmalogen) and of sphingolipids fromwhite matter. Thus, the lipid composition of whitematter resembles that of gray matter. The cerebrospinalfluid shows raised phospholipid levels.The sphingolipidoses (lipid storage diseases) are agroup of inherited diseases that are often manifested inchildhood. These diseases are part of a larger group oflysosomal disorders and exhibit several constant features:(1) Complex lipids containing ceramide accumulatein cells, particularly neurons, causing neurodegen-ACeramideSphingomyelinPhosphatidylcholineDiacylglycerolBCeramideUDPGalUDPPAPSGalactosylceramide(cerebroside)Sulfogalactosylceramide(sulfatide)Figure 24–8. Biosynthesis of sphingomyelin (A),galactosylceramide and its sulfo derivative (B). (PAPS,“active sulfate,” adenosine 3′-phosphate-5′-phosphosulfate.)

METABOLISM OF ACYLGLYCEROLS & SPHINGOLIPIDS / 203UDPGlcUDPUDPGalUDPCMP-NeuAcCMPCeramideGlucosylceramide(Cer-Glc)Cer-Glc-GalCer-Glc-GalNeuAc(G M3 )UDP-N-acetylgalactosamineUDPUDPGalUDPHigher gangliosides(disialo- and trisialogangliosides)Cer-Glc-Gal-GalNAc-GalNeuAc(G M1 )Cer-Glc-Gal-GalNAcNeuAc(G M2 )Figure 24–9.Biosynthesis of gangliosides. (NeuAc, N-acetylneuraminic acid.)eration and shortening the life span. (2) The rate ofsynthesis of the stored lipid is normal. (3) The enzymaticdefect is in the lysosomal degradation pathwayof sphingolipids. (4) The extent to which the activity ofthe affected enzyme is decreased is similar in all tissues.There is no effective treatment for many of the diseases,though some success has been achieved with enzymesthat have been chemically modified to ensure bindingto receptors of target cells, eg, to macrophages in theliver in order to deliver β-glucosidase (glucocerebrosidase)in the treatment of Gaucher’s disease. A recentpromising approach is substrate reduction therapy toinhibit the synthesis of sphingolipids, and gene therapyfor lysosomal disorders is currently under investigation.Some examples of the more important lipid storage diseasesare shown in Table 24–1.Multiple sulfatase deficiency results in accumulationof sulfogalactosylceramide, steroid sulfates, andproteoglycans owing to a combined deficiency of arylsulfatasesA, B, and C and steroid sulfatase.Table 24–1. Examples of sphingolipidoses.Disease Enzyme Deficiency Lipid Accumulating 1 Clinical SymptomsTay-Sachs disease Hexosaminidase A Cer—Glc—Gal(NeuAc)— : GalNAc Mental retardation, blindness, muscular weakness.G M2 GangliosideFabry’s disease α-Galactosidase Cer—Glc—Gal— : Gal Skin rash, kidney failure (full symptoms only inGlobotriaosylceramide males; X-linked recessive).Metachromatic Arylsulfatase A Cer—Gal— : OSO 3 Mental retardation and psychologic disturbances inleukodystrophy 3-Sulfogalactosylceramide adults; demyelination.Krabbe’s disease β-Galactosidase Cer— : Gal Mental retardation; myelin almost absent.GalactosylceramideGaucher’s disease β-Glucosidase Cer— : Glc Enlarged liver and spleen, erosion of long bones,Glucosylceramidemental retardation in infants.Niemann-Pick Sphingomyelinase Cer— : P—choline Enlarged liver and spleen, mental retardation; fatal indisease Sphingomyelin early life.Farber’s disease Ceramidase Acyl— : Sphingosine Hoarseness, dermatitis, skeletal deformation, mentalCeramideretardation; fatal in early life.1 NeuAc, N-acetylneuraminic acid; Cer, ceramide; Glc, glucose; Gal, galactose. — : , site of deficient enzyme reaction.

204 / CHAPTER 24SUMMARY• Triacylglycerols are the major energy-storing lipids,whereas phosphoglycerols, sphingomyelin, and glycosphingolipidsare amphipathic and have structuralfunctions in cell membranes as well as other specializedroles.• Triacylglycerols and some phosphoglycerols are synthesizedby progressive acylation of glycerol 3-phosphate.The pathway bifurcates at phosphatidate,forming inositol phospholipids and cardiolipin onthe one hand and triacylglycerol and choline andethanolamine phospholipids on the other.• Plasmalogens and platelet-activating factor (PAF) areether phospholipids formed from dihydroxyacetonephosphate.• Sphingolipids are formed from ceramide (N-acylsphingosine).Sphingomyelin is present in membranesof organelles involved in secretory processes(eg, Golgi apparatus). The simplest glycosphingolipidsare a combination of ceramide plus a sugarresidue (eg, GalCer in myelin). Gangliosides aremore complex glycosphingolipids containing moresugar residues plus sialic acid. They are present in theouter layer of the plasma membrane, where they contributeto the glycocalyx and are important as antigensand cell receptors.• Phospholipids and sphingolipids are involved in severaldisease processes, including respiratory distresssyndrome (lack of lung surfactant), multiple sclerosis(demyelination), and sphingolipidoses (inability tobreak down sphingolipids in lysosomes due to inheriteddefects in hydrolase enzymes).REFERENCESGriese M: Pulmonary surfactant in health and human lung diseases:state of the art. Eur Respir J 1999;13:1455.Merrill AH, Sweeley CC: Sphingolipids: metabolism and cell signaling.In: Biochemistry of Lipids, Lipoproteins and Membranes.Vance DE, Vance JE (editors). Elsevier, 1996.Prescott SM et al: Platelet-activating factor and related lipid mediators.Annu Rev Biochem 2000;69:419.Ruvolo PP: Ceramide regulates cellular homeostasis via diversestress signaling pathways. Leukemia 2001;15:1153.Schuette CG et al: The glycosphingolipidoses—from disease tobasic principles of metabolism. Biol Chem 1999;380:759.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Tijburg LBM, Geelen MJH, van Golde LMG: Regulation of thebiosynthesis of triacylglycerol, phosphatidylcholine and phosphatidylethanolaminein the liver. Biochim Biophys Acta1989;1004:1.Vance DE: Glycerolipid biosynthesis in eukaryotes. In: Biochemistryof Lipids, Lipoproteins and Membranes. Vance DE, VanceJE (editors). Elsevier, 1996.van Echten G, Sandhoff K: Ganglioside metabolism. Enzymology,topology, and regulation. J Biol Chem 1993;268:5341.Waite M: Phospholipases. In: Biochemistry of Lipids, Lipoproteinsand Membranes. Vance DE, Vance JE (editors). Elsevier,1996.

Lipid Transport & Storage 25Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCEFat absorbed from the diet and lipids synthesized by theliver and adipose tissue must be transported betweenthe various tissues and organs for utilization and storage.Since lipids are insoluble in water, the problem ofhow to transport them in the aqueous blood plasma issolved by associating nonpolar lipids (triacylglyceroland cholesteryl esters) with amphipathic lipids (phospholipidsand cholesterol) and proteins to make watermisciblelipoproteins.In a meal-eating omnivore such as the human, excesscalories are ingested in the anabolic phase of thefeeding cycle, followed by a period of negative caloricbalance when the organism draws upon its carbohydrateand fat stores. Lipoproteins mediate this cycle bytransporting lipids from the intestines as chylomicrons—andfrom the liver as very low density lipoproteins(VLDL)—to most tissues for oxidation and toadipose tissue for storage. Lipid is mobilized from adiposetissue as free fatty acids (FFA) attached to serumalbumin. Abnormalities of lipoprotein metabolismcause various hypo- or hyperlipoproteinemias. Themost common of these is diabetes mellitus, where insulindeficiency causes excessive mobilization of FFAand underutilization of chylomicrons and VLDL, leadingto hypertriacylglycerolemia. Most other pathologicconditions affecting lipid transport are due primarilyto inherited defects, some of which causehypercholesterolemia, and premature atherosclerosis.Obesity—particularly abdominal obesity—is a risk factorfor increased mortality, hypertension, type 2 diabetesmellitus, hyperlipidemia, hyperglycemia, and variousendocrine dysfunctions.LIPIDS ARE TRANSPORTED IN THEPLASMA AS LIPOPROTEINSFour Major Lipid Classes Are Presentin LipoproteinsPlasma lipids consist of triacylglycerols (16%), phospholipids(30%), cholesterol (14%), and cholesterylesters (36%) and a much smaller fraction of unesterifiedlong-chain fatty acids (free fatty acids) (4%). Thislatter fraction, the free fatty acids (FFA), is metabolicallythe most active of the plasma lipids.205Four Major Groups of Plasma LipoproteinsHave Been IdentifiedBecause fat is less dense than water, the density of alipoprotein decreases as the proportion of lipid to proteinincreases (Table 25–1). In addition to FFA, fourmajor groups of lipoproteins have been identified thatare important physiologically and in clinical diagnosis.These are (1) chylomicrons, derived from intestinalabsorption of triacylglycerol and other lipids; (2) verylow density lipoproteins (VLDL, or pre-β-lipoproteins),derived from the liver for the export of triacylglycerol;(3) low-density lipoproteins (LDL, or β-lipoproteins), representing a final stage in the catabolismof VLDL; and (4) high-density lipoproteins (HDL, orα-lipoproteins), involved in VLDL and chylomicronmetabolism and also in cholesterol transport. Triacylglycerolis the predominant lipid in chylomicrons andVLDL, whereas cholesterol and phospholipid are thepredominant lipids in LDL and HDL, respectively(Table 25–1). Lipoproteins may be separated accordingto their electrophoretic properties into -, -, and pre--lipoproteins.Lipoproteins Consist of a NonpolarCore & a Single Surface Layer ofAmphipathic LipidsThe nonpolar lipid core consists of mainly triacylglyceroland cholesteryl ester and is surrounded by a singlesurface layer of amphipathic phospholipid andcholesterol molecules (Figure 25–1). These are orientedso that their polar groups face outward to the aqueousmedium, as in the cell membrane (Chapter 14). Theprotein moiety of a lipoprotein is known as an apolipoproteinor apoprotein, constituting nearly 70% ofsome HDL and as little as 1% of chylomicrons. Someapolipoproteins are integral and cannot be removed,whereas others are free to transfer to other lipoproteins.The Distribution of ApolipoproteinsCharacterizes the LipoproteinOne or more apolipoproteins (proteins or polypeptides)are present in each lipoprotein. The major apolipoproteinsof HDL (α-lipoprotein) are designated A (Table

206 / CHAPTER 25Table 25–1. Composition of the lipoproteins in plasma of humans.CompositionDiameter Density Protein Lipid Main LipidLipoprotein Source (nm) (g/mL) (%) (%) Components ApolipoproteinsChylomicrons Intestine 90–1000 < 0.95 1–2 98–99 Triacylglycerol A-I, A-II, A-IV, 1 B-48, C-I, C-II, C-III,EChylomicron Chylomicrons 45–150 < 1.006 6–8 92–94 Triacylglycerol, B-48, Eremnantsphospholipids,cholesterolVLDL Liver (intestine) 30–90 0.95–1.006 7–10 90–93 Triacylglycerol B-100, C-I, C-II, C-IIIIDL VLDL 25–35 1.006–1.019 11 89 Triacylglycerol, B-100, EcholesterolLDL VLDL 20–25 1.019–1.063 21 79 Cholesterol B-100HDL Liver, intestine, Phospholipids, A-I, A-II, A-IV, C-I, C-II, C-III, D, 2 EHDL 1 VLDL, chylo- 20–25 1.019–1.063 32 68 cholesterolHDL 2microns10–20 1.063–1.125 33 67HDL 3 5–10 1.125–1.210 57 43Preβ-HDL 3 < 5 > 1.210 A-IAlbumin/free Adipose > 1.281 99 1 Free fatty acidsfatty acids tissueAbbreviations: HDL, high-density lipoproteins; IDL, intermediate-density lipoproteins; LDL, low-density lipoproteins; VLDL, very lowdensity lipoproteins.1 Secreted with chylomicrons but transfers to HDL.2 Associated with HDL 2 and HDL 3 subfractions.3 Part of a minor fraction known as very high density lipoproteins (VHDL).25–1). The main apolipoprotein of LDL (β-lipoprotein)is apolipoprotein B (B-100) and is found also inVLDL. Chylomicrons contain a truncated form of apoB (B-48) that is synthesized in the intestine, whileB-100 is synthesized in the liver. Apo B-100 is one ofthe longest single polypeptide chains known, having4536 amino acids and a molecular mass of 550,000 Da.Apo B-48 (48% of B-100) is formed from the samemRNA as apo B-100 after the introduction of a stop signalby an RNA editing enzyme. Apo C-I, C-II, andC-III are smaller polypeptides (molecular mass 7000–9000 Da) freely transferable between several differentlipoproteins. Apo E is found in VLDL, HDL, chylomicrons,and chylomicron remnants; it accounts for 5–10% of total VLDL apolipoproteins in normal subjects.Apolipoproteins carry out several roles: (1) they canform part of the structure of the lipoprotein, eg, apo B;(2) they are enzyme cofactors, eg, C-II for lipoproteinlipase, A-I for lecithin:cholesterol acyltransferase, or enzymeinhibitors, eg, apo A-II and apo C-III for lipoproteinlipase, apo C-I for cholesteryl ester transfer protein;and (3) they act as ligands for interaction with lipoproteinreceptors in tissues, eg, apo B-100 and apo E forthe LDL receptor, apo E for the LDL receptor-relatedprotein (LRP), which has been identified as the remnantreceptor, and apo A-I for the HDL receptor. Thefunctions of apo A-IV and apo D, however, are not yetclearly defined.FREE FATTY ACIDS ARERAPIDLY METABOLIZEDThe free fatty acids (FFA, nonesterified fatty acids, unesterifiedfatty acids) arise in the plasma from lipolysisof triacylglycerol in adipose tissue or as a result of theaction of lipoprotein lipase during uptake of plasma triacylglycerolsinto tissues. They are found in combinationwith albumin, a very effective solubilizer, in concentrationsvarying between 0.1 and 2.0 µeq/mL ofplasma. Levels are low in the fully fed condition andrise to 0.7–0.8 µeq/mL in the starved state. In uncontrolleddiabetes mellitus, the level may rise to as muchas 2 µeq/mL.

LIPID TRANSPORT & STORAGE / 207FreecholesterolIntegralapoprotein(eg, apo B)Peripheral apoprotein(eg, apo C)PhospholipidCholesterylesterTriacylglycerolCore of mainlynonpolar lipidsMonolayer of mainlyamphipathic lipidsFigure 25–1. Generalized structure of a plasmalipoprotein. The similarities with the structure of theplasma membrane are to be noted. Small amounts ofcholesteryl ester and triacylglycerol are to be found inthe surface layer and a little free cholesterol in the core.Free fatty acids are removed from the blood extremelyrapidly and oxidized (fulfilling 25–50% of energyrequirements in starvation) or esterified to formtriacylglycerol in the tissues. In starvation, esterifiedlipids from the circulation or in the tissues are oxidizedas well, particularly in heart and skeletal muscle cells,where considerable stores of lipid are to be found.The free fatty acid uptake by tissues is related directlyto the plasma free fatty acid concentration, whichin turn is determined by the rate of lipolysis in adiposetissue. After dissociation of the fatty acid-albumin complexat the plasma membrane, fatty acids bind to amembrane fatty acid transport protein that acts as atransmembrane cotransporter with Na + . On enteringthe cytosol, free fatty acids are bound by intracellularfatty acid-binding proteins. The role of these proteinsin intracellular transport is thought to be similar to thatof serum albumin in extracellular transport of longchainfatty acids.TRIACYLGLYCEROL IS TRANSPORTEDFROM THE INTESTINES INCHYLOMICRONS & FROM THE LIVER INVERY LOW DENSITY LIPOPROTEINSBy definition, chylomicrons are found in chyle formedonly by the lymphatic system draining the intestine.They are responsible for the transport of all dietarylipids into the circulation. Small quantities of VLDLare also to be found in chyle; however, most of theplasma VLDL are of hepatic origin. They are the vehiclesof transport of triacylglycerol from the liver tothe extrahepatic tissues.There are striking similarities in the mechanisms offormation of chylomicrons by intestinal cells and ofVLDL by hepatic parenchymal cells (Figure 25–2), perhapsbecause—apart from the mammary gland—theintestine and liver are the only tissues from which particulatelipid is secreted. Newly secreted or “nascent”chylomicrons and VLDL contain only a small amountof apolipoproteins C and E, and the full complement isacquired from HDL in the circulation (Figures 25–3and 25–4). Apo B is essential for chylomicron andVLDL formation. In abetalipoproteinemia (a rare disease),lipoproteins containing apo B are not formed andlipid droplets accumulate in the intestine and liver.A more detailed account of the factors controllinghepatic VLDL secretion is given below.CHYLOMICRONS & VERY LOWDENSITY LIPOPROTEINS ARERAPIDLY CATABOLIZEDThe clearance of labeled chylomicrons from the bloodis rapid, the half-time of disappearance being under 1hour in humans. Larger particles are catabolized morequickly than smaller ones. Fatty acids originating fromchylomicron triacylglycerol are delivered mainly to adiposetissue, heart, and muscle (80%), while about 20%goes to the liver. However, the liver does not metabolizenative chylomicrons or VLDL significantly; thus,the fatty acids in the liver must be secondary to theirmetabolism in extrahepatic tissues.Triacylglycerols of Chylomicrons & VLDLAre Hydrolyzed by Lipoprotein LipaseLipoprotein lipase is located on the walls of blood capillaries,anchored to the endothelium by negativelycharged proteoglycan chains of heparan sulfate. It hasbeen found in heart, adipose tissue, spleen, lung, renalmedulla, aorta, diaphragm, and lactating mammarygland, though it is not active in adult liver. It is notnormally found in blood; however, following injectionof heparin, lipoprotein lipase is released from its heparansulfate binding into the circulation. Hepatic lipaseis bound to the sinusoidal surface of liver cells andis released by heparin. This enzyme, however, does notreact readily with chylomicrons or VLDL but is concernedwith chylomicron remnant and HDL metabolism.Both phospholipids and apo C-II are required ascofactors for lipoprotein lipase activity, while apo A-II

•••208 / CHAPTER 25AIntestinal lumenB•••••••••••••••••••••••••••••••••••RER•• ••••••• ••••••••••••••••••••••••••••••••• ••••••••••••••••••••••••••••••••••SERG•••••••••••••• ••••••••••••••••••••••••••••••••••• • ••••••• •• •••••••• • • • • • • •RERSERNNCFenestraGVLDLBilecanaliculusSDEndothelialcellBloodcapillaryLymph vessel leadingto thoracic ductELumen of blood sinusoidFigure 25–2. The formation and secretion of (A) chylomicrons by an intestinal cell and (B) very low densitylipoproteins by a hepatic cell. (RER, rough endoplasmic reticulum; SER, smooth endoplasmic reticulum; G, Golgiapparatus; N, nucleus; C, chylomicrons; VLDL, very low density lipoproteins; E, endothelium; SD, space of Disse,containing blood plasma.) Apolipoprotein B, synthesized in the RER, is incorporated into lipoproteins in the SER,the main site of synthesis of triacylglycerol. After addition of carbohydrate residues in G, they are released fromthe cell by reverse pinocytosis. Chylomicrons pass into the lymphatic system. VLDL are secreted into the spaceof Disse and then into the hepatic sinusoids through fenestrae in the endothelial lining.and apo C-III act as inhibitors. Hydrolysis takes placewhile the lipoproteins are attached to the enzyme onthe endothelium. Triacylglycerol is hydrolyzed progressivelythrough a diacylglycerol to a monoacylglycerolthat is finally hydrolyzed to free fatty acid plus glycerol.Some of the released free fatty acids return to the circulation,attached to albumin, but the bulk is transportedinto the tissue (Figures 25–3 and 25–4). Heart lipoproteinlipase has a low K m for triacylglycerol, about onetenthof that for the enzyme in adipose tissue. This enablesthe delivery of fatty acids from triacylglycerol tobe redirected from adipose tissue to the heart in thestarved state when the plasma triacylglycerol decreases.A similar redirection to the mammary gland occursduring lactation, allowing uptake of lipoprotein triacylglycerolfatty acid for milk fat synthesis. The VLDL receptorplays an important part in the delivery of fattyacids from VLDL triacylglycerol to adipocytes by bindingVLDL and bringing it into close contact withlipoprotein lipase. In adipose tissue, insulin enhanceslipoprotein lipase synthesis in adipocytes and itstranslocation to the luminal surface of the capillary endothelium.The Action of Lipoprotein Lipase FormsRemnant LipoproteinsReaction with lipoprotein lipase results in the loss ofapproximately 90% of the triacylglycerol of chylomicronsand in the loss of apo C (which returns to HDL)but not apo E, which is retained. The resulting chylomicronremnant is about half the diameter of theparent chylomicron and is relatively enriched in cholesteroland cholesteryl esters because of the loss of triacylglycerol(Figure 25–3). Similar changes occur toVLDL, with the formation of VLDL remnants or IDL(intermediate-density lipoprotein) (Figure 25–4).The Liver Is Responsible for the Uptakeof Remnant LipoproteinsChylomicron remnants are taken up by the liver by receptor-mediatedendocytosis, and the cholesteryl estersand triacylglycerols are hydrolyzed and metabolized.Uptake is mediated by a receptor specific for apo E(Figure 25–3), and both the LDL (apo B-100, E) receptorand the LRP (LDL receptor-related protein)

LIPID TRANSPORT & STORAGE / 209Dietary TGSMALLINTESTINELymphaticsLDL(apo B-100, E)receptorCholesterolFatty acidsNascentchylomicronAB-48TGCEApo C, Apo EAPCHDLCApoB-48A,Apo CEChylomicronB-48TGCACLIPOPROTEIN LIPASEEXTRAHEPATICTISSUESLIVERHLLRP receptorTGCEChylomicronremnantGlycerolFatty acidsFigure 25–3. Metabolic fate of chylomicrons. (A, apolipoprotein A; B-48, apolipoprotein B-48; C ,apolipoprotein C; E, apolipoprotein E; HDL, high-density lipoprotein; TG, triacylglycerol; C, cholesterol andcholesteryl ester; P, phospholipid; HL, hepatic lipase; LRP, LDL receptor-related protein.) Only the predominantlipids are shown.are believed to take part. Hepatic lipase has a dual role:(1) in acting as a ligand to the lipoprotein and (2) inhydrolyzing its triacylglycerol and phospholipid.VLDL is the precursor of IDL, which is then convertedto LDL. Only one molecule of apo B-100 ispresent in each of these lipoprotein particles, and this isconserved during the transformations. Thus, each LDLparticle is derived from only one VLDL particle (Figure25–4). Two possible fates await IDL. It can be taken upby the liver directly via the LDL (apo B-100, E) receptor,or it is converted to LDL. In humans, a relativelylarge proportion forms LDL, accounting for the increasedconcentrations of LDL in humans comparedwith many other mammals.LDL IS METABOLIZED VIATHE LDL RECEPTORThe liver and many extrahepatic tissues express theLDL (B-100, E) receptor. It is so designated because itis specific for apo B-100 but not B-48, which lacks thecarboxyl terminal domain of B-100 containing theLDL receptor ligand, and it also takes up lipoproteinsrich in apo E. This receptor is defective in familial hypercholesterolemia.Approximately 30% of LDL is degradedin extrahepatic tissues and 70% in the liver. Apositive correlation exists between the incidence ofcoronary atherosclerosis and the plasma concentrationof LDL cholesterol. For further discussion of theregulation of the LDL receptor, see Chapter 26.HDL TAKES PART IN BOTHLIPOPROTEIN TRIACYLGLYCEROL& CHOLESTEROL METABOLISMHDL is synthesized and secreted from both liver andintestine (Figure 25–5). However, apo C and apo E aresynthesized in the liver and transferred from liver HDLto intestinal HDL when the latter enters the plasma. Amajor function of HDL is to act as a repository for theapo C and apo E required in the metabolism of chylomicronsand VLDL. Nascent HDL consists of discoidphospholipid bilayers containing apo A and free cholesterol.These lipoproteins are similar to the particlesfound in the plasma of patients with a deficiency of theplasma enzyme lecithin:cholesterol acyltransferase(LCAT) and in the plasma of patients with obstructivejaundice. LCAT—and the LCAT activator apo A-I—bind to the disk, and the surface phospholipid and freecholesterol are converted into cholesteryl esters and

210 / CHAPTER 25NascentVLDLB-100ELDL(apo B-100, E)receptorTGCCEApo C, Apo EAPC CEVLDLB-100TGCCEXTRAHEPATICTISSUESFatty acidsHDLApo CLIPOPROTEIN LIPASEB-100CholesterolLIVERFinal destruction inliver, extrahepatictissues (eg, lymphocytes,fibroblasts)via endocytosisLDL(apo B-100, E)receptor?B-100CLDLTGCEIDL(VLDL remnant)EXTRAHEPATICTISSUESGlycerolFatty acidsFigure 25–4. Metabolic fate of very low density lipoproteins (VLDL) and production of low-densitylipoproteins (LDL). (A, apolipoprotein A; B-100, apolipoprotein B-100; C , apolipoprotein C; E, apolipoproteinE; HDL, high-density lipoprotein; TG, triacylglycerol; IDL, intermediate-density lipoprotein; C, cholesterol andcholesteryl ester; P, phospholipid.) Only the predominant lipids are shown. It is possible that some IDL is alsometabolized via the LRP.lysolecithin (Chapter 24). The nonpolar cholesteryl estersmove into the hydrophobic interior of the bilayer,whereas lysolecithin is transferred to plasma albumin.Thus, a nonpolar core is generated, forming a spherical,pseudomicellar HDL covered by a surface film of polarlipids and apolipoproteins. In this way, the LCAT systemis involved in the removal of excess unesterifiedcholesterol from lipoproteins and tissues. The class Bscavenger receptor B1 (SR-B1) has recently beenidentified as an HDL receptor in the liver and insteroidogenic tissues. HDL binds to the receptor viaapo A-I and cholesteryl ester is selectively delivered tothe cells, but the particle itself, including apo A-I, is nottaken up. The transport of cholesterol from the tissuesto the liver is known as reverse cholesterol transportand is mediated by an HDL cycle (Figure 25–5). Thesmaller HDL 3 accepts cholesterol from the tissues viathe ATP-binding cassette transporter-1 (ABC-1).ABC-1 is a member of a family of transporter proteinsthat couple the hydrolysis of ATP to the binding of asubstrate, enabling it to be transported across the membrane.After being accepted by HDL 3 , the cholesterol isthen esterified by LCAT, increasing the size of the particlesto form the less dense HDL 2 . The cycle is completedby the re-formation of HDL 3 , either after selectivedelivery of cholesteryl ester to the liver via theSR-B1 or by hydrolysis of HDL 2 phospholipid and triacylglycerolby hepatic lipase. In addition, free apo A-Iis released by these processes and forms pre-HDLafter associating with a minimum amount of phospholipidand cholesterol. Preβ-HDL is the most potentform of HDL in inducing cholesterol efflux from thetissues to form discoidal HDL. Surplus apo A-I is destroyedin the kidney.HDL concentrations vary reciprocally with plasmatriacylglycerol concentrations and directly with the activityof lipoprotein lipase. This may be due to surplussurface constituents, eg, phospholipid and apo A-Ibeing released during hydrolysis of chylomicrons andVLDL and contributing toward the formation of preβ-HDL and discoidal HDL. HDL 2 concentrations are inverselyrelated to the incidence of coronary atherosclerosis,possibly because they reflect the efficiency ofreverse cholesterol transport. HDL c (HDL 1 ) is found in

LIPID TRANSPORT & STORAGE / 211LIVERCC CE PLSR-B1HEPATICLIPASEA-1CCEPLHDL 2KidneyA-1Bile C andbile acidsSynthesisPL CA-1Preβ-HDLA-1CCE LCATPLHDL 3PLCDiscoidalHDLA-1LCATSynthesisABC-1SMALL INTESTINEPhospholipidbilayerTISSUESCFigure 25–5. Metabolism of high-density lipoprotein (HDL) in reverse cholesterol transport.(LCAT, lecithin:cholesterol acyltransferase; C, cholesterol; CE, cholesteryl ester; PL, phospholipid;A-I, apolipoprotein A-I; SR-B1, scavenger receptor B1; ABC-1, ATP binding cassette transporter 1.)Preβ-HDL, HDL 2 , HDL 3 —see Table 25–1. Surplus surface constituents from the action of lipoproteinlipase on chylomicrons and VLDL are another source of preβ-HDL. Hepatic lipase activity isincreased by androgens and decreased by estrogens, which may account for higher concentrationsof plasma HDL 2 in women.the blood of diet-induced hypercholesterolemic animals.It is rich in cholesterol, and its sole apolipoproteinis apo E. It appears that all plasma lipoproteins areinterrelated components of one or more metabolic cyclesthat together are responsible for the complexprocess of plasma lipid transport.THE LIVER PLAYS A CENTRAL ROLE INLIPID TRANSPORT & METABOLISMThe liver carries out the following major functions inlipid metabolism: (1) It facilitates the digestion and absorptionof lipids by the production of bile, which containscholesterol and bile salts synthesized within theliver de novo or from uptake of lipoprotein cholesterol(Chapter 26). (2) The liver has active enzyme systemsfor synthesizing and oxidizing fatty acids (Chapters 21and 22) and for synthesizing triacylglycerols and phospholipids(Chapter 24). (3) It converts fatty acids to ketonebodies (ketogenesis) (Chapter 22). (4) It plays anintegral part in the synthesis and metabolism of plasmalipoproteins (this chapter).Hepatic VLDL Secretion Is Relatedto Dietary & Hormonal StatusThe cellular events involved in VLDL formation andsecretion have been described above. Hepatic triacylglycerolsynthesis provides the immediate stimulus forthe formation and secretion of VLDL. The fatty acidsused are derived from two possible sources: (1) synthesiswithin the liver from acetyl-CoA derived mainlyfrom carbohydrate (perhaps not so important in humans)and (2) uptake of free fatty acids from the circulation.The first source is predominant in the well-fedcondition, when fatty acid synthesis is high and thelevel of circulating free fatty acids is low. As triacylglyceroldoes not normally accumulate in the liver underthis condition, it must be inferred that it is transportedfrom the liver in VLDL as rapidly as it is synthesizedand that the synthesis of apo B-100 is not rate-limiting.Free fatty acids from the circulation are the main sourceduring starvation, the feeding of high-fat diets, or in diabetesmellitus, when hepatic lipogenesis is inhibited.Factors that enhance both the synthesis of triacylglyceroland the secretion of VLDL by the liver include (1)

212 / CHAPTER 25the fed state rather than the starved state; (2) the feedingof diets high in carbohydrate (particularly if theycontain sucrose or fructose), leading to high rates of lipogenesisand esterification of fatty acids; (3) high levelsof circulating free fatty acids; (4) ingestion ofethanol; and (5) the presence of high concentrations ofinsulin and low concentrations of glucagon, which enhancefatty acid synthesis and esterification and inhibittheir oxidation (Figure 25–6).CLINICAL ASPECTSImbalance in the Rate of TriacylglycerolFormation & Export Causes Fatty LiverFor a variety of reasons, lipid—mainly as triacylglycerol—canaccumulate in the liver (Figure 25–6). Extensiveaccumulation is regarded as a pathologic condition.When accumulation of lipid in the liver becomeschronic, fibrotic changes occur in the cells that progressto cirrhosis and impaired liver function.Fatty livers fall into two main categories. The firsttype is associated with raised levels of plasma freefatty acids resulting from mobilization of fat from adiposetissue or from the hydrolysis of lipoprotein triacylglycerolby lipoprotein lipase in extrahepatic tissues.The production of VLDL does not keep pace with theincreasing influx and esterification of free fatty acids, allowingtriacylglycerol to accumulate, causing a fattyliver. This occurs during starvation and the feeding ofhigh-fat diets. The ability to secrete VLDL may also beimpaired (eg, in starvation). In uncontrolled diabetesmellitus, twin lamb disease, and ketosis in cattle,fatty infiltration is sufficiently severe to cause visiblepallor (fatty appearance) and enlargement of the liverwith possible liver dysfunction.The second type of fatty liver is usually due to ametabolic block in the production of plasma lipoproteins,thus allowing triacylglycerol to accumulate.Theoretically, the lesion may be due to (1) a block inapolipoprotein synthesis, (2) a block in the synthesis ofthe lipoprotein from lipid and apolipoprotein, (3) afailure in provision of phospholipids that are found inlipoproteins, or (4) a failure in the secretory mechanismitself.One type of fatty liver that has been studied extensivelyin rats is due to a deficiency of choline, whichhas therefore been called a lipotropic factor. The antibioticpuromycin, ethionine (α-amino-γ-mercaptobutyricacid), carbon tetrachloride, chloroform, phosphorus,lead, and arsenic all cause fatty liver and a markedreduction in concentration of VLDL in rats. Cholinewill not protect the organism against these agents butappears to aid in recovery. The action of carbon tetrachlorideprobably involves formation of free radicalscausing lipid peroxidation. Some protection against thisis provided by the antioxidant action of vitamin E-supplementeddiets. The action of ethionine is thought tobe due to a reduction in availability of ATP due to itsreplacing methionine in S-adenosylmethionine, trappingavailable adenine and preventing synthesis ofATP. Orotic acid also causes fatty liver; it is believed tointerfere with glycosylation of the lipoprotein, thus inhibitingrelease, and may also impair the recruitment oftriacylglycerol to the particles. A deficiency of vitaminE enhances the hepatic necrosis of the choline deficiencytype of fatty liver. Added vitamin E or a sourceof selenium has a protective effect by combating lipidperoxidation. In addition to protein deficiency, essentialfatty acid and vitamin deficiencies (eg, linoleic acid,pyridoxine, and pantothenic acid) can cause fatty infiltrationof the liver.Ethanol Also Causes Fatty LiverAlcoholism leads to fat accumulation in the liver, hyperlipidemia,and ultimately cirrhosis. The exactmechanism of action of ethanol in the long term is stilluncertain. Ethanol consumption over a long periodleads to the accumulation of fatty acids in the liver thatare derived from endogenous synthesis rather than fromincreased mobilization from adipose tissue. There is noimpairment of hepatic synthesis of protein after ethanolingestion. Oxidation of ethanol by alcohol dehydrogenaseleads to excess production of NADH.CH 3 CH 2 OHEthanolALCOHOLDEHYDROGENASENAD + NADH + H +CH 3CHOAcetaldehydeThe NADH generated competes with reducingequivalents from other substrates, including fatty acids,for the respiratory chain, inhibiting their oxidation, anddecreasing activity of the citric acid cycle. The net effectof inhibiting fatty acid oxidation is to cause increasedesterification of fatty acids in triacylglycerol, resultingin the fatty liver. Oxidation of ethanol leads to the formationof acetaldehyde, which is oxidized by aldehydedehydrogenase, producing acetate. Other effects ofethanol may include increased lipogenesis and cholesterolsynthesis from acetyl-CoA, and lipid peroxidation.The increased [NADH]/[NAD + ] ratio also causes increased[lactate]/[pyruvate], resulting in hyperlacticacidemia,which decreases excretion of uric acid, aggravatinggout. Some metabolism of ethanol takes placevia a cytochrome P450-dependent microsomal ethanoloxidizing system (MEOS) involving NADPH and O 2 .This system increases in activity in chronic alcoholism

LIPID TRANSPORT & STORAGE / 213VLDLBLOODNascentVLDLApo CApo EHDLLIVERHEPATOCYTENascentVLDLGolgi complex–GlycosylresiduesCholesterolCholesterylesterSmoothendoplasmicreticulum–Triacylglycerol*TRIACYLGLYCEROLMembranesynthesisOrotic acidMPhospholipidCarbontetrachloride–EFADestructionof surplus apo B-100Apo B-100Apo CApo ECholesterol feedingEFA deficiencyPolyribosomesCarbon tetrachloridePuromycinEthionineRoughendoplasmicreticulumCholinedeficiency–LipidMAminoacidsProteinsynthesisNascentpolypeptidechains ofapo B-100Insulin+1,2-DiacylglycerolCDP-choline Phosphocholine CholineInsulinEthanolGlucagon+–Acyl-CoA–OxidationFFA+Lipogenesis fromcarbohydrateInsulinFigure 25–6. The synthesis of very low density lipoprotein (VLDL) in the liver and the possible loci of action offactors causing accumulation of triacylglycerol and a fatty liver. (EFA, essential fatty acids; FFA, free fatty acids;HDL, high-density lipoproteins; Apo, apolipoprotein; M, microsomal triacylglycerol transfer protein.) The pathwaysindicated form a basis for events depicted in Figure 25–2. The main triacylglycerol pool in liver is not on the directpathway of VLDL synthesis from acyl-CoA. Thus, FFA, insulin, and glucagon have immediate effects on VLDL secretionas their effects impinge directly on the small triacylglycerol* precursor pool. In the fully fed state, apo B-100 issynthesized in excess of requirements for VLDL secretion and the surplus is destroyed in the liver. During translationof apo B-100, microsomal transfer protein-mediated lipid transport enables lipid to become associated withthe nascent polypeptide chain. After release from the ribosomes, these particles fuse with more lipids from thesmooth endoplasmic reticulum, producing nascent VLDL.

214 / CHAPTER 25and may account for the increased metabolic clearancein this condition. Ethanol will also inhibit the metabolismof some drugs, eg, barbiturates, by competing forcytochrome P450-dependent enzymes.MEOSCH 3 CH + NADPH + H + 2 OH+ O 2Ethanol CH + NADP + 3 CHO+ 2H 2 OAcetaldehydeIn some Asian populations and Native Americans,alcohol consumption results in increased adverse reactionsto acetaldehyde owing to a genetic defect of mitochondrialaldehyde dehydrogenase.ADIPOSE TISSUE IS THE MAIN STOREOF TRIACYLGLYCEROL IN THE BODYThe triacylglycerol stores in adipose tissue are continuallyundergoing lipolysis (hydrolysis) and reesterification(Figure 25–7). These two processes are entirely differentpathways involving different reactants andenzymes. This allows the processes of esterification orlipolysis to be regulated separately by many nutritional,metabolic, and hormonal factors. The resultant of thesetwo processes determines the magnitude of the freefatty acid pool in adipose tissue, which in turn determinesthe level of free fatty acids circulating in theplasma. Since the latter has most profound effects uponthe metabolism of other tissues, particularly liver andmuscle, the factors operating in adipose tissue that regulatethe outflow of free fatty acids exert an influencefar beyond the tissue itself.The Provision of Glycerol 3-PhosphateRegulates Esterification: Lipolysis IsControlled by Hormone-Sensitive Lipase(Figure 25–7)Triacylglycerol is synthesized from acyl-CoA and glycerol3-phosphate (Figure 24–2). Because the enzymeglycerol kinase is not expressed in adipose tissue, glycerolcannot be utilized for the provision of glycerol3-phosphate, which must be supplied by glucose viaglycolysis.Triacylglycerol undergoes hydrolysis by a hormonesensitivelipase to form free fatty acids and glycerol.This lipase is distinct from lipoprotein lipase that catalyzeslipoprotein triacylglycerol hydrolysis before itsuptake into extrahepatic tissues (see above). Since glycerolcannot be utilized, it diffuses into the blood,whence it is utilized by tissues such as those of the liverand kidney, which possess an active glycerol kinase.FFA(pool 2)Insulin +BLOODADIPOSE TISSUE Glucose 6-phosphateCO2 PPPGlycolysisAcetyl-CoANADPH + H +Acyl-CoABLOODATPCoAACYL-CoASYNTHETASEFFA(pool 1)FFAFFAEsterificationTGLipolysisLIPOPROTEINLIPASEGlucoseHORMONE-SENSITIVELIPASETG(chylomicrons, VLDL)CO2Glycerol3-phosphateGlycerolGlycerolGlycerolFigure 25–7. Metabolism of adipose tissue. Hormone-sensitivelipase is activated by ACTH, TSH,glucagon, epinephrine, norepinephrine, and vasopressinand inhibited by insulin, prostaglandin E 1 , andnicotinic acid. Details of the formation of glycerol3-phosphate from intermediates of glycolysis areshown in Figure 24–2. (PPP, pentose phosphate pathway;TG, triacylglycerol; FFA, free fatty acids; VLDL, verylow density lipoprotein.)

LIPID TRANSPORT & STORAGE / 215The free fatty acids formed by lipolysis can be reconvertedin the tissue to acyl-CoA by acyl-CoA synthetaseand reesterified with glycerol 3-phosphate toform triacylglycerol. Thus, there is a continuous cycleof lipolysis and reesterification within the tissue.However, when the rate of reesterification is not sufficientto match the rate of lipolysis, free fatty acids accumulateand diffuse into the plasma, where they bind toalbumin and raise the concentration of plasma free fattyacids.Increased Glucose Metabolism Reducesthe Output of Free Fatty AcidsWhen the utilization of glucose by adipose tissue is increased,the free fatty acid outflow decreases. However,the release of glycerol continues, demonstrating that theeffect of glucose is not mediated by reducing the rate oflipolysis. The effect is due to the provision of glycerol3-phosphate, which enhances esterification of free fattyacids. Glucose can take several pathways in adipose tissue,including oxidation to CO 2 via the citric acidcycle, oxidation in the pentose phosphate pathway,conversion to long-chain fatty acids, and formation ofacylglycerol via glycerol 3-phosphate (Figure 25–7).When glucose utilization is high, a larger proportion ofthe uptake is oxidized to CO 2 and converted to fattyacids. However, as total glucose utilization decreases,the greater proportion of the glucose is directed to theformation of glycerol 3-phosphate for the esterificationof acyl-CoA, which helps to minimize the efflux of freefatty acids.HORMONES REGULATEFAT MOBILIZATIONInsulin Reduces the Outputof Free Fatty AcidsThe rate of release of free fatty acids from adipose tissueis affected by many hormones that influence either therate of esterification or the rate of lipolysis. Insulin inhibitsthe release of free fatty acids from adipose tissue,which is followed by a fall in circulating plasma freefatty acids. It enhances lipogenesis and the synthesis ofacylglycerol and increases the oxidation of glucose toCO 2 via the pentose phosphate pathway. All of these effectsare dependent on the presence of glucose and canbe explained, to a large extent, on the basis of the abilityof insulin to enhance the uptake of glucose into adiposecells via the GLUT 4 transporter. Insulin also increasesthe activity of pyruvate dehydrogenase, acetyl-CoA carboxylase, and glycerol phosphate acyltransferase,reinforcing the effects of increased glucose uptakeon the enhancement of fatty acid and acylglycerolsynthesis. These three enzymes are now known to beregulated in a coordinate manner by phosphorylationdephosphorylationmechanisms.A principal action of insulin in adipose tissue is toinhibit the activity of hormone-sensitive lipase, reducingthe release not only of free fatty acids but of glycerolas well. Adipose tissue is much more sensitive to insulinthan are many other tissues, which points to adiposetissue as a major site of insulin action in vivo.Several Hormones Promote LipolysisOther hormones accelerate the release of free fatty acidsfrom adipose tissue and raise the plasma free fatty acidconcentration by increasing the rate of lipolysis of thetriacylglycerol stores (Figure 25–8). These include epinephrine,norepinephrine, glucagon, adrenocorticotropichormone (ACTH), α- and β-melanocyte-stimulatinghormones (MSH), thyroid-stimulating hormone(TSH), growth hormone (GH), and vasopressin. Manyof these activate the hormone-sensitive lipase. For anoptimal effect, most of these lipolytic processes requirethe presence of glucocorticoids and thyroid hormones.These hormones act in a facilitatory or permissivecapacity with respect to other lipolytic endocrinefactors.The hormones that act rapidly in promoting lipolysis,ie, catecholamines, do so by stimulating the activityof adenylyl cyclase, the enzyme that converts ATP tocAMP. The mechanism is analogous to that responsiblefor hormonal stimulation of glycogenolysis (Chapter18). cAMP, by stimulating cAMP-dependent proteinkinase, activates hormone-sensitive lipase. Thus,processes which destroy or preserve cAMP influencelipolysis. cAMP is degraded to 5′-AMP by the enzymecyclic 3,5-nucleotide phosphodiesterase. This enzymeis inhibited by methylxanthines such as caffeineand theophylline. Insulin antagonizes the effect of thelipolytic hormones. Lipolysis appears to be more sensitiveto changes in concentration of insulin than are glucoseutilization and esterification. The antilipolytic effectsof insulin, nicotinic acid, and prostaglandin E 1 areaccounted for by inhibition of the synthesis of cAMP atthe adenylyl cyclase site, acting through a G i protein.Insulin also stimulates phosphodiesterase and the lipasephosphatase that inactivates hormone-sensitive lipase.The effect of growth hormone in promoting lipolysis isdependent on synthesis of proteins involved in the formationof cAMP. Glucocorticoids promote lipolysis viasynthesis of new lipase protein by a cAMP-independentpathway, which may be inhibited by insulin, and alsoby promoting transcription of genes involved in thecAMP signal cascade. These findings help to explainthe role of the pituitary gland and the adrenal cortex inenhancing fat mobilization. The recently discoveredbody weight regulatory hormone, leptin, stimulates

216 / CHAPTER 25Epinephrine,norepinephrineACTH,TSH,( )glucagonInsulin, prostaglandin E 1 ,nicotinic acidβ-AdrenergicblockersThyroid hormone–++ +–ATPGrowth hormoneInhibitors ofprotein synthesisGTP+––ADENYLYLCYCLASE–AdenosinePPi–FFAcAMP+ATPcAMPdependentproteinkinaseHormone-sensitivelipase b(inactive)Mg 2+PiInsulin+LipasephosphataseMethylxanthines(eg, caffeine)Thyroid hormone?––PHOSPHODI-ESTERASEInsulin+ +5′ AMPGlucocorticoids–ADP–cAMP-independent pathwayInsulinInhibitors ofprotein synthesisHormone-sensitivelipase a(active)PHormone-sensitivelipase2-MonoacylglycerollipaseTRIACYL-GLYCEROL–FFA +DiacylglycerolFFA +2-MonoacylglycerolFFA + glycerolFigure 25–8. Control of adipose tissue lipolysis. (TSH, thyroid-stimulating hormone; FFA, free fatty acids.)Note the cascade sequence of reactions affording amplification at each step. The lipolytic stimulus is “switchedoff” by removal of the stimulating hormone; the action of lipase phosphatase; the inhibition of the lipase andadenylyl cyclase by high concentrations of FFA; the inhibition of adenylyl cyclase by adenosine; and the removalof cAMP by the action of phosphodiesterase. ACTH, TSH, and glucagon may not activate adenylyl cyclase in vivo,since the concentration of each hormone required in vitro is much higher than is found in the circulation. Positive(+ ) and negative (− ) regulatory effects are represented by broken lines and substrate flow by solid lines.lipolysis and inhibits lipogenesis by influencing the activityof the enzymes in the pathways for the breakdownand synthesis of fatty acids.The sympathetic nervous system, through liberationof norepinephrine in adipose tissue, plays a central rolein the mobilization of free fatty acids. Thus, the increasedlipolysis caused by many of the factors describedabove can be reduced or abolished by denervationof adipose tissue or by ganglionic blockade.A Variety of Mechanisms Have Evolved forFine Control of Adipose Tissue MetabolismHuman adipose tissue may not be an important site oflipogenesis. There is no significant incorporation ofglucose or pyruvate into long-chain fatty acids; ATPcitratelyase, a key enzyme in lipogenesis, does not appearto be present, and other lipogenic enzymes—eg,glucose-6-phosphate dehydrogenase and the malic enzyme—donot undergo adaptive changes. Indeed, it hasbeen suggested that in humans there is a “carbohydrateexcess syndrome” due to a unique limitation in abilityto dispose of excess carbohydrate by lipogenesis. Inbirds, lipogenesis is confined to the liver, where it isparticularly important in providing lipids for egg formation,stimulated by estrogens. Human adipose tissueis unresponsive to most of the lipolytic hormones apartfrom the catecholamines.On consideration of the profound derangement ofmetabolism in diabetes mellitus (due in large part toincreased release of free fatty acids from the depots) andthe fact that insulin to a large extent corrects the condi-

LIPID TRANSPORT & STORAGE / 217+++OUTSIDENorepinephinecAMPHormonesensitivelipaseTriacylglycerolFFA+Acyl-CoAF 0F 1ATPsynthaseH +H + HeatF 0+H +–PurinenucleotidesINNERMITOCHONDRIALMEMBRANERespiratorychainThermogeninCarnitinetransporterH +INSIDEReducingequivalentsHeatFigure 25–9. Thermogenesis in brown adipose tissue.Activity of the respiratory chain produces heat inaddition to translocating protons (Chapter 12). Theseprotons dissipate more heat when returned to theinner mitochondrial compartment via thermogenin insteadof generating ATP when returning via the F 1 ATPsynthase. The passage of H + via thermogenin is inhibitedby purine nucleotides when brown adipose tissueis unstimulated. Under the influence of norepinephrine,the inhibition is removed by the production of freefatty acids (FFA) and acyl-CoA. Note the dual role ofacyl-CoA in both facilitating the action of thermogeninand supplying reducing equivalents for the respiratorychain. + and − signify positive or negative regulatoryeffects.β-Oxidationtion, it must be concluded that insulin plays a prominentrole in the regulation of adipose tissue metabolism.BROWN ADIPOSE TISSUEPROMOTES THERMOGENESISBrown adipose tissue is involved in metabolism particularlyat times when heat generation is necessary. Thus,the tissue is extremely active in some species in arousalfrom hibernation, in animals exposed to cold (nonshiveringthermogenesis), and in heat production in thenewborn animal. Though not a prominent tissue in humans,it is present in normal individuals, where it couldbe responsible for “diet-induced thermogenesis.” It isnoteworthy that brown adipose tissue is reduced or absentin obese persons. The tissue is characterized by awell-developed blood supply and a high content of mitochondriaand cytochromes but low activity of ATPsynthase. Metabolic emphasis is placed on oxidation ofboth glucose and fatty acids. Norepinephrine liberatedfrom sympathetic nerve endings is important in increasinglipolysis in the tissue and increasing synthesis oflipoprotein lipase to enhance utilization of triacylglycerol-richlipoproteins from the circulation. Oxidationand phosphorylation are not coupled in mitochondriaof this tissue, and the phosphorylation that does occuris at the substrate level, eg, at the succinate thiokinasestep and in glycolysis. Thus, oxidation produces muchheat, and little free energy is trapped in ATP. A thermogenicuncoupling protein, thermogenin, acts as aproton conductance pathway dissipating the electrochemicalpotential across the mitochondrial membrane(Figure 25–9).SUMMARY• Since nonpolar lipids are insoluble in water, fortransport between the tissues in the aqueous bloodplasma they are combined with amphipathic lipidsand proteins to make water-miscible lipoproteins.• Four major groups of lipoproteins are recognized:Chylomicrons transport lipids resulting from digestionand absorption. Very low density lipoproteins(VLDL) transport triacylglycerol from the liver. Lowdensitylipoproteins (LDL) deliver cholesterol to thetissues, and high-density lipoproteins (HDL) removecholesterol from the tissues in the process known asreverse cholesterol transport.• Chylomicrons and VLDL are metabolized by hydrolysisof their triacylglycerol, and lipoprotein remnantsare left in the circulation. These are taken up by liver,but some of the remnants (IDL) resulting fromVLDL form LDL which is taken up by the liver andother tissues via the LDL receptor.

218 / CHAPTER 25• Apolipoproteins constitute the protein moiety oflipoproteins. They act as enzyme activators (eg, apoC-II and apo A-I) or as ligands for cell receptors (eg,apo A-I, apo E, and apo B-100).• Triacylglycerol is the main storage lipid in adiposetissue. Upon mobilization, free fatty acids and glycerolare released. Free fatty acids are an importantfuel source.• Brown adipose tissue is the site of “nonshiveringthermogenesis.” It is found in hibernating and newbornanimals and is present in small quantity in humans.Thermogenesis results from the presence of anuncoupling protein, thermogenin, in the inner mitochondrialmembrane.REFERENCESChappell DA, Medh JD: Receptor-mediated mechanisms oflipoprotein remnant catabolism. Prog Lipid Res 1998;37:393.Eaton S et al: Multiple biochemical effects in the pathogenesis offatty liver. Eur J Clin Invest 1997;27:719.Goldberg IJ, Merkel M: Lipoprotein lipase: physiology, biochemistryand molecular biology. Front Biosci 2001;6:D388.Holm C et al: Molecular mechanisms regulating hormone sensitivelipase and lipolysis. Annu Rev Nutr 2000;20:365.Kaikans RM, Bass NM, Ockner RK: Functions of fatty acid bindingproteins. Experientia 1990;46:617.Lardy H, Shrago E: Biochemical aspects of obesity. Annu RevBiochem 1990;59:689.Rye K-A et al: Overview of plasma lipid transport. In: PlasmaLipids and Their Role in Disease. Barter PJ, Rye K-A (editors).Harwood Academic Publishers, 1999.Shelness GS, Sellers JA: Very-low-density lipoprotein assembly andsecretion. Curr Opin Lipidol 2001;12:151.Various authors: Biochemistry of Lipids, Lipoproteins and Membranes.Vance DE, Vance JE (editors). Elsevier, 1996.Various authors: Brown adipose tissue—role in nutritional energetics.(Symposium.) Proc Nutr Soc 1989;48:165.

Cholesterol Synthesis,Transport,& Excretion 26Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DScBIOMEDICAL IMPORTANCECholesterol is present in tissues and in plasma either asfree cholesterol or as a storage form, combined with along-chain fatty acid as cholesteryl ester. In plasma,both forms are transported in lipoproteins (Chapter25). Cholesterol is an amphipathic lipid and as such isan essential structural component of membranes and ofthe outer layer of plasma lipoproteins. It is synthesizedin many tissues from acetyl-CoA and is the precursor ofall other steroids in the body such as corticosteroids, sexhormones, bile acids, and vitamin D. As a typical productof animal metabolism, cholesterol occurs in foodsof animal origin such as egg yolk, meat, liver, andbrain. Plasma low-density lipoprotein (LDL) is the vehicleof uptake of cholesterol and cholesteryl ester intomany tissues. Free cholesterol is removed from tissuesby plasma high-density lipoprotein (HDL) and transportedto the liver, where it is eliminated from the bodyeither unchanged or after conversion to bile acids in theprocess known as reverse cholesterol transport. Cholesterolis a major constituent of gallstones. However,its chief role in pathologic processes is as a factor in thegenesis of atherosclerosis of vital arteries, causing cerebrovascular,coronary, and peripheral vascular disease.CHOLESTEROL IS DERIVEDABOUT EQUALLY FROM THE DIET& FROM BIOSYNTHESISA little more than half the cholesterol of the body arisesby synthesis (about 700 mg/d), and the remainder isprovided by the average diet. The liver and intestine accountfor approximately 10% each of total synthesis inhumans. Virtually all tissues containing nucleated cellsare capable of cholesterol synthesis, which occurs in theendoplasmic reticulum and the cytosol.Acetyl-CoA Is the Source of All CarbonAtoms in CholesterolThe biosynthesis of cholesterol may be divided into fivesteps: (1) Synthesis of mevalonate occurs from acetyl-CoA (Figure 26–1). (2) Isoprenoid units are formedfrom mevalonate by loss of CO 2 (Figure 26–2). (3) Sixisoprenoid units condense to form squalene. (4) Squalenecyclizes to give rise to the parent steroid, lanosterol.(5) Cholesterol is formed from lanosterol (Figure26–3).Step 1—Biosynthesis of Mevalonate: HMG-CoA(3-hydroxy-3-methylglutaryl-CoA) is formed by the reactionsused in mitochondria to synthesize ketone bodies(Figure 22–7). However, since cholesterol synthesisis extramitochondrial, the two pathways are distinct.Initially, two molecules of acetyl-CoA condense toform acetoacetyl-CoA catalyzed by cytosolic thiolase.Acetoacetyl-CoA condenses with a further molecule ofacetyl-CoA catalyzed by HMG-CoA synthase to formHMG-CoA, which is reduced to mevalonate byNADPH catalyzed by HMG-CoA reductase. This isthe principal regulatory step in the pathway of cholesterolsynthesis and is the site of action of the most effectiveclass of cholesterol-lowering drugs, the HMG-CoAreductase inhibitors (statins) (Figure 26–1).Step 2—Formation of Isoprenoid Units: Mevalonateis phosphorylated sequentially by ATP by threekinases, and after decarboxylation (Figure 26–2) the activeisoprenoid unit, isopentenyl diphosphate, isformed.Step 3—Six Isoprenoid Units Form Squalene:Isopentenyl diphosphate is isomerized by a shift of thedouble bond to form dimethylallyl diphosphate, thencondensed with another molecule of isopentenyldiphosphate to form the ten-carbon intermediate geranyldiphosphate (Figure 26–2). A further condensationwith isopentenyl diphosphate forms farnesyldiphosphate. Two molecules of farnesyl diphosphatecondense at the diphosphate end to form squalene. Initially,inorganic pyrophosphate is eliminated, formingpresqualene diphosphate, which is then reduced byNADPH with elimination of a further inorganic pyrophosphatemolecule.Step 4—Formation of Lanosterol: Squalene canfold into a structure that closely resembles the steroidnucleus (Figure 26–3). Before ring closure occurs, squaleneis converted to squalene 2,3-epoxide by a mixed-219

220 / CHAPTER 26OCH 3 C S CoA2 Acetyl-CoATHIOLASEOCCH 2– OOC C CH2 CH 2 OHCoA SHCH 3C CH 2OC S CoAOAcetoacetyl-CoA OH 2 OCH 3 C S CoAAcetyl-CoAHMG-CoA SYNTHASECoA SHCH 3– OOC CH 2 CH 2 C S CoAOH3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA)Bile acid, cholesterol2NADPH + 2H +HMG-CoA REDUCTASEStatins, eg,simvastatin2NADP + + CoA SHMevalonateCH 3OHMevalonateFigure 26–1. Biosynthesis of mevalonate. HMG-CoAreductase is inhibited by atorvastatin, pravastatin, andsimvastatin. The open and solid circles indicate the fateof each of the carbons in the acetyl moiety of acetyl-CoA.function oxidase in the endoplasmic reticulum, squaleneepoxidase. The methyl group on C 14 is transferredto C 13 and that on C 8 to C 14 as cyclization occurs, catalyzedby oxidosqualene:lanosterol cyclase.Step 5—Formation of Cholesterol: The formationof cholesterol from lanosterol takes place in themembranes of the endoplasmic reticulum and involveschanges in the steroid nucleus and side chain (Figure26–3). The methyl groups on C 14 and C 4 are removedto form 14-desmethyl lanosterol and then zymosterol.The double bond at C 8 –C 9 is subsequently moved toC 5 –C 6 in two steps, forming desmosterol. Finally, thedouble bond of the side chain is reduced, producingcholesterol. The exact order in which the steps describedactually take place is not known with certainty.Farnesyl Diphosphate Gives Riseto Dolichol & UbiquinoneThe polyisoprenoids dolichol (Figure 14–20 andChapter 47) and ubiquinone (Figure 12–5) are formedfrom farnesyl diphosphate by the further addition of upto 16 (dolichol) or 3–7 (ubiquinone) isopentenyldiphosphate residues, respectively. Some GTP-bindingproteins in the cell membrane are prenylated with farnesylor geranylgeranyl (20 carbon) residues. Proteinprenylation is believed to facilitate the anchoring ofproteins into lipoid membranes and may also be involvedin protein-protein interactions and membraneassociatedprotein trafficking.CHOLESTEROL SYNTHESIS ISCONTROLLED BY REGULATIONOF HMG-CoA REDUCTASERegulation of cholesterol synthesis is exerted near thebeginning of the pathway, at the HMG-CoA reductasestep. The reduced synthesis of cholesterol in starvinganimals is accompanied by a decrease in the activity ofthe enzyme. However, it is only hepatic synthesis that isinhibited by dietary cholesterol. HMG-CoA reductasein liver is inhibited by mevalonate, the immediate productof the pathway, and by cholesterol, the main product.Cholesterol (or a metabolite, eg, oxygenated sterol)represses transcription of the HMG-CoA reductasegene and is also believed to influence translation. A diurnalvariation occurs in both cholesterol synthesisand reductase activity. In addition to these mechanismsregulating the rate of protein synthesis, the enzyme activityis also modulated more rapidly by posttranslationalmodification (Figure 26–4). Insulin or thyroidhormone increases HMG-CoA reductase activity,whereas glucagon or glucocorticoids decrease it. Activityis reversibly modified by phosphorylation-dephosphorylationmechanisms, some of which may becAMP-dependent and therefore immediately responsiveto glucagon. Attempts to lower plasma cholesterol inhumans by reducing the amount of cholesterol in thediet produce variable results. Generally, a decrease of100 mg in dietary cholesterol causes a decrease of approximately0.13 mmol/L of serum.MANY FACTORS INFLUENCE THECHOLESTEROL BALANCE IN TISSUESIn tissues, cholesterol balance is regulated as follows (Figure26–5): Cell cholesterol increase is due to uptake ofcholesterol-containing lipoproteins by receptors, eg, theLDL receptor or the scavenger receptor; uptake of freecholesterol from cholesterol-rich lipoproteins to the cell

CH 3COHATPMg 2+ADPCH 3 OH– OOC C CH 2CH 2– OOCCH 2CH 2OHCH 2MEVALONATEKINASECH 2OPMevalonateMevalonate 5-phosphateATPPHOSPHOMEVALONATEKINASEMg 2+ ADPCH 3 O P– OOC C CH 2CH 2ADPMg 2+ATPCH 3 OH– OOC C CH 2CH 2CH 2OPPDIPHOSPHOMEVALONATEKINASECH 2OPPMevalonate 3-phospho-5-diphosphateMevalonate 5-diphosphateCO 2 + P itrans-MethylglutaconateshuntHMG-CoACH 3CH 3CCHCH 23,3-DimethylallyldiphosphateDIPHOSPHO-MEVALONATEDECARBOXYLASEO P PISOPENTENYL-DIPHOSPHATEISOMERASECH 3C CH 2CH 2 CH 2IsopentenyldiphosphateO P PIsopentenyl tRNACIS-PRENYLTRANSFERASEPP iCH 3CH 3Prenylated proteinsCH 3CCHCH 2CCH 2CHCH 2O P PGeranyl diphosphateCIS-PRENYLTRANSFERASESide chain ofubiquinoneTRANS-PRENYLTRANSFERASEPP iCIS-PRENYLTRANSFERASECH * 2DolicholO P PHeme aFarnesyl diphosphateNADPH + H +SQUALENE SYNTHETASE Mg 2+ , Mn 2+2PP i NADP +CH *2*CH 2SqualeneFigure 26–2. Biosynthesis of squalene, ubiquinone, dolichol, and other polyisoprene derivatives. (HMG,3-hydroxy-3-methylglutaryl; ⋅×⋅⋅, cytokinin.) A farnesyl residue is present in heme a of cytochrome oxidase.The carbon marked with asterisk becomes C 11 or C 12 in squalene. Squalene synthetase is a microsomal enzyme;all other enzymes indicated are soluble cytosolic proteins, and some are found in peroxisomes.221

222 / CHAPTER 26OCH 3CH 3CH 3CSCoA– OOC C CH 2 CH 2 OH CH3 C CH CH 2 –CH 2CO 2 H 2 OOHAcetyl-CoA Mevalonate Isoprenoid unitCH 3CH 2CCH 2CCH 2121224 CH 324 CH 3CH 2 13 CH HC C* CH2 13 CH HC CSqualene*11CH 11CHCH CH CH 3CH 2 CH CH 3epoxide 2221 CH 1 CHCH 3CH 32 CH14CCH 2 2 CH14CCH 28SQUALENE8H 2 CC CCH 3 EPOXIDASE H 2 CC CCH 3CH 3CH 3HC 3 CH CHNADPH 1 HC 3 X62/2 O CH CHFAD 22CCH 2 CCH 2 SqualeneOOXIDOSQUALENE:CH 3LANOSTEROLCH 3 CH 3CH 3CYCLASECH 3CH 2HO4814LanosterolNADPHO 2HCOOHHO1414-DesmethyllanosterolO 2 , NADPHNAD +2CO 2HO8ZymosterolISOMERASE2122HO1 24 251711 12 NADPH19 13 1627C D14 1510∆ 24 -REDUCTASE818 20 23262A B34567Cholesterol –HOTriparanol3524Desmosterol(24-dehydrocholesterol)NADPHO 2HO724∆ 7,24 -CholestadienolFigure 26–3. Biosynthesis of cholesterol. The numbered positions are those of the steroid nucleus and theopen and solid circles indicate the fate of each of the carbons in the acetyl moiety of acetyl-CoA. Asterisks: Referto labeling of squalene in Figure 26–2.

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION / 223ATPREDUCTASEKINASE(inactive)PiREDUCTASEKINASEKINASEPPROTEINPHOSPHATASES+?InsulinADPREDUCTASEKINASE(active)H 2 O–Glucagon+HMG-CoALDL-cholesterolCholesterolOxysterols–ATPHMG-CoAREDUCTASE(active)PiEnzyme synthesisPROTEINPHOSPHATASESP?+–ADPHMG-CoAREDUCTASE(inactive)H 2 OInsulinInhibitor-1-phosphate*cAMP+Figure 26–4. Possible mechanisms in the regulation of cholesterol synthesis by HMG-CoA reductase. Insulinhas a dominant role compared with glucagon. Asterisk: See Figure 18–6.membrane; cholesterol synthesis; and hydrolysis of cholesterylesters by the enzyme cholesteryl ester hydrolase.Decrease is due to efflux of cholesterol from the membraneto HDL, promoted by LCAT (lecithin:cholesterolacyltransferase) (Chapter 25); esterification of cholesterolby ACAT (acyl-CoA:cholesterol acyltransferase); and utilizationof cholesterol for synthesis of other steroids, suchas hormones, or bile acids in the liver.The LDL Receptor Is Highly RegulatedLDL (apo B-100, E) receptors occur on the cell surfacein pits that are coated on the cytosolic side of the cellmembrane with a protein called clathrin. The glycoproteinreceptor spans the membrane, the B-100 bindingregion being at the exposed amino terminal end. Afterbinding, LDL is taken up intact by endocytosis. Theapoprotein and cholesteryl ester are then hydrolyzed inthe lysosomes, and cholesterol is translocated into thecell. The receptors are recycled to the cell surface. Thisinflux of cholesterol inhibits in a coordinated mannerHMG-CoA synthase, HMG-CoA reductase, and,therefore, cholesterol synthesis; stimulates ACAT activity;and down-regulates synthesis of the LDL receptor.Thus, the number of LDL receptors on the cell surfaceis regulated by the cholesterol requirement for membranes,steroid hormones, or bile acid synthesis (Figure26–5). The apo B-100, E receptor is a “high-affinity”LDL receptor, which may be saturated under most circumstances.Other “low-affinity” LDL receptors alsoappear to be present in addition to a scavenger pathway,which is not regulated.CHOLESTEROL IS TRANSPORTEDBETWEEN TISSUES IN PLASMALIPOPROTEINS(Figure 26–6)In Western countries, the total plasma cholesterol inhumans is about 5.2 mmol/L, rising with age, thoughthere are wide variations between individuals. Thegreater part is found in the esterified form. It is transportedin lipoproteins of the plasma, and the highestproportion of cholesterol is found in the LDL. Dietarycholesterol equilibrates with plasma cholesterol in days

224 / CHAPTER 26CELL MEMBRANELDL (apo B-100, E)receptors(in coated pits)LDLLDLCECoatedvesicleScavenger receptor ornonregulated pathwayRecyclingvesicleEndosomeCECECE CLysosomeReceptorsynthesisLysosomeCEC–Down-regulationUnesterifiedcholesterolpool(mainly in membranes)Cholesterolsynthesis–+ACATCEHYDROLASECELDLVLDLCSynthesisof steroidsABC-1A-1PL CPreβ-HDLFigure 26–5. Factors affecting cholesterol balance at the cellular level. Reverse cholesterol transport maybe initiated by preβ HDL binding to the ABC-1 transporter protein via apo A-I. Cholesterol is then moved outof the cell via the transporter, lipidating the HDL, and the larger particles then dissociate from the ABC-1 molecule.(C, cholesterol; CE, cholesteryl ester; PL, phospholipid; ACAT, acyl-CoA:cholesterol acyltransferase; LCAT,lecithin:cholesterol acyltransferase; A-I, apolipoprotein A-I; LDL, low-density lipoprotein; VLDL, very low densitylipoprotein.) LDL and HDL are not shown to scale.LCATCEPLHDL3A-1and with tissue cholesterol in weeks. Cholesteryl esterin the diet is hydrolyzed to cholesterol, which is thenabsorbed by the intestine together with dietary unesterifiedcholesterol and other lipids. With cholesterol synthesizedin the intestines, it is then incorporated intochylomicrons. Of the cholesterol absorbed, 80–90% isesterified with long-chain fatty acids in the intestinalmucosa. Ninety-five percent of the chylomicron cholesterolis delivered to the liver in chylomicron remnants,and most of the cholesterol secreted by the liver inVLDL is retained during the formation of IDL and ultimatelyLDL, which is taken up by the LDL receptorin liver and extrahepatic tissues (Chapter 25).Plasma LCAT Is Responsible for VirtuallyAll Plasma Cholesteryl Ester in HumansLCAT activity is associated with HDL containing apoA-I. As cholesterol in HDL becomes esterified, it createsa concentration gradient and draws in cholesterolfrom tissues and from other lipoproteins (Figures 26–5and 26–6), thus enabling HDL to function in reversecholesterol transport (Figure 25–5).Cholesteryl Ester Transfer ProteinFacilitates Transfer of Cholesteryl EsterFrom HDL to Other LipoproteinsThis protein is found in plasma of humans and manyother species, associated with HDL. It facilitates transferof cholesteryl ester from HDL to VLDL, IDL, and LDLin exchange for triacylglycerol, relieving product inhibitionof LCAT activity in HDL. Thus, in humans, muchof the cholesteryl ester formed by LCAT finds its way tothe liver via VLDL remnants (IDL) or LDL (Figure26–6). The triacylglycerol-enriched HDL 2 delivers itscholesterol to the liver in the HDL cycle (Figure 25–5).

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION / 225LIVER–CLRP receptorChylomicronremnantSynthesisUnesterifiedcholesterolpoolTGCECC–Bile acids(total pool, 3–5 g)ACATCCEHLTG, CELDL(apo B-100, E)receptorTGCECCECLDLTGCETGCEIDL(VLDL remnant)ENTEROHEPATIC CIRCULATIONTGCECHEPATIC PORTAL VEINCEVLDLTGLCATA-IHDLCECCETPGALLBLADDERBILE DUCTChylomicronTGCECCECDiet (0.4 g/d)CCECEBileacidsC(0.6 g/d)Bile acids(0.4 g/d)FecesILEUM9 8–99 %CLDL(apo B-100, E)receptorLPLCEXTRAHEPATICTISSUESCSynthesisCEFigure 26–6. Transport of cholesterol between the tissues in humans. (C, unesterified cholesterol; CE, cholesterylester; TG, triacylglycerol; VLDL, very low density lipoprotein; IDL, intermediate-density lipoprotein; LDL,low-density lipoprotein; HDL, high-density lipoprotein; ACAT, acyl-CoA:cholesterol acyltransferase; LCAT,lecithin:cholesterol acyltransferase; A-I, apolipoprotein A-I; CETP, cholesteryl ester transfer protein; LPL, lipoproteinlipase; HL, hepatic lipase; LRP, LDL receptor-related protein.)CHOLESTEROL IS EXCRETED FROM THEBODY IN THE BILE AS CHOLESTEROL ORBILE ACIDS (SALTS)About 1 g of cholesterol is eliminated from the bodyper day. Approximately half is excreted in the feces afterconversion to bile acids. The remainder is excreted ascholesterol. Coprostanol is the principal sterol in thefeces; it is formed from cholesterol by the bacteria inthe lower intestine.Bile Acids Are Formed From CholesterolThe primary bile acids are synthesized in the liver fromcholesterol. These are cholic acid (found in the largestamount) and chenodeoxycholic acid (Figure 26–7).

226 / CHAPTER 2612 17Vitamin CNADP +HO3 77α-HYDROXYLASEHO7OHCholesterol7α-HydroxycholesterolBileacidsVitamin CdeficiencyNADPH + H +O 2O 2NADPH + H +12α-HYDROX-YLASEO 2NADPH + H +2 CoA SH(Severalsteps)2 CoA SHPropionyl-CoAPropionyl-CoAHOHO*HOHCOTaurocholic acid(primary bile acid)CoAHOHOHOHGlycocholic acid(primary bile acid)COHNCoASHHNDeconjugation+ 7α-dehydroxylation(CH 2 ) 2GlycineSHTaurineHOHOCH 2 COOHSO 3 HHHOHCholyl-CoAOHOH12Deoxycholic acid(secondary bile acid)COCOOHSCoAHOHOHOHCOChenodeoxycholyl-CoATauro- and glycochenodeoxycholicacid(primary bile acids)H*Lithocholic acid(secondary bile acid)SCoADeconjugation+ 7α-dehydroxylationFigure 26–7. Biosynthesis and degradation of bile acids. A second pathway in mitochondria involves hydroxylationof cholesterol by sterol 27-hydroxylase. Asterisk: Catalyzed by microbial enzymes.COOHThe 7α-hydroxylation of cholesterol is the first andprincipal regulatory step in the biosynthesis of bile acidscatalyzed by 7-hydroxylase, a microsomal enzyme. Atypical monooxygenase, it requires oxygen, NADPH,and cytochrome P450. Subsequent hydroxylation stepsare also catalyzed by monooxygenases. The pathway ofbile acid biosynthesis divides early into one subpathwayleading to cholyl-CoA, characterized by an extra α-OHgroup on position 12, and another pathway leading tochenodeoxycholyl-CoA (Figure 26–7). A second pathwayin mitochondria involving the 27-hydroxylation ofcholesterol by sterol 27-hydroxylase as the first step isresponsible for a significant proportion of the primarybile acids synthesized. The primary bile acids (Figure26–7) enter the bile as glycine or taurine conjugates.Conjugation takes place in peroxisomes. In humans, theratio of the glycine to the taurine conjugates is normally3:1. In the alkaline bile, the bile acids and their conju-

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION / 227gates are assumed to be in a salt form—hence the term“bile salts.”A portion of the primary bile acids in the intestine issubjected to further changes by the activity of the intestinalbacteria. These include deconjugation and 7αdehydroxylation,which produce the secondary bileacids, deoxycholic acid and lithocholic acid.Most Bile Acids Return to the Liverin the Enterohepatic CirculationAlthough products of fat digestion, including cholesterol,are absorbed in the first 100 cm of small intestine,the primary and secondary bile acids are absorbed almostexclusively in the ileum, and 98–99% are returnedto the liver via the portal circulation. This isknown as the enterohepatic circulation (Figure 26–6).However, lithocholic acid, because of its insolubility, isnot reabsorbed to any significant extent. Only a smallfraction of the bile salts escapes absorption and is thereforeeliminated in the feces. Nonetheless, this representsa major pathway for the elimination of cholesterol.Each day the small pool of bile acids (about 3–5 g) iscycled through the intestine six to ten times and anamount of bile acid equivalent to that lost in the feces issynthesized from cholesterol, so that a pool of bile acidsof constant size is maintained. This is accomplished bya system of feedback controls.Bile Acid Synthesis Is Regulatedat the 7-Hydroxylase StepThe principal rate-limiting step in the biosynthesis ofbile acids is at the cholesterol 7-hydroxylase reaction(Figure 26–7). The activity of the enzyme is feedback-regulatedvia the nuclear bile acid-binding receptorfarnesoid X receptor (FXR). When the size of thebile acid pool in the enterohepatic circulation increases,FXR is activated and transcription of the cholesterol7α-hydroxylase gene is suppressed. Chenodeoxycholicacid is particularly important in activating FXR. Cholesterol7α-hydroxylase activity is also enhanced bycholesterol of endogenous and dietary origin and regulatedby insulin, glucagon, glucocorticoids, and thyroidhormone.CLINICAL ASPECTSThe Serum Cholesterol Is Correlated Withthe Incidence of Atherosclerosis &Coronary Heart DiseaseWhile cholesterol is believed to be chiefly concerned inthe relationship, other serum lipids such as triacylglycerolsmay also play a role. Atherosclerosis is characterizedby the deposition of cholesterol and cholesterylester from the plasma lipoproteins into the artery wall.Diseases in which prolonged elevated levels of VLDL,IDL, chylomicron remnants, or LDL occur in theblood (eg, diabetes mellitus, lipid nephrosis, hypothyroidism,and other conditions of hyperlipidemia) areoften accompanied by premature or more severe atherosclerosis.There is also an inverse relationship betweenHDL (HDL 2 ) concentrations and coronary heart disease,and some consider that the most predictive relationshipis the LDL:HDL cholesterol ratio. This isconsistent with the function of HDL in reverse cholesteroltransport. Susceptibility to atherosclerosis varieswidely among species, and humans are one of the fewin which the disease can be induced by diets high incholesterol.Diet Can Play an Important Role inReducing Serum CholesterolHereditary factors play the greatest role in determiningindividual serum cholesterol concentrations; however,dietary and environmental factors also play a part, andthe most beneficial of these is the substitution in thediet of polyunsaturated and monounsaturated fattyacids for saturated fatty acids. Plant oils such as corn oiland sunflower seed oil contain a high proportion ofpolyunsaturated fatty acids, while olive oil contains ahigh concentration of monounsaturated fatty acids. Onthe other hand, butterfat, beef fat, and palm oil containa high proportion of saturated fatty acids. Sucrose andfructose have a greater effect in raising blood lipids, particularlytriacylglycerols, than do other carbohydrates.The reason for the cholesterol-lowering effect ofpolyunsaturated fatty acids is still not fully understood.It is clear, however, that one of the mechanisms involvedis the up-regulation of LDL receptors by polyandmonounsaturated as compared with saturated fattyacids, causing an increase in the catabolic rate of LDL,the main atherogenic lipoprotein. In addition, saturatedfatty acids cause the formation of smaller VLDL particlesthat contain relatively more cholesterol, and theyare utilized by extrahepatic tissues at a slower rate thanare larger particles—tendencies that may be regarded asatherogenic.Lifestyle Affects the SerumCholesterol LevelAdditional factors considered to play a part in coronaryheart disease include high blood pressure, smoking,male gender, obesity (particularly abdominal obesity),lack of exercise, and drinking soft as opposed to hardwater. Factors associated with elevation of plasma FFAfollowed by increased output of triacylglycerol and cho-

228 / CHAPTER 26lesterol into the circulation in VLDL include emotionalstress and coffee drinking. Premenopausal women appearto be protected against many of these deleteriousfactors, and this is thought to be related to the beneficialeffects of estrogen. There is an association betweenmoderate alcohol consumption and a lower incidenceof coronary heart disease. This may be due to elevationof HDL concentrations resulting from increased synthesisof apo A-I and changes in activity of cholesterylester transfer protein. It has been claimed that red wineis particularly beneficial, perhaps because of its contentof antioxidants. Regular exercise lowers plasma LDLTable 26–1. Primary disorders of plasma lipoproteins (dyslipoproteinemias).Name Defect RemarksHypolipoproteinemias No chylomicrons, VLDL, or LDL are Rare; blood acylglycerols low; intestine and liverAbetalipoproteinemia formed because of defect in the accumulate acylglycerols. Intestinal malabsorploadingof apo B with lipid.tion. Early death avoidable by administration oflarge doses of fat-soluble vitamins, particularlyvitamin E.Familial alpha-lipoprotein deficiency All have low or near absence of HDL. Tendency toward hypertriacylglycerolemia as aTangier diseaseresult of absence of apo C-II, causing inactiveFish-eye diseaseLPL. Low LDL levels. Atherosclerosis in the el-Apo-A-I deficienciesderly.Hyperlipoproteinemias Hypertriacylglycerolemia due to de- Slow clearance of chylomicrons and VLDL. LowFamilial lipoprotein lipase ficiency of LPL, abnormal LPL, or apo levels of LDL and HDL. No increased risk of corodeficiency(type I) C-II deficiency causing inactive LPL. nary disease.Familial hypercholesterolemia Defective LDL receptors or mutation Elevated LDL levels and hypercholesterolemia,(type IIa) in ligand region of apo B-100. resulting in atherosclerosis and coronary disease.Familial type III hyperlipoprotein- Deficiency in remnant clearance by Increase in chylomicron and VLDL remnants ofemia (broad beta disease, rem- the liver is due to abnormality in apo density < 1.019 (β-VLDL). Causes hypercholesnantremoval disease, familial E. Patients lack isoforms E3 and E4 terolemia, xanthomas, and atherosclerosis.dysbetalipoproteinemia)and have only E2, which does notreact with the E receptor. 1Familial hypertriacylglycerolemia Overproduction of VLDL often Cholesterol levels rise with the VLDL concentra-(type IV) associated with glucose intolerance tion. LDL and HDL tend to be subnormal. Thisand hyperinsulinemia.type of pattern is commonly associated withcoronary heart disease, type II diabetes mellitus,obesity, alcoholism, and administration ofprogestational hormones.Familial hyperalphalipoproteinemia Increased concentrations of HDL. A rare condition apparently beneficial to healthand longevity.Hepatic lipase deficiency Deficiency of the enzyme leads to Patients have xanthomas and coronary heartaccumulation of large triacylgly- disease.cerol-rich HDL and VLDL remnants.Familial lecithin:cholesterol Absence of LCAT leads to block in Plasma concentrations of cholesteryl esters andacyltransferase (LCAT) deficiency reverse cholesterol transport. HDL lysolecithin are low. Present is an abnormal LDLremains as nascent disks incapable fraction, lipoprotein X, found also in patientsof taking up and esterifying choles- with cholestasis. VLDL is abnormal (β-VLDL).terol.Familial lipoprotein(a) excess Lp(a) consists of 1 mol of LDL Premature coronary heart disease due to atheroattachedto 1 mol of apo(a). Apo(a) sclerosis, plus thrombosis due to inhibition ofshows structural homologies to plas- fibrinolysis.minogen.1 There is an association between patients possessing the apo E4 allele and the incidence of Alzheimer’s disease. Apparently, apo E4 bindsmore avidly to β-amyloid found in neuritic plaques.

CHOLESTEROL SYNTHESIS, TRANSPORT, & EXCRETION / 229but raises HDL. Triacylglycerol concentrations are alsoreduced, due most likely to increased insulin sensitivity,which enhances expression of lipoprotein lipase.When Diet Changes Fail, HypolipidemicDrugs Will Reduce Serum Cholesterol& TriacylglycerolSignificant reductions of plasma cholesterol can be effectedmedically by the use of cholestyramine resin orsurgically by the ileal exclusion operations. Both proceduresblock the reabsorption of bile acids, causing increasedbile acid synthesis in the liver. This increasescholesterol excretion and up-regulates LDL receptors,lowering plasma cholesterol. Sitosterol is a hypocholesterolemicagent that acts by blocking the absorption ofcholesterol from the gastrointestinal tract.Several drugs are known to block the formation ofcholesterol at various stages in the biosynthetic pathway.The statins inhibit HMG-CoA reductase, thusup-regulating LDL receptors. Statins currently in useinclude atorvastatin, simvastatin, and pravastatin. Fibratessuch as clofibrate and gemfibrozil act mainly tolower plasma triacylglycerols by decreasing the secretionof triacylglycerol and cholesterol-containing VLDL bythe liver. In addition, they stimulate hydrolysis ofVLDL triacylglycerols by lipoprotein lipase. Probucolappears to increase LDL catabolism via receptorindependentpathways, but its antioxidant propertiesmay be more important in preventing accumulation ofoxidized LDL, which has enhanced atherogenic properties,in arterial walls. Nicotinic acid reduces the flux ofFFA by inhibiting adipose tissue lipolysis, thereby inhibitingVLDL production by the liver.Primary Disorders of the PlasmaLipoproteins (Dyslipoproteinemias)Are InheritedInherited defects in lipoprotein metabolism lead to theprimary condition of either hypo- or hyperlipoproteinemia(Table 26–1). In addition, diseases such asdiabetes mellitus, hypothyroidism, kidney disease(nephrotic syndrome), and atherosclerosis are associatedwith secondary abnormal lipoprotein patterns thatare very similar to one or another of the primary inheritedconditions. Virtually all of the primary conditionsare due to a defect at a stage in lipoprotein formation,transport, or destruction (see Figures 25–4, 26–5, and26–6). Not all of the abnormalities are harmful.SUMMARY• Cholesterol is the precursor of all other steroids inthe body, eg, corticosteroids, sex hormones, bileacids, and vitamin D. It also plays an importantstructural role in membranes and in the outer layer oflipoproteins.• Cholesterol is synthesized in the body entirely fromacetyl-CoA. Three molecules of acetyl-CoA formmevalonate via the important regulatory reaction forthe pathway, catalyzed by HMG-CoA reductase.Next, a five-carbon isoprenoid unit is formed, andsix of these condense to form squalene. Squalene undergoescyclization to form the parent steroid lanosterol,which, after the loss of three methyl groups,forms cholesterol.• Cholesterol synthesis in the liver is regulated partlyby cholesterol in the diet. In tissues, cholesterol balanceis maintained between the factors causing gainof cholesterol (eg, synthesis, uptake via the LDL orscavenger receptors) and the factors causing loss ofcholesterol (eg, steroid synthesis, cholesteryl ester formation,excretion). The activity of the LDL receptoris modulated by cellular cholesterol levels to achievethis balance. In reverse cholesterol transport, HDL(preβ-HDL, discoidal, or HDL 3 ) takes up cholesterolfrom the tissues and LCAT esterifies it and depositsit in the core of HDL, which is converted to HDL 2 .The cholesteryl ester in HDL 2 is taken up by theliver, either directly or after transfer to VLDL, IDL,or LDL via the cholesteryl ester transfer protein.• Excess cholesterol is excreted from the liver in thebile as cholesterol or bile salts. A large proportion ofbile salts is absorbed into the portal circulation andreturned to the liver as part of the enterohepatic circulation.• Elevated levels of cholesterol present in VLDL, IDL,or LDL are associated with atherosclerosis, whereashigh levels of HDL have a protective effect.• Inherited defects in lipoprotein metabolism lead to aprimary condition of hypo- or hyperlipoproteinemia.Conditions such as diabetes mellitus, hypothyroidism,kidney disease, and atherosclerosis exhibitsecondary abnormal lipoprotein patterns that resemblecertain primary conditions.REFERENCESIllingworth DR: Management of hypercholesterolemia. Med ClinNorth Am 2000;84:23.Ness GC, Chambers CM: Feedback and hormonal regulation ofhepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase:the concept of cholesterol buffering capacity. Proc Soc ExpBiol Med 2000;224:8.Parks DJ et al: Bile acids: natural ligands for a nuclear orphan receptor.Science 1999;284:1365.Princen HMG: Regulation of bile acid synthesis. Curr Pharm Design1997;3:59.

230 / CHAPTER 26Russell DW: Cholesterol biosynthesis and metabolism. CardiovascularDrugs Therap 1992;6:103.Spady DK, Woollett LA, Dietschy JM: Regulation of plasma LDLcholesterollevels by dietary cholesterol and fatty acids. AnnuRev Nutr 1993;13:355.Tall A: Plasma lipid transfer proteins. Annu Rev Biochem 1995;64:235.Various authors: Biochemistry of Lipids, Lipoproteins and Membranes.Vance DE, Vance JE (editors). Elsevier, 1996.Various authors: The cholesterol facts. A summary of the evidencerelating dietary fats, serum cholesterol, and coronary heartdisease. Circulation 1990;81:1721.Zhang FL, Casey PJ: Protein prenylation: Molecular mechanismsand functional consequences. Annu Rev Biochem 1996;65:241.

Integration of Metabolism—The Provision of Metabolic Fuels 27David A Bender, PhD, & Peter A. Mayes, PhD, DScBIOMEDICAL IMPORTANCEAn adult human weighing 70 kg requires about 10–12MJ (2400–2900 kcal) from metabolic fuels each day.This requirement is met from carbohydrates (40–60%),lipids (mainly triacylglycerol, 30–40%), protein (10–15%), and alcohol if consumed. The mix being oxidizedvaries depending on whether the subject is in thefed or starving state and on the intensity of physicalwork. The requirement for metabolic fuels is relativelyconstant throughout the day, since average physical activityonly increases metabolic rate by about 40–50%over the basal metabolic rate. However, most peopleconsume their daily intake of metabolic fuels in two orthree meals, so there is a need to form reserves of carbohydrate(glycogen in liver and muscle) and lipid (triacylglycerolin adipose tissue) for use between meals.If the intake of fuels is consistently greater than energyexpenditure, the surplus is stored, largely as fat,leading to the development of obesity and its associatedhealth hazards. If the intake of fuels is consistentlylower than energy expenditure, there will be negligiblefat and carbohydrate reserves, and amino acids arisingfrom protein turnover will be used for energy ratherthan replacement protein synthesis, leading to emaciationand eventually death.After a normal meal there is an ample supply of carbohydrate,and the fuel for most tissues is glucose. Inthe starving state, glucose must be spared for use by thecentral nervous system (which is largely dependent onglucose) and the erythrocytes (which are wholly relianton glucose). Other tissues can utilize alternative fuelssuch as fatty acids and ketone bodies. As glycogen reservesbecome depleted, so amino acids arising fromprotein turnover and glycerol arising from lipolysis areused for gluconeogenesis. These events are largely controlledby the hormones insulin and glucagon. In diabetesmellitus there is either impaired synthesis andsecretion of insulin (type 1 diabetes mellitus) or impairedsensitivity of tissues to insulin action (type 2 diabetesmellitus), leading to severe metabolic derangement.In cattle the demands of heavy lactation can leadto ketosis, as can the demands of twin pregnancy insheep.MANY METABOLIC FUELSARE INTERCONVERTIBLECarbohydrate in excess of immediate requirements asfuel or for synthesis of glycogen in muscle and liver maybe used for lipogenesis (Chapter 21) and hence triacylglycerolsynthesis in both adipose tissue and liver(whence it is exported in very low density lipoprotein).The importance of lipogenesis in human beings is unclear;in Western countries, dietary fat provides35–45% of energy intake, while in less developed countrieswhere carbohydrate may provide 60–75% of energyintake the total intake of food may be so low thatthere is little surplus for lipogenesis. A high intake of fatinhibits lipogenesis.Fatty acids (and ketone bodies formed from them)cannot be used for the synthesis of glucose. The reactionof pyruvate dehydrogenase, forming acetyl-CoA, isirreversible, and for every two-carbon unit from acetyl-CoA that enters the citric acid cycle there is a loss oftwo carbon atoms as carbon dioxide before only onemolecule of oxaloacetate is re-formed—ie, there is nonet increase. This means that acetyl-CoA (and thereforeany substrates that yield acetyl-CoA) can never be usedfor gluconeogenesis (Chapter 19). The (relatively rare)fatty acids with an odd number of carbon atoms yieldpropionyl-CoA as the product of the final cycle of β-oxidation (Chapter 22), and this can be a substrate forgluconeogenesis, as can the glycerol released by lipolysisof adipose tissue triacylglycerol reserves. Most of theamino acids in excess of requirements for protein synthesis(arising from the diet or from tissue proteinturnover) yield pyruvate, or five- and four-carbon intermediatesof the citric acid cycle. Pyruvate can becarboxylated to oxaloacetate, which is the primarysubstrate for gluconeogenesis, and the five- andfour-carbon intermediates also result in a net increase inthe formation of oxaloacetate, which is then availablefor gluconeogenesis. These amino acids are classified asglucogenic. Lysine and leucine yield only acetyl-CoAon oxidation and thus cannot be used for gluconeogenesis,while phenylalanine, tyrosine, tryptophan, andisoleucine give rise to both acetyl-CoA and to intermediatesof the citric acid cycle that can be used for gluco-231

232 / CHAPTER 27neogenesis. Those amino acids that give rise to acetyl-CoA are classified as ketogenic because in the starvingstate much of the acetyl-CoA will be used for synthesisof ketone bodies in the liver.A SUPPLY OF METABOLIC FUELSIS PROVIDED IN BOTH THE FED& STARVING STATES(Figure 27–1)Glucose Is Always Required by the CentralNervous System & ErythrocytesErythrocytes lack mitochondria and hence are whollyreliant on glycolysis and the pentose phosphate pathway.The brain can metabolize ketone bodies to meetabout 20% of its energy requirements; the remaindermust be supplied by glucose. The metabolic changesthat occur in starvation are the consequences of theneed to preserve glucose and the limited reserves ofglycogen in liver for use by the brain and erythrocytesand to ensure the provision of alternative fuels for othertissues. The fetus and synthesis of lactose in milk alsorequire a significant amount of glucose.In the Fed State, Metabolic FuelReserves Are Laid DownFor several hours after a meal, while the products of digestionare being absorbed, there is an abundant supplyof metabolic fuels. Under these conditions, glucose isthe major fuel for oxidation in most tissues; this is observedas an increase in the respiratory quotient (theratio of carbon dioxide produced to oxygen consumed)from about 0.8 in the starved state to near 1 (Table27–1).Glucose uptake into muscle and adipose tissue iscontrolled by insulin, which is secreted by the B isletcells of the pancreas in response to an increased concentrationof glucose in the portal blood. An early responseto insulin in muscle and adipose tissue is the migrationof glucose transporter vesicles to the cell surface, exposingactive glucose transporters (GLUT 4). These insulin-sensitivetissues will only take up glucose from theblood stream to any significant extent in the presence ofthe hormone. As insulin secretion falls in the starvedstate, so the transporters are internalized again, reducingglucose uptake.The uptake of glucose into the liver is independentof insulin, but liver has an isoenzyme of hexokinase(glucokinase) with a high K m , so that as the concentrationof glucose entering the liver increases, so does therate of synthesis of glucose 6-phosphate. This is in excessof the liver’s requirement for energy and is usedmainly for synthesis of glycogen. In both liver andskeletal muscle, insulin acts to stimulate glycogen synthaseand inhibit glycogen phosphorylase. Some of theglucose entering the liver may also be used for lipogenesisand synthesis of triacylglycerol. In adipose tissue, insulinstimulates glucose uptake, its conversion to fattyacids, and their esterification; and inhibits intracellularlipolysis and the release of free fatty acids.The products of lipid digestion enter the circulationas triacylglycerol-rich chylomicrons (Chapter 25). Inadipose tissue and skeletal muscle, lipoprotein lipase isactivated in response to insulin; the resultant free fattyacids are largely taken up to form triacylglycerol reserves,while the glycerol remains in the blood streamand is taken up by the liver and used for glycogen synthesisor lipogenesis. Free fatty acids remaining in theblood stream are taken up by the liver and reesterified.The lipid-depleted chylomicron remnants are alsocleared by the liver, and surplus liver triacylglycerol—including that from lipogenesis—is exported in verylow density lipoprotein.Under normal feeding patterns the rate of tissueprotein catabolism is more or less constant throughoutthe day; it is only in cachexia that there is an increasedrate of protein catabolism. There is net protein catabolismin the postabsorptive phase of the feeding cycleand net protein synthesis in the absorptive phase, whenthe rate of synthesis increases by about 20–25%. Theincreased rate of protein synthesis is, again, a responseto insulin action. Protein synthesis is an energy-expensiveprocess, accounting for up to almost 20% of energyexpenditure in the fed state, when there is an amplesupply of amino acids from the diet, but under 9% inthe starved state.Metabolic Fuel Reserves Are Mobilizedin the Starving StateThere is a small fall in plasma glucose upon starvation,then little change as starvation progresses (Table 27–2;Figure 27–2). Plasma free fatty acids increase withonset of starvation but then plateau. There is an initialdelay in ketone body production, but as starvation progressesthe plasma concentration of ketone bodies increasesmarkedly.In the postabsorptive state, as the concentration ofglucose in the portal blood falls, so insulin secretion decreases,resulting in skeletal muscle and adipose tissuetaking up less glucose. The increase in secretion ofglucagon from the A cells of the pancreas inhibitsglycogen synthase and activates glycogen phosphorylasein liver. The resulting glucose 6-phosphate in liver is

INTEGRATION OF METABOLISM—THE PROVISION OF METABOLIC FUELS / 233Glucose 6-phosphateAcyl-CoAGlycerol 3-phosphateADIPOSETISSUETRIACYLGLYCEROL (TG)cAMPFFAGlycerolEXTRAHEPATICTISSUE (eg,heart muscle)FFALPLGlycerolBLOODGlycerolOxidationLPLFFAFFAChylomicronsTG(lipoproteins)GASTRO-INTESTINALTRACTGlucoseVLDLExtraglucosedrain (eg,diabetes,pregnancy,lactation)GlucoseKetone bodiesFFATGLIVERGlucoseAcyl-CoAGlycerol 3-phosphateAcetyl-CoACitricacidcycle2CO 2Amino acids,lactateGluconeogenesisGlucose 6-phosphateGlycogenFigure 27–1. Metabolic interrelationships between adipose tissue, the liver, and extrahepatictissues. In extrahepatic tissues such as heart, metabolic fuels are oxidized in the following order ofpreference: (1) ketone bodies, (2) fatty acids, (3) glucose. (LPL, lipoprotein lipase; FFA, free fattyacids; VLDL, very low density lipoproteins.)

234 / CHAPTER 27Table 27–1. Energy yields, oxygen consumption, and carbondioxide production in the oxidation of metabolic fuels.Energy Yield O 2 Consumed CO 2 Produced Oxygen(kJ/g) (L/g) (L/g) RQ (kJ/L)Carbohydrate 16 0.829 0.829 1.00 20Protein 17 0.966 0.782 0.81 20Fat 37 2.016 1.427 0.71 20hydrolyzed by glucose-6-phosphatase, and glucose is releasedinto the blood stream for use by other tissues,particularly the brain and erythrocytes.Muscle glycogen cannot contribute directly toplasma glucose, since muscle lacks glucose-6-phosphatase,and the primary purpose of muscle glycogen isto provide a source of glucose 6-phosphate for energyyieldingmetabolism in the muscle itself. However,acetyl-CoA formed by oxidation of fatty acids in muscleinhibits pyruvate dehydrogenase and leads to citrate accumulation,which in turn inhibits phosphofructokinaseand therefore glycolysis, thus sparing glucose. Anyaccumulated pyruvate is transaminated to alanine at theexpense of amino acids arising from breakdown of proteinreserves. The alanine—and much of the keto acidsresulting from this transamination—are exported frommuscle and taken up by the liver, where the alanine istransaminated to yield pyruvate. The resultant aminoacids are largely exported back to muscle to provideamino groups for formation of more alanine, while thepyruvate is a major substrate for gluconeogenesis in theliver.In adipose tissue, the effect of the decrease in insulinand increase in glucagon results in inhibition of lipogenesis,inactivation of lipoprotein lipase, and activationof hormone-sensitive lipase (Chapter 25). Thisleads to release of increased amounts of glycerol (a substratefor gluconeogenesis in the liver) and free fattyacids, which are used by skeletal muscle and liver astheir preferred metabolic fuels, so sparing glucose.Although muscle takes up and preferentially oxidizesfree fatty acids in the starving state, it cannot meet all ofits energy requirements by β-oxidation. By contrast, theliver has a greater capacity for β-oxidation than it requiresto meet its own energy needs and forms moreacetyl-CoA than can be oxidized. This acetyl-CoA isused to synthesize ketone bodies (Chapter 22), whichare major metabolic fuels for skeletal and heart muscleand can meet some of the brain’s energy needs. In prolongedstarvation, glucose may represent less than 10%of whole body energy-yielding metabolism. Furthermore,as a result of protein catabolism, an increasingnumber of amino acids are released and utilized in theliver and kidneys for gluconeogenesis.Relative changePlasmainsulinPlasma freefatty acidsPlasma glucagonBlood glucoseTable 27–2. Plasma concentrations of metabolicfuels (mmol/L) in the fed and starving states.40 Hours 7 DaysFed Starvation StarvationGlucose 5.5 3.6 3.5Free fatty acids 0.30 1.15 1.19Ketone bodies Negligible 2.9 4.5BloodketonebodiesLiver glycogen012–24Hours of starvationFigure 27–2. Relative changes in metabolic parametersduring the onset of starvation.

Table 27–3. Summary of the major and unique features of metabolism of the principal organs.Organ Major Function Major Pathways Main Substrates Major Products Specialist EnzymesLiver Service for the other Most represented, inclu- Free fatty acids, glucose (well Glucose, VLDL (triacylglyc- Glucokinase, glucose-6-phosphatase,organs and tissues ding gluconeogenesis; fed), lactate, glycerol, fructose, erol), HDL, ketone bodies, glycerol kinase, phosphoenolpyruvateβ-oxidation; ketogenesis; amino acids urea, uric acid, bile acids, carboxykinase, fructokinase, arginase,lipoprotein formation; plasma proteins HMG-CoA synthase and lyase, 7αurea,uric acid, and bilehydroxylaseacid formation; cholesterolsynthesis; lipogenesis 1 (Ethanol) (Acetate) (Alcohol dehydrogenase)Brain Coordination of the Glycolysis, amino acid me- Glucose, amino acid, ketone Lactatenervous system tabolism bodies (in starvation)Polyunsaturated fatty acidsin neonateHeart Pumping of blood Aerobic pathways, eg, Free fatty acids, lactate, ke- Lipoprotein lipase. Respiratory chainβ-oxidation and citric acid tone bodies, VLDL and chylo- well developed.cyclemicron triacylglycerol, someglucoseAdipose tissue Storage and break- Esterification of fatty acids Glucose, lipoprotein triacyl- Free fatty acids, glycerol Lipoprotein lipase, hormone-sensitivedown of triacylglyc- and lipolysis; lipogenesis 1 glycerol lipaseerolMuscleFast twitch Rapid movement Glycolysis Glucose LactateSlow twitch Sustained movement Aerobic pathways, eg, Ketone bodies, triacylglycerol Lipoprotein lipase.β-oxidation and citric in VLDL and chylomicrons, Respiratory chain well developed.acid cyclefree fatty acidsKidney Excretion and glu- Gluconeogenesis Free fatty acids, lactate, Glucose Glycerol kinase, phosphoenolpyruvateconeogenesis glycerol carboxykinaseErythrocytes Transport of O 2 Glycolysis, pentose phos- Glucose Lactate (Hemoglobin)phate pathway. No mitochondriaand therefore noβ-oxidation or citric acidcycle.1 In many species but not very active in humans.

236 / CHAPTER 27CLINICAL ASPECTSIn prolonged starvation, as adipose tissue reserves aredepleted there is a very considerable increase in the netrate of protein catabolism to provide amino acids notonly as substrates for gluconeogenesis but also as themain metabolic fuel of the tissues. Death results whenessential tissue proteins are catabolized beyond thepoint at which they can sustain this metabolic drain. Inpatients with cachexia as a result of release of cytokinesin response to tumors and a number of other pathologicconditions, there is an increase in the rate of tissueprotein catabolism as well as a considerably increasedmetabolic rate, resulting in a state of advanced starvation.Again, death results when essential tissue proteinshave been catabolized.The high demand for glucose by the fetus and forsynthesis of lactose in lactation can lead to ketosis. Thismay be seen as mild ketosis with hypoglycemia inwomen, but in lactating cattle and in ewes carryingtwins there may be very pronounced ketosis and profoundhypoglycemia.In poorly controlled type 1 diabetes mellitus, patientsmay become hyperglycemic, partly as a result oflack of insulin to stimulate uptake and utilization ofglucose and partly because of increased gluconeogenesisfrom amino acids in the liver. At the same time, thelack of insulin results in increased lipolysis in adiposetissue, and the resultant free fatty acids are substratesfor ketogenesis in the liver. It is possible that in very severediabetes utilization of ketone bodies in muscle(and other tissues) is impaired because of lack of oxaloacetate(most tissues have a requirement for someglucose metabolism to maintain an adequate amount ofoxaloacetate for citric acid cycle activity). In uncontrolleddiabetes, the magnitude of ketosis may be suchas to result in severe acidosis (ketoacidosis) since acetoaceticacid and 3-hydroxybutyric acid are relativelystrong acids. Coma results from both the acidosis andthe considerably increased osmolarity of extracellularfluid (mainly due to the hyperglycemia).A summary of the major and unique metabolic featuresof the principal tissues is presented in Table 27–3.SUMMARY• The body can interconvert the majority of foodstuffs.However, there is no net conversion of most fattyacids (or other acetyl-CoA-forming substances) toglucose. Most amino acids, arising from the diet orfrom tissue protein, can be used for gluconeogenesis,as can the glycerol from triacylglycerol.• In starvation, glucose must be provided for the brainand erythrocytes; initially, this is supplied from liverglycogen reserves. To spare glucose, muscle and othertissues reduce glucose uptake in response to loweredinsulin secretion; they also oxidize fatty acids and ketonebodies preferentially to glucose.• Adipose tissue releases free fatty acids in starvation,and these are used by many tissues as fuel. Furthermore,in the liver they are the substrate for synthesisof ketone bodies.• Ketosis, a metabolic adaptation to starvation, is exacerbatedin pathologic conditions such as diabetesmellitus and ruminant ketosis.REFERENCESBender DA: Introduction to Nutrition and Metabolism, 3rd edition.Taylor & Francis, 2002.Caprio S et al: Oxidative fuel metabolism during mild hypoglycemia:critical role of free fatty acids. Am J Physiol1989;256:E413.Fell D: Understanding the Control of Metabolism. Portland Press,1997.Frayn KN: Metabolic Regulation—A Human Perspective. PortlandPress, 1996.McNamara JP: Role and regulation of metabolism in adipose tissueduring lactation. J Nutr Biochem 1995;6:120.Randle PJ: The glucose-fatty acid cycle—biochemical aspects. AtherosclerosisRev 1991;22:183.

SECTION IIIMetabolism of Proteins & Amino AcidsBiosynthesis of the NutritionallyNonessential Amino Acids 28Victor W. Rodwell, PhDBIOMEDICAL IMPORTANCEAll 20 of the amino acids present in proteins are essentialfor health. While comparatively rare in the Westernworld, amino acid deficiency states are endemic in certainregions of West Africa where the diet relies heavilyon grains that are poor sources of amino acids such astryptophan and lysine. These disorders include kwashiorkor,which results when a child is weaned onto astarchy diet poor in protein; and marasmus, in whichboth caloric intake and specific amino acids are deficient.Humans can synthesize 12 of the 20 common aminoacids from the amphibolic intermediates of glycolysis andof the citric acid cycle (Table 28–1). While nutritionallynonessential, these 12 amino acids are not “nonessential.”All 20 amino acids are biologically essential. Of the 12 nutritionallynonessential amino acids, nine are formed fromamphibolic intermediates and three (cysteine, tyrosineand hydroxylysine) from nutritionally essential aminoacids. Identification of the twelve amino acids that humanscan synthesize rested primarily on data derived fromfeeding diets in which purified amino acids replaced protein.This chapter considers only the biosynthesis of thetwelve amino acids that are synthesized in human tissues,not the other eight that are synthesized by plants.NUTRITIONALLY NONESSENTIALAMINO ACIDS HAVE SHORTBIOSYNTHETIC PATHWAYSThe enzymes glutamate dehydrogenase, glutamine synthetase,and aminotransferases occupy central positionsin amino acid biosynthesis. The combined effect ofthose three enzymes is to transform ammonium ioninto the α-amino nitrogen of various amino acids.Glutamate and Glutamine. Reductive amination ofα-ketoglutarate is catalyzed by glutamate dehydrogenase(Figure 28–1). Amination of glutamate to glutamine iscatalyzed by glutamine synthetase (Figure 28–2).Alanine. Transamination of pyruvate forms alanine(Figure 28–3).Aspartate and Asparagine. Transamination ofoxaloacetate forms aspartate. The conversion of aspartateTable 28–1. Amino acid requirementsof humans.Nutritionally Essential Nutritionally NonessentialArginine 1AlanineHistidineAsparagineIsoleucineAspartateLeucineCysteineLysineGlutamateMethionineGlutaminePhenylalanineGlycineThreonine Hydroxyproline 2Tryptophan Hydroxylysine 2ValineProlineSerineTyrosine1 “Nutritionally semiessential.” Synthesized at rates inadequateto support growth of children.2 Not necessary for protein synthesis but formed during posttranslationalprocessing of collagen.237

238 / CHAPTER 28O– O O–NH+ 3– O O–OO –NH 3+O –OOα-KetoglutarateOOL-GlutamatePyruvateOOAlanineNH 4+H 2 OGlu or Aspα-Ketoglutarate or oxaloacetateNAD(P)H + H + NAD(P) +Figure 28–1. The glutamate dehydrogenasereaction.Figure 28–3. Formation of alanine by transaminationof pyruvate. The amino donor may be glutamate oraspartate. The other product thus is α-ketoglutarate oroxaloacetate.to asparagine is catalyzed by asparagine synthetase (Figure28–4), which resembles glutamine synthetase (Figure28–2) except that glutamine, not ammonium ion,provides the nitrogen. Bacterial asparagine synthetasescan, however, also use ammonium ion. Coupled hydrolysisof PP i to P i by pyrophosphatase ensures thatthe reaction is strongly favored.Serine. Oxidation of the α-hydroxyl group of theglycolytic intermediate 3-phosphoglycerate converts itto an oxo acid, whose subsequent transamination anddephosphorylation leads to serine (Figure 28–5).Glycine. Glycine aminotransferases can catalyze thesynthesis of glycine from glyoxylate and glutamate oralanine. Unlike most aminotransferase reactions, thesestrongly favor glycine synthesis. Additional importantmammalian routes for glycine formation are fromcholine (Figure 28–6) and from serine (Figure 28–7).Proline. Proline is formed from glutamate by reversalof the reactions of proline catabolism (Figure 28–8).Cysteine. Cysteine, while not nutritionally essential,is formed from methionine, which is nutritionallyessential. Following conversion of methionine to ho-NH 3+– O O–OOL-GlutamateFigure 28–2.NH 4+Mg-ATPNH 3+H 2 N O –OOL-GlutamineMg-ADP + P iThe glutamine synthetase reaction.–OO NH 3+OL-AspartateGlnMg-ATPO –H 2 NO NH 3+OL-AsparagineGluMg-AMP + PP iFigure 28–4. The asparagine synthetase reaction.Note similarities to and differences from the glutaminesynthetase reaction (Figure 28–2).POHOOHNH 3+OOL-SerineO −O −D-3-PhosphoglycerateNADHNH +3P i H 2 OPPOOOOOO –O −Phosphohydroxypyruvateα-AAα-KAO −Phospho-L-serineFigure 28–5. Serine biosynthesis. (α-AA, α-aminoacids; α-KA, α-keto acids.)

BIOSYNTHESIS OF THE NUTRITIONALLY NONESSENTIAL AMINO ACIDS / 239H 3 CCholineH 3 CHDimethylglycineCH 32HCH 3N + CH 3H N + 3 C CH 3+NOHCH 3OO −[CH 2 O]Betaine aldehyde[CH 3 ]H 3 CNAD +BetaineOCH3N + CH 3H + CH 3 [CH 2 O]NH +N3HO −Sarcosine OOGlycine OO −O −OO −– O O NH+ 3L-GlutamateONADHH 2 OOOO −L-Glutamateγ-semialdehydeH 2 ONH 3+O − NADHO −NH+ 2NH +L-Proline∆ 2 -Pyrrolidine-5-carboxylateFigure 28–8. Biosynthesis of proline from glutamateby reversal of reactions of proline catabolism.OFigure 28–6.Formation of glycine from choline.NH 3+mocysteine (see Chapter 30), homocysteine and serineform cysteine and homoserine (Figure 28–9).Tyrosine. Phenylalanine hydroxylase convertsphenylalanine to tyrosine (Figure 28–10). Providedthat the diet contains adequate nutritionally essentialphenylalanine, tyrosine is nutritionally nonessential.But since the reaction is irreversible, dietary tyrosinecannot replace phenylalanine. Catalysis by this mixedfunctionoxygenase incorporates one atom of O 2 intophenylalanine and reduces the other atom to water. Reducingpower, provided as tetrahydrobiopterin, derivesultimately from NADPH.HONH 3+OSerineO –H 4 folateMethyleneH 4 folateNH 3+OO –GlycineFigure 28–7. The serine hydroxymethyltransferasereaction. The reaction is freely reversible. (H 4 folate,tetrahydrofolate.)O −H 3 N +OL-HomocysteineH 3 N +OO −HO O+ L-SerineS HH 2 ONH+ 3H 2 OO −O −L-HomoserineNH 3+H 3 N +OO −HS OOHL-Cysteine+SCystathionineFigure 28–9. Conversion of homocysteine and serineto homoserine and cysteine. The sulfur of cysteinederives from methionine and the carbon skeleton fromserine.OO −

240 / CHAPTER 28CH 2 CH COO –+ NH 3HOH 2 NDihydrobiopterinTetrahydrobiopterinO 2L-PhenylalanineNADP + NADPH + H +NHNHN CHOHCHOHCH 3TetrahydrobiopterinHOIIIHNH 2 OCH 2L-TyrosineCH COO −+ NH 3Figure 28–10. The phenylalanine hydroxylase reaction.Two distinct enzymatic activities are involved. ActivityII catalyzes reduction of dihydrobiopterin byNADPH, and activity I the reduction of O 2 to H 2 O and ofphenylalanine to tyrosine. This reaction is associatedwith several defects of phenylalanine metabolism discussedin Chapter 30.Hydroxyproline and Hydroxylysine. Hydroxyprolineand hydroxylysine are present principally incollagen. Since there is no tRNA for either hydroxylatedamino acid, neither dietary hydroxyproline norhydroxylysine is incorporated into protein. Both arecompletely degraded (see Chapter 30). Hydroxyprolineand hydroxylysine arise from proline and lysine, butonly after these amino acids have been incorporatedinto peptides. Hydroxylation of peptide-bound prolyland lysyl residues is catalyzed by prolyl hydroxylase andlysyl hydroxylase of tissues, including skin and skeletalmuscle, and of granulating wounds (Figure 28–11).The hydroxylases are mixed-function oxygenases thatrequire substrate, molecular O 2 , ascorbate, Fe 2+ , andα-ketoglutarate. For every mole of proline or lysine hydroxylated,one mole of α-ketoglutarate is decarboxylatedto succinate. One atom of O 2 is incorporated intoproline or lysine, the other into succinate (Figure28–11). A deficiency of the vitamin C required forthese hydroxylases results in scurvy.Valine, Leucine, and Isoleucine. While leucine,valine, and isoleucine are all nutritionally essentialα-KetoglutarateFe 2+18 O 2Ascorbate18 OHPro[ 18 O] SuccinateProFigure 28–11. The prolyl hydroxylase reaction. Thesubstrate is a proline-rich peptide. During the course ofthe reaction, molecular oxygen is incorporated intoboth succinate and proline. Lysyl hydroxylase catalyzesan analogous reaction.amino acids, tissue aminotransferases reversibly interconvertall three amino acids and their correspondingα-keto acids. These α-keto acids thus can replace theiramino acids in the diet.Selenocysteine. While not normally consideredan amino acid present in proteins, selenocysteine occursat the active sites of several enzymes. Examples includethe human enzymes thioredoxin reductase, glutathioneperoxidase, and the deiodinase that convertsthyroxine to triiodothyronine. Unlike hydroxyprolineor hydroxylysine, selenocysteine arises co-translationallyduring its incorporation into peptides. The UGAanticodon of the unusual tRNA designated tRNA Secnormally signals STOP. The ability of the protein syntheticapparatus to identify a selenocysteine-specificUGA codon involves the selenocysteine insertion element,a stem-loop structure in the untranslated regionof the mRNA. Selenocysteine-tRNA Sec is first chargedwith serine by the ligase that charges tRNA Ser . Subsequentreplacement of the serine oxygen by seleniuminvolves selenophosphate formed by selenophosphatesynthase (Figure 28–12).Se + ATPHSeHCH 2 C COO –+NH 3AMP + P i + HSeOPO –Figure 28–12. Selenocysteine (top) and the reactioncatalyzed by selenophosphate synthetase (bottom).O –

BIOSYNTHESIS OF THE NUTRITIONALLY NONESSENTIAL AMINO ACIDS / 241SUMMARY• All vertebrates can form certain amino acids fromamphibolic intermediates or from other dietaryamino acids. The intermediates and the amino acidsto which they give rise are α-ketoglutarate (Glu, Gln,Pro, Hyp), oxaloacetate (Asp, Asn) and 3-phosphoglycerate(Ser, Gly).• Cysteine, tyrosine, and hydroxylysine are formedfrom nutritionally essential amino acids. Serine providesthe carbon skeleton and homocysteine the sulfurfor cysteine biosynthesis. Phenylalanine hydroxylaseconverts phenylalanine to tyrosine.• Neither dietary hydroxyproline nor hydroxylysine isincorporated into proteins because no codon ortRNA dictates their insertion into peptides.• Peptidyl hydroxyproline and hydroxylysine areformed by hydroxylation of peptidyl proline or lysinein reactions catalyzed by mixed-function oxidasesthat require vitamin C as cofactor. The nutritionaldisease scurvy reflects impaired hydroxylation due toa deficiency of vitamin C.• Selenocysteine, an essential active site residue in severalmammalian enzymes, arises by co-translationalinsertion of a previously modified tRNA.REFERENCESBrown KM, Arthur JR: Selenium, selenoproteins and humanhealth: a review. Public Health Nutr 2001;4:593.Combs GF, Gray WP: Chemopreventive agents—selenium. PharmacolTher 1998;79:179.Mercer LP, Dodds SJ, Smith DI: Dispensable, indispensable, andconditionally indispensable amino acid ratios in the diet. In:Absorption and Utilization of Amino Acids. Friedman M (editor).CRC Press, 1989.Nordberg J et al: Mammalian thioredoxin reductase is irreversiblyinhibited by dinitrohalobenzenes by alkylation of both theredox active selenocysteine and its neighboring cysteineresidue. J Biol Chem 1998;273:10835.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.St Germain DL, Galton VA: The deiodinase family of selenoproteins.Thyroid 1997;7:655.

Catabolism of Proteins& of Amino Acid Nitrogen 29Victor W. Rodwell, PhDBIOMEDICAL IMPORTANCEThis chapter describes how the nitrogen of amino acids isconverted to urea and the rare disorders that accompanydefects in urea biosynthesis. In normal adults, nitrogenintake matches nitrogen excreted. Positive nitrogen balance,an excess of ingested over excreted nitrogen, accompaniesgrowth and pregnancy. Negative nitrogenbalance, where output exceeds intake, may followsurgery, advanced cancer, and kwashiorkor or marasmus.While ammonia, derived mainly from the α-aminonitrogen of amino acids, is highly toxic, tissues convertammonia to the amide nitrogen of nontoxic glutamine.Subsequent deamination of glutamine in the liver releasesammonia, which is then converted to nontoxicurea. If liver function is compromised, as in cirrhosis orhepatitis, elevated blood ammonia levels generate clinicalsigns and symptoms. Rare metabolic disorders involveeach of the five urea cycle enzymes.PROTEIN TURNOVER OCCURSIN ALL FORMS OF LIFEThe continuous degradation and synthesis of cellularproteins occur in all forms of life. Each day humansturn over 1–2% of their total body protein, principallymuscle protein. High rates of protein degradation occurin tissues undergoing structural rearrangement—eg,uterine tissue during pregnancy, tadpole tail tissue duringmetamorphosis, or skeletal muscle in starvation. Ofthe liberated amino acids, approximately 75% are reutilized.The excess nitrogen forms urea. Since excessamino acids are not stored, those not immediately incorporatedinto new protein are rapidly degraded.242PROTEASES & PEPTIDASES DEGRADEPROTEINS TO AMINO ACIDSThe susceptibility of a protein to degradation is expressedas its half-life (t 1/2 ), the time required to lowerits concentration to half the initial value. Half-lives ofliver proteins range from under 30 minutes to over 150hours. Typical “housekeeping” enzymes have t 1/2 valuesof over 100 hours. By contrast, many key regulatory enzymeshave a t 1/2 of 0.5–2 hours. PEST sequences, regionsrich in proline (P), glutamate (E), serine (S), andthreonine (T), target some proteins for rapid degradation.Intracellular proteases hydrolyze internal peptidebonds. The resulting peptides are then degraded toamino acids by endopeptidases that cleave internalbonds and by aminopeptidases and carboxypeptidasesthat remove amino acids sequentially from the aminoand carboxyl terminals, respectively. Degradation ofcirculating peptides such as hormones follows loss of asialic acid moiety from the nonreducing ends of theiroligosaccharide chains. Asialoglycoproteins are internalizedby liver cell asialoglycoprotein receptors and degradedby lysosomal proteases termed cathepsins.Extracellular, membrane-associated, and long-livedintracellular proteins are degraded in lysosomes byATP-independent processes. By contrast, degradationof abnormal and other short-lived proteins occurs inthe cytosol and requires ATP and ubiquitin. Ubiquitin,so named because it is present in all eukaryotic cells, is asmall (8.5 kDa) protein that targets many intracellularproteins for degradation. The primary structure ofubiquitin is highly conserved. Only 3 of 76 residuesdiffer between yeast and human ubiquitin. Several moleculesof ubiquitin are attached by non-α-peptidebonds formed between the carboxyl terminal of ubiquitinand the ε-amino groups of lysyl residues in the targetprotein (Figure 29–1). The residue present at itsamino terminal affects whether a protein is ubiquitinated.Amino terminal Met or Ser retards whereas Aspor Arg accelerates ubiquitination. Degradation occursin a multicatalytic complex of proteases known as theproteasome.ANIMALS CONVERT -AMINO NITROGENTO VARIED END PRODUCTSDifferent animals excrete excess nitrogen as ammonia,uric acid, or urea. The aqueous environment ofteleostean fish, which are ammonotelic (excrete ammonia),compels them to excrete water continuously, facilitatingexcretion of highly toxic ammonia. Birds, whichmust conserve water and maintain low weight, are uricotelicand excrete uric acid as semisolid guano. Many

CATABOLISM OF PROTEINS & OF AMINO ACID NITROGEN / 243OO1.2.3.UB C O – + E 1 SH + ATP AMP + PP i + UB C S E 1OOUB C S E 1 + E 2 SH E 1 SH + UB C S E 2OE 3O HUB C S E 2 + H 2 N ε Protein E 2 SH + UB C N ε ProteinFigure 29–1. Partial reactions in the attachment of ubiquitin (UB) toproteins. (1) The terminal COOH of ubiquitin forms a thioester bond withan -SH of E 1 in a reaction driven by conversion of ATP to AMP and PP i . Subsequenthydrolysis of PP i by pyrophosphatase ensures that reaction 1 willproceed readily. (2) A thioester exchange reaction transfers activated ubiquitinto E 2 . (3) E 3 catalyzes transfer of ubiquitin to ε-amino groups of lysylresidues of target proteins.land animals, including humans, are ureotelic and excretenontoxic, water-soluble urea. High blood urea levelsin renal disease are a consequence—not a cause—ofimpaired renal function.BIOSYNTHESIS OF UREAUrea biosynthesis occurs in four stages: (1) transamination,(2) oxidative deamination of glutamate, (3) ammoniatransport, and (4) reactions of the urea cycle(Figure 29–2).Transamination Transfers α-AminoNitrogen to α-Ketoglutarate,Forming GlutamateTransamination interconverts pairs of α-amino acidsand α-keto acids (Figure 29–3). All the protein aminoacids except lysine, threonine, proline, and hydroxyprolineparticipate in transamination. Transamination isreadily reversible, and aminotransferases also functionin amino acid biosynthesis. The coenzyme pyridoxalphosphate (PLP) is present at the catalytic site ofaminotransferases and of many other enzymes that acton amino acids. PLP, a derivative of vitamin B 6 , formsan enzyme-bound Schiff base intermediate that can rearrangein various ways. During transamination, boundPLP serves as a carrier of amino groups. Rearrangementforms an α-keto acid and enzyme-bound pyridoxaminephosphate, which forms a Schiff base with a secondketo acid. Following removal of α-amino nitrogen bytransamination, the remaining carbon “skeleton” is degradedby pathways discussed in Chapter 30.Alanine-pyruvate aminotransferase (alanine aminotransferase)and glutamate-α-ketoglutarate aminotransferase(glutamate aminotransferase) catalyze the transferα-Amino acidα-Keto acidTRANSAMINATIONα-KetoglutarateL-GlutamateR 1NH+3CHCO –R 1OCCO –OXIDATIVEDEAMINATIONOOFigure 29–2.catabolism.NH 3 CO 2UREA CYCLEUreaOverall flow of nitrogen in amino acidONH+3C O –CH O –R 2 C R 2 COOFigure 29–3. Transamination. The reaction is freelyreversible with an equilibrium constant close to unity.

244 / CHAPTER 29of amino groups to pyruvate (forming alanine) or to α-ketoglutarate (forming glutamate) (Figure 29–4). Eachaminotransferase is specific for one pair of substratesbut nonspecific for the other pair. Since alanine is also asubstrate for glutamate aminotransferase, all the aminonitrogen from amino acids that undergo transaminationcan be concentrated in glutamate. This is importantbecause L-glutamate is the only amino acid thatundergoes oxidative deamination at an appreciable ratein mammalian tissues. The formation of ammoniafrom α-amino groups thus occurs mainly via the α-amino nitrogen of L-glutamate.Transamination is not restricted to α-amino groups.The δ-amino group of ornithine—but not the ε-aminogroup of lysine—readily undergoes transamination.Serum levels of aminotransferases are elevated in somedisease states (see Figure 7–11).L-GLUTAMATE DEHYDROGENASEOCCUPIES A CENTRAL POSITIONIN NITROGEN METABOLISMTransfer of amino nitrogen to α-ketoglutarate forms L-glutamate. Release of this nitrogen as ammonia is thencatalyzed by hepatic L-glutamate dehydrogenase(GDH), which can use either NAD + or NADP + (Figure29–5). Conversion of α-amino nitrogen to ammoniaby the concerted action of glutamate aminotransferaseand GDH is often termed “transdeamination.”Liver GDH activity is allosterically inhibited by ATP,GTP, and NADH and activated by ADP. The reactioncatalyzed by GDH is freely reversible and functions alsoin amino acid biosynthesis (see Figure 28–1).NAD(P) + NAD(P)H + H +L-GlutamateNH 3α-KetoglutarateFigure 29–5. The L-glutamate dehydrogenase reaction.NAD(P) + means that either NAD + or NADP + canserve as co-substrate. The reaction is reversible but favorsglutamate formation.drogen peroxide (H 2 O 2 ), which then is split to O 2 andH 2 O by catalase.Ammonia Intoxication Is Life-ThreateningThe ammonia produced by enteric bacteria and absorbedinto portal venous blood and the ammonia producedby tissues are rapidly removed from circulationby the liver and converted to urea. Only traces (10–20µg/dL) thus normally are present in peripheral blood.This is essential, since ammonia is toxic to the centralnervous system. Should portal blood bypass the liver,systemic blood ammonia levels may rise to toxic levels.This occurs in severely impaired hepatic function or thedevelopment of collateral links between the portal andsystemic veins in cirrhosis. Symptoms of ammonia intoxicationinclude tremor, slurred speech, blurred vision,coma, and ultimately death. Ammonia may betoxic to the brain in part because it reacts with α-ketoglutarateto form glutamate. The resulting depleted levelsof α-ketoglutarate then impair function of the tricarboxylicacid (TCA) cycle in neurons.Amino Acid Oxidases Also RemoveNitrogen as AmmoniaWhile their physiologic role is uncertain, L-amino acidoxidases of liver and kidney convert amino acids to anα-imino acid that decomposes to an α-keto acid withrelease of ammonium ion (Figure 29–6). The reducedflavin is reoxidized by molecular oxygen, forming hy-RNH 3+CHCO –Oα-Amino acidFlavinAMINO ACIDOXIDASEFlavin-H 2+NH 2C O –R COα-Imino acidH 2 O+NH 4Pyruvateα-Amino acidL-Alanineα-Keto acidα-Ketoglutarateα-Amino acidL-Glutamateα-Keto acidFigure 29–4. Alanine aminotransferase (top) andglutamate aminotransferase (bottom).CATALASEH 2 O 2 O 2H 2 O1 /2O 2ROCCOα-Keto acidFigure 29–6. Oxidative deamination catalyzed byL-amino acid oxidase (L-α-amino acid:O 2 oxidoreductase).The α-imino acid, shown in brackets, is not astable intermediate.O –

CATABOLISM OF PROTEINS & OF AMINO ACID NITROGEN / 245Glutamine Synthase Fixes Ammoniaas GlutamineFormation of glutamine is catalyzed by mitochondrialglutamine synthase (Figure 29–7). Since amide bondsynthesis is coupled to the hydrolysis of ATP to ADPand P i , the reaction strongly favors glutamine synthesis.One function of glutamine is to sequester ammonia ina nontoxic form.Glutaminase & Asparaginase DeamidateGlutamine & AsparagineHydrolytic release of the amide nitrogen of glutamineas ammonia, catalyzed by glutaminase (Figure 29–8),strongly favors glutamate formation. The concerted actionof glutamine synthase and glutaminase thus catalyzesthe interconversion of free ammonium ion andglutamine. An analogous reaction is catalyzed by L-asparaginase.Formation & Secretion of AmmoniaMaintains Acid-Base BalanceExcretion into urine of ammonia produced by renal tubularcells facilitates cation conservation and regulation ofacid-base balance. Ammonia production from intracellularrenal amino acids, especially glutamine, increases inmetabolic acidosis and decreases in metabolic alkalosis.UREA IS THE MAJOR END PRODUCT OFNITROGEN CATABOLISM IN HUMANSSynthesis of 1 mol of urea requires 3 mol of ATP plus1 mol each of ammonium ion and of the α-amino nitrogenof aspartate. Five enzymes catalyze the numberedON H 2–COCH 2NH 3+CHCH 2COL-GlutamateMg-ATPNH+4O –GLUTAMINESYNTHASEMg-ADPH 2 O+ P iNH+3COCH 2CHCH 2L-GlutamineFigure 29–7. The glutamine synthase reactionstrongly favors glutamine synthesis.COO –NH 3+H CH O –2 N CH C 2CH 2 COOL-GlutamineH 2 OGLUTAMINASENH+4NH+3O–COCH 2CHCH 2L-GlutamateFigure 29–8. The glutaminase reaction proceeds essentiallyirreversibly in the direction of glutamate andNH + 4 formation. Note that the amide nitrogen, not theα-amino nitrogen, is removed.reactions of Figure 29–9. Of the six participatingamino acids, N-acetylglutamate functions solely as anenzyme activator. The others serve as carriers of theatoms that ultimately become urea. The major metabolicrole of ornithine, citrulline, and argininosuccinatein mammals is urea synthesis. Urea synthesis is acyclic process. Since the ornithine consumed in reaction2 is regenerated in reaction 5, there is no net lossor gain of ornithine, citrulline, argininosuccinate, orarginine. Ammonium ion, CO 2 , ATP, and aspartateare, however, consumed. Some reactions of urea synthesisoccur in the matrix of the mitochondrion, otherreactions in the cytosol (Figure 29–9).Carbamoyl Phosphate Synthase IInitiates Urea BiosynthesisCondensation of CO 2 , ammonia, and ATP to formcarbamoyl phosphate is catalyzed by mitochondrialcarbamoyl phosphate synthase I (reaction 1, Figure29–9). A cytosolic form of this enzyme, carbamoylphosphate synthase II, uses glutamine rather than ammoniaas the nitrogen donor and functions in pyrimidinebiosynthesis (see Chapter 34). Carbamoyl phosphatesynthase I, the rate-limiting enzyme of the ureacycle, is active only in the presence of its allosteric activatorN-acetylglutamate, which enhances the affinityof the synthase for ATP. Formation of carbamoyl phosphaterequires 2 mol of ATP, one of which serves as aphosphate donor. Conversion of the second ATP toAMP and pyrophosphate, coupled to the hydrolysis ofpyrophosphate to orthophosphate, provides the drivingCOO –

246 / CHAPTER 29CO 22Mg-ATPN-Acetylglutamate2Mg-ADP + P iCO 2 + NH 4+CARBAMOYLPHOSPHATESYNTHASE IO OH 2 N C O P O −CarbamoylphosphateO −P i21NH 4+CH 2 NH + 3CH 2CH 2H C NH + 3COO −L-OrnithineORNITHINETRANSCARBAMOYLASENH 2Urea C ONH 25ARGINASEH 2 ONH + 3C NHCH 2 NHCH 2CH 2H C NH + 3COO −L-Arginine4ARGININOSUCCINASEHC COO −−OOC CHFumarateNH 2C OCH 2 NHCH 2CH 2H C NH + 3COO −L-Citrulline3ARGININOSUCCINIC ACIDSYNTHASENHCNHCOO −CHCH 2 NH CH 2CH 2 COO −CH 2 ArgininosuccinateH C NH + 3COO −Mg-ATPAMP + Mg-PP iH 2 NCOO −CHCH 2COO −L-AspartateFigure 29–9. Reactions and intermediates of urea biosynthesis. The nitrogen-containing groups thatcontribute to the formation of urea are shaded. Reactions 1 and 2 occur in the matrix of liver mitochondriaand reactions 3 , 4 , and 5 in liver cytosol. CO 2 (as bicarbonate), ammonium ion, ornithine, and citrullineenter the mitochondrial matrix via specific carriers (see heavy dots) present in the inner membrane ofliver mitochondria.force for synthesis of the amide bond and the mixedacid anhydride bond of carbamoyl phosphate. The concertedaction of GDH and carbamoyl phosphate synthaseI thus shuttles nitrogen into carbamoyl phosphate,a compound with high group transfer potential.The reaction proceeds stepwise. Reaction of bicarbonatewith ATP forms carbonyl phosphate and ADP.Ammonia then displaces ADP, forming carbamate andorthophosphate. Phosphorylation of carbamate by thesecond ATP then forms carbamoyl phosphate.Carbamoyl Phosphate Plus OrnithineForms CitrullineL-Ornithine transcarbamoylase catalyzes transfer ofthe carbamoyl group of carbamoyl phosphate to ornithine,forming citrulline and orthophosphate (reaction2, Figure 29–9). While the reaction occurs in themitochondrial matrix, both the formation of ornithineand the subsequent metabolism of citrulline take placein the cytosol. Entry of ornithine into mitochondria

CATABOLISM OF PROTEINS & OF AMINO ACID NITROGEN / 247and exodus of citrulline from mitochondria thereforeinvolve mitochondrial inner membrane transport systems(Figure 29–9).Citrulline Plus AspartateForms ArgininosuccinateArgininosuccinate synthase links aspartate and citrullinevia the amino group of aspartate (reaction 3,Figure 29–9) and provides the second nitrogen of urea.The reaction requires ATP and involves intermediateformation of citrullyl-AMP. Subsequent displacementof AMP by aspartate then forms citrulline.Cleavage of ArgininosuccinateForms Arginine & FumarateCleavage of argininosuccinate, catalyzed by argininosuccinase,proceeds with retention of nitrogen inarginine and release of the aspartate skeleton as fumarate(reaction 4, Figure 29–9). Addition of water tofumarate forms L-malate, and subsequent NAD + -dependent oxidation of malate forms oxaloacetate.These two reactions are analogous to reactions of thecitric acid cycle (see Figure 16–3) but are catalyzed bycytosolic fumarase and malate dehydrogenase. Transaminationof oxaloacetate by glutamate aminotransferasethen re-forms aspartate. The carbon skeleton of aspartatefumaratethus acts as a carrier of the nitrogen of glutamateinto a precursor of urea.Cleavage of Arginine Releases Urea& Re-forms OrnithineHydrolytic cleavage of the guanidino group of arginine,catalyzed by liver arginase, releases urea (reaction 5,Figure 29–9). The other product, ornithine, reentersliver mitochondria for additional rounds of urea synthesis.Ornithine and lysine are potent inhibitors ofarginase, competitive with arginine. Arginine also servesas the precursor of the potent muscle relaxant nitricoxide (NO) in a Ca 2+ -dependent reaction catalyzed byNO synthase (see Figure 49–15).Carbamoyl Phosphate Synthase I Is thePacemaker Enzyme of the Urea CycleThe activity of carbamoyl phosphate synthase I is determinedby N-acetylglutamate, whose steady-state level isdictated by its rate of synthesis from acetyl-CoA andglutamate and its rate of hydrolysis to acetate and glutamate.These reactions are catalyzed by N-acetylglutamatesynthase and N-acetylglutamate hydrolase, respectively.Major changes in diet can increase theconcentrations of individual urea cycle enzymes 10-foldto 20-fold. Starvation, for example, elevates enzyme levels,presumably to cope with the increased productionof ammonia that accompanies enhanced protein degradation.METABOLIC DISORDERS AREASSOCIATED WITH EACH REACTIONOF THE UREA CYCLEMetabolic disorders of urea biosynthesis, while extremelyrare, illustrate four important principles: (1)Defects in any of several enzymes of a metabolic pathwayenzyme can result in similar clinical signs andsymptoms. (2) The identification of intermediates andof ancillary products that accumulate prior to a metabolicblock provides insight into the reaction that is impaired.(3) Precise diagnosis requires quantitative assayof the activity of the enzyme thought to be defective.(4) Rational therapy must be based on an understandingof the underlying biochemical reactions in normaland impaired individuals.All defects in urea synthesis result in ammonia intoxication.Intoxication is more severe when the metabolicblock occurs at reactions 1 or 2 since some covalentlinking of ammonia to carbon has already occurredif citrulline can be synthesized. Clinical symptomscommon to all urea cycle disorders include vomiting,avoidance of high-protein foods, intermittent ataxia, irritability,lethargy, and mental retardation. The clinicalfeatures and treatment of all five disorders discussedbelow are similar. Significant improvement and minimizationof brain damage accompany a low-proteindiet ingested as frequent small meals to avoid suddenincreases in blood ammonia levels.Hyperammonemia Type 1. A consequence ofcarbamoyl phosphate synthase I deficiency (reaction1, Figure 29–9), this relatively infrequent condition(estimated frequency 1:62,000) probably is familial.Hyperammonemia Type 2. A deficiency of ornithinetranscarbamoylase (reaction 2, Figure 29–9)produces this X chromosome–linked deficiency. Themothers also exhibit hyperammonemia and an aversionto high-protein foods. Levels of glutamine are elevatedin blood, cerebrospinal fluid, and urine, probably dueto enhanced glutamine synthesis in response to elevatedlevels of tissue ammonia.Citrullinemia. In this rare disorder, plasma andcerebrospinal fluid citrulline levels are elevated and1–2 g of citrulline are excreted daily. One patient lackeddetectable argininosuccinate synthase activity (reaction3, Figure 29–9). In another, the K m for citrullinewas 25 times higher than normal. Citrulline and argininosuccinate,which contain nitrogen destined for ureasynthesis, serve as alternative carriers of excess nitrogen.Feeding arginine enhanced excretion of citrulline in thesepatients. Similarly, feeding benzoate diverts ammonianitrogen to hippurate via glycine (see Figure 31–1).

248 / CHAPTER 29Argininosuccinicaciduria. A rare disease characterizedby elevated levels of argininosuccinate in blood,cerebrospinal fluid, and urine is associated with friable,tufted hair (trichorrhexis nodosa). Both early-onset andlate-onset types are known. The metabolic defect isthe absence of argininosuccinase (reaction 4, Figure29–9). Diagnosis by measurement of erythrocyte argininosuccinaseactivity can be performed on umbilicalcord blood or amniotic fluid cells. As for citrullinemia,feeding arginine and benzoate promotes nitrogen excretion.Hyperargininemia. This defect is characterized byelevated blood and cerebrospinal fluid arginine levels,low erythrocyte levels of arginase (reaction 5, Figure29–9), and a urinary amino acid pattern resemblingthat of lysine-cystinuria. This pattern may reflect competitionby arginine with lysine and cystine for reabsorptionin the renal tubule. A low-protein diet lowersplasma ammonia levels and abolishes lysine-cystinuria.Gene Therapy Offers Promise forCorrecting Defects in Urea BiosynthesisGene therapy for rectification of defects in the enzymesof the urea cycle is an area of active investigation. Encouragingpreliminary results have been obtained, forexample, in animal models using an adenoviral vectorto treat citrullinemia.SUMMARY• Human subjects degrade 1–2% of their body proteindaily at rates that vary widely between proteins andwith physiologic state. Key regulatory enzymes oftenhave short half-lives.• Proteins are degraded by both ATP-dependent andATP-independent pathways. Ubiquitin targets manyintracellular proteins for degradation. Liver cell surfacereceptors bind and internalize circulating asialoglycoproteinsdestined for lysosomal degradation.• Ammonia is highly toxic. Fish excrete NH 3 directly;birds convert NH 3 to uric acid. Higher vertebratesconvert NH 3 to urea.• Transamination channels α-amino acid nitrogen intoglutamate. L-Glutamate dehydrogenase (GDH) occupiesa central position in nitrogen metabolism.• Glutamine synthase converts NH 3 to nontoxic glutamine.Glutaminase releases NH 3 for use in urea synthesis.• NH 3 , CO 2 , and the amide nitrogen of aspartate providethe atoms of urea.• Hepatic urea synthesis takes place in part in the mitochondrialmatrix and in part in the cytosol. Inbornerrors of metabolism are associated with each reactionof the urea cycle.• Changes in enzyme levels and allosteric regulation ofcarbamoyl phosphate synthase by N-acetylglutamateregulate urea biosynthesis.REFERENCESBrooks P et al: Subcellular localization of proteasomes and theirregulatory complexes in mammalian cells. Biochem J 2000;346:155.Curthoys NP, Watford M: Regulation of glutaminase activity andglutamine metabolism. Annu Rev Nutr 1995;15:133.Hershko A, Ciechanover A: The ubiquitin system. Annu RevBiochem 1998;67:425.Iyer R et al: The human arginases and arginase deficiency. J InheritMetab Dis 1998;21:86.Lim CB et al: Reduction in the rates of protein and amino acid catabolismto slow down the accumulation of endogenous ammonia:a strategy potentially adopted by mudskippers duringaerial exposure in constant darkness. J Exp Biol 2001;204:1605.Patejunas G et al: Evaluation of gene therapy for citrullinaemiausing murine and bovine models. J Inherit Metab Dis1998;21:138.Pickart CM. Mechanisms underlying ubiquitination. Annu RevBiochem 2001;70:503.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Tuchman M et al: The biochemical and molecular spectrum of ornithinetranscarbamoylase deficiency. J Inherit Metab Dis1998;21:40.Turner MA et al: Human argininosuccinate lyase: a structural basisfor intragenic complementation. Proc Natl Acad Sci U S A1997;94:9063.

Catabolism of the Carbon Skeletonsof Amino Acids 30Victor W. Rodwell, PhDBIOMEDICAL IMPORTANCEThis chapter considers conversion of the carbon skeletonsof the common L-amino acids to amphibolic intermediatesand the metabolic diseases or “inborn errors ofmetabolism” associated with these processes. Left untreated,they can result in irreversible brain damage andearly mortality. Prenatal or early postnatal detectionand timely initiation of treatment thus are essential.Many of the enzymes concerned can be detected in culturedamniotic fluid cells, which facilitates early diagnosisby amniocentesis. Treatment consists primarily offeeding diets low in the amino acids whose catabolismis impaired. While many changes in the primary structureof enzymes have no adverse effects, others modifythe three-dimensional structure of catalytic or regulatorysites, lower catalytic efficiency (lower V max or elevateK m ), or alter the affinity for an allosteric regulatorof activity. A variety of mutations thus may give rise tothe same clinical signs and symptoms.TRANSAMINATION TYPICALLY INITIATESAMINO ACID CATABOLISMRemoval of α-amino nitrogen by transamination (seeFigure 28–3) is the first catabolic reaction of aminoacids except in the case of proline, hydroxyproline,threonine, and lysine. The residual hydrocarbon skeletonis then degraded to amphibolic intermediates asoutlined in Figure 30–1.Asparagine, Aspartate, Glutamine, and Glutamate.All four carbons of asparagine and aspartateform oxaloacetate (Figure 30–2, top). Analogous reactionsconvert glutamine and glutamate to -ketoglutarate(Figure 30–2, bottom). Since the enzymes alsofulfill anabolic functions, no metabolic defects are associatedwith the catabolism of these four amino acids.Proline. Proline forms dehydroproline, glutamateγ-semialdehyde,glutamate, and, ultimately, -ketoglutarate(Figure 30–3, top). The metabolic block in typeI hyperprolinemia is at proline dehydrogenase.AlaCysGlyHypSerThrα-KetoglutarateGlutamateArgHisGlnProlleLeuTrpPyruvateCitrateCitratecycleSuccinyl-CoAlleMetValAcetyl-CoAAcetoacetyl-CoALeu, Lys,Phe, Trp,TyrFigure 30–1.of amino acids.OxaloacetateAspartate249AsnFumarateTyrPheAmphibolic intermediates formed from the carbon skeletons

250 / CHAPTER 30CONH 2H 2 O NH 4+COO –PYRALACOO –CH 2CH 2CH 2HC NH 2ASPARAGINASEHC NH 3+TRANSAMINASECOCOO –COOCOO –L-AsparagineL-AspartateOxaloacetateCCH 2ONH 2H 2 O+NH 4CH 2H C+NH 3GLUTAMINASEHOCO –CH 2CH 2+C NH 3COO – COO –L-GlutamineL-GlutamatePYRALATRANSAMINASECOO –Figure 30–2. Catabolism of L-asparagine(top) and of L-glutamineCH 2CH 2 (bottom) to amphibolic intermediates.(PYR, pyruvate; ALA, L-alanine.)C OCOO – In this and subsequent figures, colorhighlights portions of the moleculesundergoing chemical change.α-KetoglutarateThere is no associated impairment of hydroxyprolinecatabolism. The metabolic block in type II hyperprolinemiais at glutamate--semialdehyde dehydrogenase,which also functions in hydroxyproline catabolism.Both proline and hydroxyproline catabolism thusare affected and ∆ 1 -pyrroline-3-hydroxy-5-carboxylate(see Figure 30–10) is excreted.Arginine and Ornithine. Arginine is converted toornithine, glutamate γ-semialdehyde, and then -ketoglutarate(Figure 30–3, bottom). Mutations in ornithine-aminotransferase elevate plasma and urinaryornithine and cause gyrate atrophy of the retina.Treatment involves restricting dietary arginine. In hyperornithinemia-hyperammonemiasyndrome, a defectivemitochondrial ornithine-citrulline antiporter(see Figure 29–9) impairs transport of ornithine intomitochondria for use in urea synthesis.Histidine. Catabolism of histidine proceeds viaurocanate, 4-imidazolone-5-propionate, and N-formiminoglutamate(Figlu). Formimino group transfer totetrahydrofolate forms glutamate, then -ketoglutarate(Figure 30–4). In folic acid deficiency, grouptransfer is impaired and Figlu is excreted. Excretion ofFiglu following a dose of histidine thus has been usedto detect folic acid deficiency. Benign disorders of histidinecatabolism include histidinemia and urocanicaciduria associated with impaired histidase.SIX AMINO ACIDS FORM PYRUVATEAll of the carbons of glycine, serine, alanine, and cysteineand two carbons of threonine form pyruvate andsubsequently acetyl-CoA.Glycine. The glycine synthase complex of liver mitochondriasplits glycine to CO 2 and NH 4 + and formsN 5 ,N 10 -methylene tetrahydrofolate (Figure 30–5).Glycinuria results from a defect in renal tubular reabsorption.The defect in primary hyperoxaluria is thefailure to catabolize glyoxylate formed by deaminationof glycine. Subsequent oxidation of glyoxylate to oxalateresults in urolithiasis, nephrocalcinosis, and earlymortality from renal failure or hypertension.Serine. Following conversion to glycine, catalyzedby serine hydroxymethyltransferase (Figure 30–5),serine catabolism merges with that of glycine (Figure30–6).Alanine. Transamination of alanine forms pyruvate.Perhaps for the reason advanced under glutamateand aspartate catabolism, there is no known metabolicdefect of alanine catabolism. Cysteine. Cystine is firstreduced to cysteine by cystine reductase (Figure30–7). Two different pathways then convert cysteine topyruvate (Figure 30–8).There are numerous abnormalities of cysteine metabolism.Cystine, lysine, arginine, and ornithine areexcreted in cystine-lysinuria (cystinuria), a defect inrenal reabsorption. Apart from cystine calculi, cystinuriais benign. The mixed disulfide of L-cysteine andL-homocysteine (Figure 30–9) excreted by cystinuricpatients is more soluble than cystine and reduces formationof cystine calculi. Several metabolic defectsresult in vitamin B 6 -responsive or -unresponsive homocystinurias.Defective carrier-mediated transportof cystine results in cystinosis (cystine storage disease)with deposition of cystine crystals in tissuesand early mortality from acute renal failure. Despite

PROLINEDEHYDROGENASEH 2 ONAD +NADH + H +CH 2 CHHC CH 2 COGLUTAMATE SEMIALDEHYDEDEHYDROGENASENH 3+NH 3+OCH 2 CHCH 2 CH 2 CNH 3+H HH+ NHO −COL-ProlineNH +COOO −+ NH 3HH CH 2 CH O −2 N NC CH 2 CH 2 CNHO+L-Arginine3Urea1O −L-Glutamate-γ-semialdehydeH 2 Oα-KGGluL-Glutamateα-KetoglutarateL-OrnithineNAD +2NADH + H +ARGINASEO −L-Glutamate-γ-semialdehydeFigure 30–3. Top: Catabolism of proline. Numerals indicatesites of the metabolic defects in 1 type I and 2 type II hyperprolinemias.Bottom: Catabolism of arginine. Glutamate-γsemialdehydeforms α-ketoglutarate as shown above. 3 , site ofthe metabolic defect in hyperargininemia.– OH 3 N +HN– O CHC CHOL-HistidineCOCH 2CH 2CHCON-Formiminoglutamate (Figlu)H 4 folateN 5 -FormiminoH 4 folateHL-Glutamateα-Ketoglutarate+NH+HISTIDASENH 4H HN NH+– O CC CHOUrocanateH 2 OUROCANASEHN NH+– O CH 2C CH 2 OO4-Imidazolone-5-propionateH 2 OIMIDAZOLONE PROPIONATEHYDROLASE+HN NH 2O –GLUTAMATE FORMIMINOTRANSFERASEFigure 30–4. Catabolism of L-histidine to α-ketoglutarate.(H 4 folate, tetrahydrofolate.) Histidase is theprobable site of the metabolic defect in histidinemia.251

252 / CHAPTER 30H 2 CNH 3+CHCO –H 4 folateMethyleneH 4 folateNH 3+CH 2CO –CysteineH2 CHS+ NH 3CHCOO −HOOL-SerineOGlycineFigure 30–5. Interconversion of serine and glycinecatalyzed by serine hydroxymethyltransferase. (H 4 folate,tetrahydrofolate.)Figure 30–7.NH 3+CH 2− OCYSTINEREDUCTASE2OGlycineCHCH2SHCO – + NAD +H 4 folateCO 2 + NH 4+ + NADH + H +N 5 ,N 10 -CH 2 -H 4 folateFigure 30–6. Reversible cleavage of glycine by themitochondrial glycine synthase complex. (PLP, pyridoxalphosphate.)NH 3+CONH 3+CHH2 CSO SCCHCH 2NH 3+NAD +O −CONADH + H +O −L-CystineL-CysteineThe cystine reductase reaction.CYSTEINEDIOXYGENASENH 3+CHH2 CCysteine sulfinate− O 2 STRANSAMINASESulfinylpyruvateH2 C– O 2 SDESULFINASETRANSAMINASE3-Mercaptopyruvate(thiolpyruvate) H2 CPyruvateOCCOCOPyruvateCYSTEINEHS2HOCCONADH+ H +H 2 S NAD +[O]O −α-Keto acidα-Amino acidO −SO 32−α-KAα-AAO −HH 2 CHSCOHO −CO3-MercaptolactateFigure 30–8. Catabolism of L-cysteine via the cysteinesulfinate pathway (top) and by the 3-mercaptopyruvatepathway (bottom).

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS / 253Mixed disulfide of cysteine and homo-Figure 30–9.cysteine.HCH 2 S S CH 2CNH 3+CH 2+COO – H C NH 3(Cysteine)COO –(Homocysteine)H HH+N HOHO –C2HO4-Hydroxy-L-proline1HYDROXYPROLINEDEHYDROGENASE+ NH 3H 3 CCHOHCHCOL-ThreonineO −NH +OHO –COL-∆ 1 -Pyrroline-3-hydroxy-5-carboxylateTHREONINEALDOLASEGlycineH 2 ONONENZYMATICALDEHYDEDEHYDROGENASECoASHACETATETHIOKINASEH 3 CCHOAcetaldehydeH 2 O NAD+H 2 OH 3 CCOAcetateNADH+H+O −H 3 CCOS CoAAcetyl-CoAMg-ATPMg-ADPFigure 30–10. Conversion of threonine to glycine(see Figure 30–6) and acetyl-CoA.Figure 30–11. Intermediates in L-hydroxyproline catabolism. (α-KA,α-keto acid; α-AA, α-amino acid.) Numerals identify sites of metabolicdefects in 1 hyperhydroxyprolinemia and 2 type II hyperprolinemia.OH+NH 3CH CH O –HC CH 2 COOγ-Hydroxy-L-glutamate-γ-semialdehydeNAD + H 2 ONADH + H + 2 DEHYDROGENASEOH+NH 3 – O CH CH O –C CH 2 COOErythro-γ-hydroxy-L-glutamateα-KATRANSAMINASEα-AAOH O– O CH C O –C CH 2 COOα-Keto-γ-hydroxyglutarateAN ALDOLASEOO– O CHC O –CH 3 C COOGlyoxylatePyruvate

O –254+NH 3 O2CH O – 1 α-KG 1 GluC O – 13 23 2[O]CH 2CCH CAscorbateCO 2PLP2OHCu 2+444 359 OCH 2 O –59 O592C[O]Fe 2+O 3C4CH 2 O –O – 2C5C C5 4O – Glutathione6839C CH=836 8 O6 CH 2 9 CH 2 2 O – 7 CH 2 9 CH 2 24HC C C C577CC C MALEYLACETOACETATEHOMOGENTISATEOCIS, TRANS ISOMERASEO O OOXIDASEO O O6 86 86 87TYROSINE7p-HYDROXYPHENYLPYRUVATE 7OOHTRANSAMINASEOHHYDROXYLASEOHL-Tyrosinep-HydroxyphenylpyruvateHomogentisate3OOMaleylacetoacetateMaleylacetoacetate(rewritten)O –OFumarylacetoacetateO –H 2 O4OC46CHCH5FumarateC7OO –+FUMARYLACETOACETATEHYDROLASECoASHH 3 C CH 2 O –H 3 C S CoAC CC+H 3 CCO –OOβ-KETOTHIOLASEOOAcetoacetateAcetyl-CoAAcetateFigure 30–12. Intermediates in tyrosine catabolism. Carbons are numbered to emphasize their ultimate fate. (α-KG, α-ketoglutarate;Glu, glutamate; PLP, pyridoxal phosphate.) Circled numerals represent the probable sites of the metabolic defects in 1 type II tyrosinemia;2 neonatal tyrosinemia; 3 alkaptonuria; and 4 type I tyrosinemia, or tyrosinosis.

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS / 255epidemiologic data suggesting a relationship betweenplasma homocysteine and cardiovascular disease,whether homocysteine represents a causal cardiovascularrisk factor remains controversial.Threonine. Threonine is cleaved to acetaldehydeand glycine. Oxidation of acetaldehyde to acetate is followedby formation of acetyl-CoA (Figure 30–10). Catabolismof glycine is discussed above.4-Hydroxyproline. Catabolism of 4-hydroxy-L-prolineforms, successively, L-∆ 1 -pyrroline-3-hydroxy-5-carboxylate,γ-hydroxy-L-glutamate-γ-semialdehyde, erythroγ-hydroxy-L-glutamate,and α-keto-γ-hydroxyglutarate.An aldol-type cleavage then forms glyoxylate plus pyruvate(Figure 30–11). A defect in 4-hydroxyproline dehydrogenaseresults in hyperhydroxyprolinemia,which is benign. There is no associated impairment ofproline catabolism.TWELVE AMINO ACIDS FORMACETYL-CoATyrosine. Figure 30–12 diagrams the conversion oftyrosine to amphibolic intermediates. Since ascorbate isthe reductant for conversion of p-hydroxyphenylpyruvateto homogentisate, scorbutic patients excrete incompletelyoxidized products of tyrosine catabolism.Subsequent catabolism forms maleylacetoacetate, fumarylacetoacetate,fumarate, acetoacetate, and ultimatelyacetyl-CoA.The probable metabolic defect in type I tyrosinemia(tyrosinosis) is at fumarylacetoacetate hydrolase(reaction 4, Figure 30–12). Therapy employs a diet lowin tyrosine and phenylalanine. Untreated acute andchronic tyrosinosis leads to death from liver failure. Alternatemetabolites of tyrosine are also excreted in typeII tyrosinemia (Richner-Hanhart syndrome), a defectin tyrosine aminotransferase (reaction 1, Figure30–12), and in neonatal tyrosinemia, due to loweredp-hydroxyphenylpyruvate hydroxylase activity (reaction2, Figure 30–12). Therapy employs a diet low inprotein.Alkaptonuria was first described in the 16th century.Characterized in 1859, it provided the basis forGarrod’s classic ideas concerning heritable metabolicdisorders. The defect is lack of homogentisate oxidase(reaction 3, Figure 30–12). The urine darkens on exposureto air due to oxidation of excreted homogentisate.Late in the disease, there is arthritis and connective tissuepigmentation (ochronosis) due to oxidation of homogentisateto benzoquinone acetate, which polymerizesand binds to connective tissue.Phenylalanine. Phenylalanine is first converted totyrosine (see Figure 28–10). Subsequent reactions arethose of tyrosine (Figure 30–12). Hyperphenylalaninemiasarise from defects in phenylalanine hydroxylaseitself (type I, classic phenylketonuria or PKU), indihydrobiopterin reductase (types II and III), or in dihydrobiopterinbiosynthesis (types IV and V) (Figure28–10). Alternative catabolites are excreted (Figure30–13). DNA probes facilitate prenatal diagnosis of defectsin phenylalanine hydroxylase or dihydrobiopterinreductase. A diet low in phenylalanine can prevent themental retardation of PKU (frequency 1:10,000TRANSAMINASENAD +PhenylacetateHNPhenylacetylglutamineNH 3+L-PhenylalanineOPhenylpyruvateCOO –CH 2 OHα-KetoglutarateL-GlutamateNADH + H +NADH + H + CO 2NAD +L-GlutamineCH 2H 2 OCH 2CH 2CH 2 COO –CHCONH 2CH 2 COO –CCH 2COO – CH 2 COO –CHOHCOPhenyllactateFigure 30–13. Alternative pathways of phenylalaninecatabolism in phenylketonuria. The reactions alsooccur in normal liver tissue but are of minor significance.

OOCCH 2CH 2COCO –H 2 OOCCH 2CH 2COCO –NADH+ H + NAD +OCCH 2CH 2CHOCO –– OH 2 C– OSaccharopineα-Ketoglutarate+NH +NH 3+NH 2+NH 3+– OL-LysineH 2NH+NH +3H 2 C CH 2 CHCH 2 CH 2 CO –H 2 CCH 2CH 2CH 2CHCOO –1,2SACCHAROPINEDEHYDROGENASE,L-LYSINE-FORMINGCH 2CH 2CH 2CHCOO –OOO256NAD +2NADH+ H +SACCHAROPINEDEHYDROGENASE,L-GLUTAMATE-FORMING– OOCO –2OCHCHCH 2 CNH + NH+ 3HCCH 2CH 2CH 2CHCOO –H 2 O– OOHCCCH 2CH 2CHL-GlutamateNH 3+NH 3+CH 2CH 2CH 2CHCCO –NAD + NADH + H + ONH+ 3C CH– 2 CHO CH 2 CH2 CH 2 OOL-α-AminoadipateO –O –OL-α-Aminoadipateδ-semialdehydeα-KG Glu OOCoASHPLPTRANSAMINASEC CH 2 C O –– O CH 2 CH 2 Cα-KetoadipateO[O]CO 2OC CH 2 C– O CH 2 CH2Glutaryl-CoAOSCoACO 2 + H 2 OFigure 30–14. Catabolism of L-lysine. (α-KG, α-ketoglutarate; Glu, glutamate; PLP, pyridoxal phosphate.) Circled numerals indicate the probable sitesof the metabolic defects in 1 periodic hyperlysinemia with associated hyperammonemia; and 2 persistent hyperlysinemia without associated hyperammonemia.

NH 3+CHH 2 C CONHL-TryptophanO −NH +3OCH O −2 H 2 C CFe 2+ C OTRYPTOPHANO OOXYGENASE(inducible)CHNHN-L-FormylkynurenineH 2 OKYNURENINEFORMYLASEFormateNH −3CHH 2 C CC OONH 2L-KynurenineO −NH 3+NH 3+H 2 CCHCO −H 3 CCHCO −O −257O 2NADPH + H +KYNURENINEHYDROXYLASEOHCNH 2OO3-L-HydroxykynurenineH 2 OPLPOKYNURENINASEOHCNH 2O3-HydroxyanthranilateO 23-HYDROXYANTHRANILATEOXIDASECHO − OCO −CONH 3+CO 2HOCHCCH− OCCCHNH 3+NADH + H + NAD +NH 4+HOCHCCO −− OCCH 2CONAD(P)H + H + NAD(P) +OH 2 CCH 2CO −− OCCH 2COO2-Acroleyl-3-aminofumarateO2-Amino-cis, cis-muconatesemialdehydeOOxalocrotonateOα-KetoadipateFigure 30–15.Catabolism of L-tryptophan. (PLP, pyridoxal phosphate.)

258 / CHAPTER 30HOHONH 2OCNH 3+NCH 2births). Elevated blood phenylalanine may not be detectableuntil 3–4 days postpartum. False-positives inpremature infants may reflect delayed maturation of enzymesof phenylalanine catabolism. A less reliablescreening test employs FeCl 3 to detect urinaryphenylpyruvate. FeCl 3 screening for PKU of the urineCH3-HydroxykynurenineXanthurenateHONH 4+Figure 30–16. Formation of xanthurenate in vitaminB 6 deficiency. Conversion of the tryptophan metabolite3-hydroxykynurenine to 3-hydroxyanthranilate is impaired(see Figure 30–15). A large portion is thereforeconverted to xanthurenate.COCOO –O –of newborn infants is compulsory in the United Statesand many other countries.Lysine. Figure 30–14 summarizes the catabolism oflysine. Lysine first forms a Schiff base with α-ketoglutarate,which is reduced to saccharopine. In one formof periodic hyperlysinemia, elevated lysine competitivelyinhibits liver arginase (see Figure 29–9), causinghyperammonemia. Restricting dietary lysine relieves theammonemia, whereas ingestion of a lysine load precipitatessevere crises and coma. In a different periodic hyperlysinemia,lysine catabolites accumulate, but even alysine load does not trigger hyperammonemia. In additionto impaired synthesis of saccharopine, some patientscannot cleave saccharopine.Tryptophan. Tryptophan is degraded to amphibolicintermediates via the kynurenine-anthranilatepathway (Figure 30–15). Tryptophan oxygenase(tryptophan pyrrolase) opens the indole ring, incorporatesmolecular oxygen, and forms N-formylkynurenine.An iron porphyrin metalloprotein that is induciblein liver by adrenal corticosteroids and bytryptophan, tryptophan oxygenase is feedbackinhibitedby nicotinic acid derivatives, includingNADPH. Hydrolytic removal of the formyl group ofN-formylkynurenine, catalyzed by kynurenine formylase,produces kynurenine. Since kynureninase requirespyridoxal phosphate, excretion of xanthurenate(Figure 30–16) in response to a tryptophan load is diagnosticof vitamin B 6 deficiency. Hartnup disease reflectsimpaired intestinal and renal transport of tryptophanand other neutral amino acids. Indole derivativesof unabsorbed tryptophan formed by intestinal bacteriaare excreted. The defect limits tryptophan availabilityfor niacin biosynthesis and accounts for the pellagralikesigns and symptoms.COO –COO –+ H3 N C H+ H3 N C HCH 2CH 2P P PH 2 OP i + PP iCH 2CH 2S +CH 3CH 2 AdenineORiboseL-METHIONINEADENOSYLTRANSFERASE+ S CH 2 AdenineCH 3ORiboseHOOHHOOHL-MethionineATPS-Adenosyl-L-methionine(“active methionine”)Figure 30–17. Formation of S-adenosylmethionine. ~CH 3 represents the highgroup transfer potential of “active methionine.”

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS / 259H 3 CSNH+ 3CH 2 CHCH 2 COL-MethionineATPP i + PP iS-Adenosyl-L-methionineAcceptorNH 3+O –CH 3 -AcceptorS-Adenosyl-L-homocysteineH 2 OAdenosineCH 2 CH O –H S CH 2 C+OO OH L-HomocysteineC CH– 2CYSTATHIONINEO CHβ-SYNTHASENH+ 3HL-Serine2 OOS– OCCHCH 2+NH 3O SHC CH– 2O CHNH+ NH+ 4 3L-CysteineConversion of methionine to propi-Figure 30–18.onyl-CoA.CH 2CystathionineH 2 OH 3 CCoASHα-KetobutyrateCO 2CH 2CH 2NH 3+CHOOCCOCONAD +O –O –NADH + H +H 3 C CCH 2 S CoAPropionyl-CoAMethionine. Methionine reacts with ATP formingS-adenosylmethionine, “active methionine” (Figure30–17). Subsequent reactions form propionyl-CoA(Figure 30–18) and ultimately succinyl-CoA (see Figure19–2).THE INITIAL REACTIONS ARE COMMONTO ALL THREE BRANCHED-CHAINAMINO ACIDSReactions 1–3 of Figure 30–19 are analogous to thoseof fatty acid catabolism. Following transamination, allthree α-keto acids undergo oxidative decarboxylationcatalyzed by mitochondrial branched-chain -ketoacid dehydrogenase. This multimeric enzyme complexof a decarboxylase, a transacylase, and a dihydrolipoyldehydrogenase closely resembles pyruvate dehydrogenase(see Figure 17–5). Its regulation also parallels thatof pyruvate dehydrogenase, being inactivated by phosphorylationand reactivated by dephosphorylation (seeFigure 17–6).Reaction 3 is analogous to the dehydrogenation offatty acyl-CoA thioesters (see Figure 22–3). In isovalericacidemia, ingestion of protein-rich foods elevatesisovalerate, the deacylation product of isovaleryl-CoA. Figures 30–20, 30–21, and 30–22 illustrate thesubsequent reactions unique to each amino acid skeleton.METABOLIC DISORDERS OF BRANCHED-CHAIN AMINO ACID CATABOLISMAs the name implies, the odor of urine in maple syrupurine disease (branched-chain ketonuria) suggestsmaple syrup or burnt sugar. The biochemical defect involvesthe -keto acid decarboxylase complex (reaction2, Figure 30–19). Plasma and urinary levels ofleucine, isoleucine, valine, α-keto acids, and α-hydroxyacids (reduced α-keto acids) are elevated. The mechanismof toxicity is unknown. Early diagnosis, especiallyprior to 1 week of age, employs enzymatic analysis.Prompt replacement of dietary protein by an aminoacid mixture that lacks leucine, isoleucine, and valineaverts brain damage and early mortality.Mutation of the dihydrolipoate reductase componentimpairs decarboxylation of branched-chain α-keto acids, of pyruvate, and of α-ketoglutarate. In intermittentbranched-chain ketonuria, the α-ketoacid decarboxylase retains some activity, and symptomsoccur later in life. The impaired enzyme in isovalericacidemia is isovaleryl-CoA dehydrogenase(reaction 3, Figure 30–19). Vomiting, acidosis, andcoma follow ingestion of excess protein. Accumulated

260 / CHAPTER 30CH 3NH 3+NH 3+NH 3+H 3 CCHCH 2CHCOH 3 CCHCHCOH 3 CCH 2CHCHCO –L-LeucineOCH 3L-ValineOCH 3L-IsoleucineOα-Keto acidα-Keto acidα-Keto acid1α-Amino acid1α-Amino acid1α-Amino acidCH 3OOOH 3 CCHCH 2CCO –H 3 CCHCCO –H 3 CCH 2CHCCO –Oα-KetoisocaproateCoASH2CO 2CH 3 Oα-KetoisovalerateCoASH2CO 2CH 3 Oα-Keto-β-methylvalerateCoASH2CO 2CH 3OOOH 3 CCH CCH 2 SIsovaleryl-CoACoAH 3 C CCHCH 3S CoAIsobutyryl-CoACH CH 3 C CHCH 3S CoAα-Methylbutyryl-CoA3[2H]3[2H]3[2H]H 3 CCH 3CCHOCSβ-Methylcrotonyl-CoACoAH 2 CCOCSCH 3Methacrylyl-CoACoAH 3 CHCCOCSCH 3Tiglyl-CoAFigure 30–19. The analogous first three reactions in the catabolism of leucine, valine, andisoleucine. Note also the analogy of reactions 2 and 3 to reactions of the catabolism of fattyacids (see Figure 22–3). The analogy to fatty acid catabolism continues, as shown in subsequentfigures.CoA

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS / 261CH 3OOC CH 3 C CH S CoAβ-Methylcrotonyl-CoAH 3 CCHCCCH 3SCoABiotinyl-*CO 2Tiglyl-CoA4L4IH 2 OBiotinO CH 3 OC* C C− O CH 2 CH S CoAβ-Methylglutaconyl-CoAH 2 O5LOOC* H 3 C OH C− O CH 2 CH 2 S CoAβ-Hydroxy-β-methylglutaryl-CoAH 3 CH 3 COCOCHHCHCH 3α-Methyl-β-hydroxybutyryl-CoA5ICHOCOCS[2H]SCoACoAO O 6LC* C− O CH 2 CH3AcetoacetateOCH 3 C S CoAAcetyl-CoAFigure 30–20. Catabolism of the β-methylcrotonyl-CoA formed from L-leucine. Asterisks indicate carbonatoms derived from CO 2 .H 3 COCα-Methylacetoacetyl-CoASCoACH 36I+CH 2CoASHOCSCoACH 3Acetyl-CoA Propionyl-CoAFigure 30–21. Subsequent catabolism of the tiglyl-CoA formed from L-isoleucine.

262 / CHAPTER 30OOH 2 C CC S CoACH 3Methacrylyl-CoAHOH 2 C4VCHOCCH 3β-Hydroxyisobutyryl-CoAHOH 2 C5VCHOCH 2 OCoASHO –CH 3β-HydroxyisobutyrateNAD +6VNADH + H +OHCCHCH 3OCO –Methylmalonate semialdehyde8VNAD +NADH + H +α-AA7Vα-KANH+3C CH 2 C– O CH S CoACHCH 3Methylmalonyl-CoA– OOC9VCH 2H 2 COOSuccinyl-CoAB 12 COENZYMECCoASHSCoAH 2 OSCoAOCO –CH 3β-AminoisobutyrateFigure 30–22. Subsequent catabolism of themethacrylyl-CoA formed from L-valine (see Figure30–19). (α-KA, α-keto acid; α-AA, α-amino acid.)isovaleryl-CoA is hydrolyzed to isovalerate and excreted.SUMMARY• Excess amino acids are catabolized to amphibolic intermediatesused as sources of energy or for carbohydrateand lipid biosynthesis.• Transamination is the most common initial reactionof amino acid catabolism. Subsequent reactions removeany additional nitrogen and restructure the hydrocarbonskeleton for conversion to oxaloacetate,α-ketoglutarate, pyruvate, and acetyl-CoA.• Metabolic diseases associated with glycine catabolisminclude glycinuria and primary hyperoxaluria.• Two distinct pathways convert cysteine to pyruvate.Metabolic disorders of cysteine catabolism includecystine-lysinuria, cystine storage disease, and the homocystinurias.• Threonine catabolism merges with that of glycineafter threonine aldolase cleaves threonine to glycineand acetaldehyde.• Following transamination, the carbon skeleton of tyrosineis degraded to fumarate and acetoacetate.Metabolic diseases of tyrosine catabolism include tyrosinosis,Richner-Hanhart syndrome, neonatal tyrosinemia,and alkaptonuria.• Metabolic disorders of phenylalanine catabolism includephenylketonuria (PKU) and several hyperphenylalaninemias.• Neither nitrogen of lysine undergoes transamination.Metabolic diseases of lysine catabolism include periodicand persistent forms of hyperlysinemiaammonemia.• The catabolism of leucine, valine, and isoleucine presentsmany analogies to fatty acid catabolism. Metabolicdisorders of branched-chain amino acid catabolisminclude hypervalinemia, maple syrup urinedisease, intermittent branched-chain ketonuria, isovalericacidemia, and methylmalonic aciduria.REFERENCESBlacher J, Safar ME: Homocysteine, folic acid, B vitamins and cardiovascularrisk. J Nutr Health Aging 2001;5:196.Cooper AJL: Biochemistry of the sulfur-containing amino acids.Annu Rev Biochem 1983;52:187.Gjetting T et al: A phenylalanine hydroxylase amino acid polymorphismwith implications for molecular diagnostics. MolGenet Metab 2001;73:280.Harris RA et al: Molecular cloning of the branched-chain α-ketoaciddehydrogenase kinase and the CoA-dependent methyl-

CATABOLISM OF THE CARBON SKELETONS OF AMINO ACIDS / 263malonate semialdehyde dehydrogenase. Adv Enzyme Regul1993;33:255.Scriver CR: Garrod’s foresight; our hindsight. J Inherit Metab Dis2001;24:93.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Waters PJ, Scriver CR, Parniak MA: Homomeric and heteromericinteractions between wild-type and mutant phenylalanine hydroxylasesubunits: evaluation of two-hybrid approaches forfunctional analysis of mutations causing hyperphenylalaninemia.Mol Genet Metab 2001;73:230.

Conversion of Amino Acidsto Specialized Products 31Victor W. Rodwell, PhDBIOMEDICAL IMPORTANCEImportant products derived from amino acids includeheme, purines, pyrimidines, hormones, neurotransmitters,and biologically active peptides. In addition, manyproteins contain amino acids that have been modifiedfor a specific function such as binding calcium or as intermediatesthat serve to stabilize proteins—generallystructural proteins—by subsequent covalent cross-linking.The amino acid residues in those proteins serve asprecursors for these modified residues. Small peptidesor peptide-like molecules not synthesized on ribosomesfulfill specific functions in cells. Histamine plays a centralrole in many allergic reactions. Neurotransmittersderived from amino acids include γ-aminobutyrate,5-hydroxytryptamine (serotonin), dopamine, norepinephrine,and epinephrine. Many drugs used to treatneurologic and psychiatric conditions affect the metabolismof these neurotransmitters.GlycineMetabolites and pharmaceuticals excreted as watersolubleglycine conjugates include glycocholic acid(Chapter 24) and hippuric acid formed from the foodadditive benzoate (Figure 31–1). Many drugs, drugmetabolites, and other compounds with carboxylgroups are excreted in the urine as glycine conjugates.Glycine is incorporated into creatine (see Figure 31–6),the nitrogen and α-carbon of glycine are incorporatedinto the pyrrole rings and the methylene bridge carbonsof heme (Chapter 32), and the entire glycine moleculebecomes atoms 4, 5, and 7 of purines (Figure 34–1).-Alanineβ-Alanine, a metabolite of cysteine (Figure 34–9), ispresent in coenzyme A and as β-alanyl dipeptides, principallycarnosine (see below). Mammalian tissues formβ-alanine from cytosine (Figure 34–9), carnosine, andanserine (Figure 31–2). Mammalian tissues transaminateβ-alanine, forming malonate semialdehyde. Bodyfluid and tissue levels of β-alanine, taurine, and264β-aminoisobutyrate are elevated in the rare metabolicdisorder hyperbeta-alaninemia.-Alanyl DipeptidesThe β-alanyl dipeptides carnosine and anserine(N-methylcarnosine) (Figure 31–2) activate myosinATPase, chelate copper, and enhance copper uptake.β-Alanyl-imidazole buffers the pH of anaerobicallycontracting skeletal muscle. Biosynthesis of carnosine iscatalyzed by carnosine synthetase in a two-stage reactionthat involves initial formation of an enzyme-boundacyl-adenylate of β-alanine and subsequent transfer ofthe β-alanyl moiety to L-histidine.ATP + β-Alanine →β-Alanyl −AMP →+ PPiβ - Alanyl − AMP + L - Histidine → Carnosine + AMPHydrolysis of carnosine to β-alanine and L-histidine iscatalyzed by carnosinase. The heritable disordercarnosinase deficiency is characterized by carnosinuria.Homocarnosine (Figure 31–2), present in humanbrain at higher levels than carnosine, is synthesized inbrain tissue by carnosine synthetase. Serum carnosinasedoes not hydrolyze homocarnosine. Homocarnosinosis,a rare genetic disorder, is associated with progressivespastic paraplegia and mental retardation.Phosphorylated Serine, Threonine,& TyrosineThe phosphorylation and dephosphorylation of seryl,threonyl, and tyrosyl residues regulate the activity ofcertain enzymes of lipid and carbohydrate metabolismand the properties of proteins that participate in signaltransduction cascades.MethionineS-Adenosylmethionine, the principal source of methylgroups in the body, also contributes its carbon skeletonfor the biosynthesis of the 3-diaminopropane portionsof the polyamines spermine and spermidine (Figure31–4).

CONVERSION OF AMINO ACIDS TO SPECIALIZED PRODUCTS / 265OCO –NSHNH 2+CH 2+NCH(CH 3 ) 3O –CATPBenzoateCoASHOErgothioneineAMP + PP iOCSCoANONH 2+CH 2CH 2C CH 2NHCH O –CNH 3+CoASHBenzoyl-CoAGlycineOCNHHippurateCH 2 O –CFigure 31–1. Biosynthesis of hippurate. Analogousreactions occur with many acidic drugs and catabolites.ON+NOOCarnosine+O CH 2 NH 3C CH 2CH 3NHHCH O –CH 2 CCOAnserineCH 2CH 2CH 2 NH 3+CysteineL-Cysteine is a precursor of the thioethanolamine portionof coenzyme A and of the taurine that conjugateswith bile acids such as taurocholic acid (Chapter 26).HistidineDecarboxylation of histidine to histamine is catalyzed bya broad-specificity aromatic L-amino acid decarboxylasethat also catalyzes the decarboxylation of dopa, 5-hydroxytryptophan,phenylalanine, tyrosine, and tryptophan.α-Methyl amino acids, which inhibit decarboxylaseactivity, find application as antihypertensive agents.Histidine compounds present in the human body includeergothioneine, carnosine, and dietary anserine(Figure 31–2). Urinary levels of 3-methylhistidine areunusually low in patients with Wilson’s disease.NNH 2+CH 2NHCHCOO –HomocarnosineFigure 31–2. Compounds related to histidine. Theboxes surround the components not derived from histidine.The SH group of ergothioneine derives from cysteine.Ornithine & ArginineArginine is the formamidine donor for creatine synthesis(Figure 31–6) and via ornithine to putrescine, spermine,and spermidine (Figure 31–3) Arginine is alsothe precursor of the intercellular signaling molecule nitricoxide (NO) that serves as a neurotransmitter,smooth muscle relaxant, and vasodilator. Synthesis ofNO, catalyzed by NO synthase, involves the NADPHdependentreaction of L-arginine with O 2 to yield L-citrullineand NO.PolyaminesThe polyamines spermidine and spermine (Figure31–4) function in cell proliferation and growth, aregrowth factors for cultured mammalian cells, and stabilizeintact cells, subcellular organelles, and membranes.Pharmacologic doses of polyamines are hypothermic

266 / CHAPTER 31PROTEINSNITRIC OXIDEUREAPROTEINSARGININECREATINEPHOSPHATE,CREATININEPROLINEORNITHINEARGININEPHOSPHATEGlutamate-γsemialdehydePUTRESCINE,SPERMIDINE,SPERMINEGLUTAMATEFigure 31–3. Arginine, ornithine, and proline metabolism. Reactions with solid arrowsall occur in mammalian tissues. Putrescine and spermine synthesis occurs inboth mammals and bacteria. Arginine phosphate of invertebrate muscle functionsas a phosphagen analogous to creatine phosphate of mammalian muscle (seeFigure 31–6).and hypotensive. Since they bear multiple positivecharges, polyamines associate readily with DNA andRNA. Figure 31–4 summarizes polyamine biosynthesis.TryptophanFollowing hydroxylation of tryptophan to 5-hydroxytryptophanby liver tyrosine hydroxylase, subsequentdecarboxylation forms serotonin (5-hydroxytrypta-mine), a potent vasoconstrictor and stimulator ofsmooth muscle contraction. Catabolism of serotonin isinitiated by monoamine oxidase-catalyzed oxidativedeamination to 5-hydroxyindoleacetate. The psychicstimulation that follows administration of iproniazidresults from its ability to prolong the action of serotoninby inhibiting monoamine oxidase. In carcinoid(argentaffinoma), tumor cells overproduce serotonin.Urinary metabolites of serotonin in patients with carci-+ H 3 NDecarboxylatedS-adenosylmethionineH 2+ NSpermidineSPERMINESYNTHASENH 3++ H 3 NMethylthioadenosineH 2+ NSpermineNH 2+NH 3+Figure 31–4. Conversion of spermidine to spermine.Spermidine formed from putrescine (decarboxylatedL-ornithine) by transfer of a propylamine moiety fromdecarboxylated S-adenosylmethionine accepts asecond propylamine moiety to form spermidine.

CONVERSION OF AMINO ACIDS TO SPECIALIZED PRODUCTS / 267HOTYROSINEHYDROXYLASEHOOHDOPADECARBOXYLASEHOHOOHDOPAMINEβ-OXIDASEOHPHENYLETHANOL-AMINE N-METHYL-TRANSFERASEHOCH 2L-TyrosineCH 2DopaNH 3+CHPLPCu 2+H 4 • biopterinH 2 • biopterinNH 3+CHCO 2O 2COCOVitamin CO –O –CH 2+CH 2 NH 3DopamineCHOHNorepinephrineOHCHOHCH 2NH 3+S-AdenosylmethionineS-AdenosylhomocysteineCH 2 CH 3N+H 2EpinephrineFigure 31–5. Conversion of tyrosine to epinephrineand norepinephrine in neuronal and adrenal cells. (PLP,pyridoxal phosphate.)noid include N-acetylserotonin glucuronide and theglycine conjugate of 5-hydroxyindoleacetate. Serotoninand 5-methoxytryptamine are metabolized to the correspondingacids by monoamine oxidase. N-Acetylationof serotonin, followed by O-methylation in the pinealbody, forms melatonin. Circulating melatonin is takenup by all tissues, including brain, but is rapidly metabolizedby hydroxylation followed by conjugation withsulfate or with glucuronic acid.Kidney tissue, liver tissue, and fecal bacteria all converttryptophan to tryptamine, then to indole 3-acetate.The principal normal urinary catabolites of tryptophanare 5-hydroxyindoleacetate and indole 3-acetate.TyrosineNeural cells convert tyrosine to epinephrine and norepinephrine(Figure 31–5). While dopa is also an intermediatein the formation of melanin, different enzymeshydroxylate tyrosine in melanocytes. Dopa decarboxylase,a pyridoxal phosphate-dependent enzyme, formsdopamine. Subsequent hydroxylation by dopamineβ-oxidase then forms norepinephrine. In the adrenalmedulla, phenylethanolamine-N-methyltransferase utilizesS-adenosylmethionine to methylate the primaryamine of norepinephrine, forming epinephrine (Figure31–5). Tyrosine is also a precursor of triiodothyronineand thyroxine (Chapter 42).CreatinineCreatinine is formed in muscle from creatine phosphateby irreversible, nonenzymatic dehydration and loss ofphosphate (Figure 31–6). The 24-hour urinary excretionof creatinine is proportionate to muscle mass.Glycine, arginine, and methionine all participate in creatinebiosynthesis. Synthesis of creatine is completed bymethylation of guanidoacetate by S-adenosylmethionine(Figure 31–6).-Aminobutyrateγ-Aminobutyrate (GABA) functions in brain tissue asan inhibitory neurotransmitter by altering transmembranepotential differences. It is formed by decarboxylationof L-glutamate, a reaction catalyzed by L-glutamatedecarboxylase (Figure 31–7). Transamination of γ-aminobutyrate forms succinate semialdehyde (Figure31–7), which may then undergo reduction to γ-hydroxybutyrate,a reaction catalyzed by L-lactate dehydrogenase,or oxidation to succinate and thence via the citricacid cycle to CO 2 and H 2 O. A rare genetic disorderof GABA metabolism involves a defective GABA aminotransferase,an enzyme that participates in the catabolismof GABA subsequent to its postsynaptic release inbrain tissue.

+H 2 NHNHCNH 2NHCHCH 2CH 2 + H3 N COO –CH 2C+NH 32GlycineCOO –L-ArginineCHNNCreatinineCOCH 3CH 2Figure 31–6.(Kidney)ARGININE-GLYCINETRANSAMIDINASEOrnithine+H 2 NS-AdenosylmethionineS-AdenosylhomocysteineCNH 2HN CH 2 COO –Glycocyamine(guanidoacetate)(Liver)ATPGUANIDOACETATEMETHYLTRANSFERASEADPNONENZYMATICIN MUSCLENH PP i + H 2 O CH 3HN CN CH 2 COO –Biosynthesis and metabolism of creatine and creatinine.Creatine phosphateCOO –L-GLUTAMATEDECARBOXYLASEC NH 3+CH 2CH 2COO –PLPL-GlutamateCO 2CH 2 OHCH 2+ H3 N CH 2 CH 2 CH 2 COO – CH 2γ-AminobutyrateCOO –[O]Hγ-HydroxybutyratePLPNAD +LACTATEDEHYDROGENASE+[NH 4 ] NADH + H +CO 2OCCH 2Hα-KATRANSAMINASEα-AACOO –CCH 2CH 2OCOO –α-KetoglutarateSUCCINICSEMIALDEHYDEDEHYDROGENASECOO –CH 2CH 2CH 2H 2 O NAD + NADH + H +COO –COO –Succinate semialdehydeSuccinateFigure 31–7. Metabolism of γ-aminobutyrate. (α-KA, α-keto acids; α-AA, α-amino acids; PLP, pyridoxalphosphate.)268

CONVERSION OF AMINO ACIDS TO SPECIALIZED PRODUCTS / 269SUMMARY• In addition to their roles in proteins and polypeptides,amino acids participate in a wide variety of additionalbiosynthetic processes.• Glycine participates in the biosynthesis of heme,purines, and creatine and is conjugated to bile acidsand to the urinary metabolites of many drugs.• In addition to its roles in phospholipid and sphingosinebiosynthesis, serine provides carbons 2 and 8 ofpurines and the methyl group of thymine.• S-Adenosylmethionine, the methyl group donor formany biosynthetic processes, also participates directlyin spermine and spermidine biosynthesis.• Glutamate and ornithine form the neurotransmitterγ-aminobutyrate (GABA).• The thioethanolamine of coenzyme A and the taurineof taurocholic acid arise from cysteine.• Decarboxylation of histidine forms histamine, andseveral dipeptides are derived from histidine andβ-alanine.• Arginine serves as the formamidine donor for creatinebiosynthesis, participates in polyamine biosynthesis,and provides the nitrogen of nitric oxide(NO).• Important tryptophan metabolites include serotonin,melanin, and melatonin.• Tyrosine forms both epinephrine and norepinephrine,and its iodination forms thyroid hormone.REFERENCEScriver CR et al (editors): The Metabolic and Molecular Bases ofInherited Disease, 8th ed. McGraw-Hill, 2001.

Porphyrins & Bile Pigments 32Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEThe biochemistry of the porphyrins and of the bile pigmentsis presented in this chapter. These topics areclosely related, because heme is synthesized from porphyrinsand iron, and the products of degradation ofheme are the bile pigments and iron.Knowledge of the biochemistry of the porphyrinsand of heme is basic to understanding the varied functionsof hemoproteins (see below) in the body. Theporphyrias are a group of diseases caused by abnormalitiesin the pathway of biosynthesis of the various porphyrins.Although porphyrias are not very prevalent,physicians must be aware of them. A much more prevalentclinical condition is jaundice, due to elevation ofbilirubin in the plasma. This elevation is due to overproductionof bilirubin or to failure of its excretion andis seen in numerous diseases ranging from hemolyticanemias to viral hepatitis and to cancer of the pancreas.METALLOPORPHYRINS& HEMOPROTEINS AREIMPORTANT IN NATUREPorphyrins are cyclic compounds formed by the linkageof four pyrrole rings through ⎯HC ⎯ methenylbridges (Figure 32–1). A characteristic property of theporphyrins is the formation of complexes with metalions bound to the nitrogen atom of the pyrrole rings.Examples are the iron porphyrins such as heme of hemoglobinand the magnesium-containing porphyrinchlorophyll, the photosynthetic pigment of plants.Proteins that contain heme (hemoproteins) arewidely distributed in nature. Examples of their importancein humans and animals are listed in Table 32–1.Natural Porphyrins Have Substituent SideChains on the Porphin NucleusThe porphyrins found in nature are compounds inwhich various side chains are substituted for the eighthydrogen atoms numbered in the porphin nucleusshown in Figure 32–1. As a simple means of showingthese substitutions, Fischer proposed a shorthand formulain which the methenyl bridges are omitted andeach pyrrole ring is shown as indicated with the eight270substituent positions numbered as shown in Figure32–2. Various porphyrins are represented in Figures32–2, 32–3, and 32–4.The arrangement of the acetate (A) and propionate(P) substituents in the uroporphyrin shown in Figure32–2 is asymmetric (in ring IV, the expected order ofthe A and P substituents is reversed). A porphyrin withthis type of asymmetric substitution is classified as atype III porphyrin. A porphyrin with a completely symmetricarrangement of the substituents is classified as atype I porphyrin. Only types I and III are found in nature,and the type III series is far more abundant (Figure32–3)—and more important because it includes heme.Heme and its immediate precursor, protoporphyrinIX (Figure 32–4), are both type III porphyrins (ie, themethyl groups are asymmetrically distributed, as in typeIII coproporphyrin). However, they are sometimesidentified as belonging to series IX, because they weredesignated ninth in a series of isomers postulated byHans Fischer, the pioneer worker in the field of porphyrinchemistry.HEME IS SYNTHESIZED FROMSUCCINYL-COA & GLYCINEHeme is synthesized in living cells by a pathway that hasbeen much studied. The two starting materials are succinyl-CoA,derived from the citric acid cycle in mitochondria,and the amino acid glycine. Pyridoxal phosphateis also necessary in this reaction to “activate”glycine. The product of the condensation reaction betweensuccinyl-CoA and glycine is α-amino-β-ketoadipicacid, which is rapidly decarboxylated to form α-aminolevulinate(ALA) (Figure 32–5). This reaction sequenceis catalyzed by ALA synthase, the rate-controlling enzymein porphyrin biosynthesis in mammalian liver.Synthesis of ALA occurs in mitochondria. In the cytosol,two molecules of ALA are condensed by the enzymeALA dehydratase to form two molecules of waterand one of porphobilinogen (PBG) (Figure 32–5). ALAdehydratase is a zinc-containing enzyme and is sensitiveto inhibition by lead, as can occur in lead poisoning.The formation of a cyclic tetrapyrrole—ie, a porphyrin—occursby condensation of four molecules ofPBG (Figure 32–6). These four molecules condense in ahead-to-tail manner to form a linear tetrapyrrole, hy-

PORPHYRINS & BILE PIGMENTS / 271HCCH12AP8 3HCNCHNIbridges (⎯HC⎯ ) are labeled α, β, γ, and δ.HIV IIPyrrole7 III 41 2H H6 5C Cδ I αHC C C CHN38 HC CC CHIV NH HN7 HC CC CH4HC C C CHγ III βC CH H6 5Porphin(C 20 H 14 N 4 )Figure 32–1. The porphin molecule. Rings are labeledI, II, III, and IV. Substituent positions on the ringsare labeled 1, 2, 3, 4, 5, 6, 7, and 8. The methenylNdroxymethylbilane (HMB). The reaction is catalyzed byuroporphyrinogen I synthase, also named PBG deaminaseor HMB synthase. HMB cyclizes spontaneously toform uroporphyrinogen I (left-hand side of Figure32–6) or is converted to uroporphyrinogen III by theaction of uroporphyrinogen III synthase (right-hand sideof Figure 32–6). Under normal conditions, the uroporphyrinogenformed is almost exclusively the III isomer,but in certain of the porphyrias (discussed below), thetype I isomers of porphyrinogens are formed in excess.Note that both of these uroporphyrinogens havethe pyrrole rings connected by methylene bridgesTable 32–1. Examples of some important humanand animal hemoproteins. 1ProteinFunctionHemoglobin Transport of oxygen in bloodMyoglobin Storage of oxygen in muscleCytochrome c Involvement in electron transport chainCytochrome P450 Hydroxylation of xenobioticsCatalaseDegradation of hydrogen peroxideTryptophan Oxidation of trypotophanpyrrolase1 The functions of the above proteins are described in variouschapters of this text.IIFigure 32–2. Uroporphyrin III. A (acetate) =⎯CH 2 COOH; P (propionate) = ⎯CH 2 CH 2 COOH.(⎯CH 2 ⎯), which do not form a conjugated ring system.Thus, these compounds are colorless (as are allporphyrinogens). However, the porphyrinogens arereadily auto-oxidized to their respective colored porphyrins.These oxidations are catalyzed by light and bythe porphyrins that are formed.Uroporphyrinogen III is converted to coproporphyrinogenIII by decarboxylation of all of the acetate(A) groups, which changes them to methyl (M) substituents.The reaction is catalyzed by uroporphyrinogendecarboxylase, which is also capable of convertinguroporphyrinogen I to coproporphyrinogen I (Figure32–7). Coproporphyrinogen III then enters the mitochondria,where it is converted to protoporphyrinogenIII and then to protoporphyrin III. Several steps areinvolved in this conversion. The mitochondrial enzymecoproporphyrinogen oxidase catalyzes the decarboxylationand oxidation of two propionic side chains toform protoporphyrinogen. This enzyme is able to actonly on type III coproporphyrinogen, which would explainwhy type I protoporphyrins do not generally occurin nature. The oxidation of protoporphyrinogen to protoporphyrinis catalyzed by another mitochondrial enzyme,protoporphyrinogen oxidase. In mammalianliver, the conversion of coproporphyrinogen to protoporphyrinrequires molecular oxygen.Formation of Heme Involves Incorporationof Iron Into ProtoporphyrinThe final step in heme synthesis involves the incorporationof ferrous iron into protoporphyrin in a reactioncatalyzed by ferrochelatase (heme synthase), anothermitochondrial enzyme (Figure 32–4).A summary of the steps in the biosynthesis of theporphyrin derivatives from PBG is given in Figure32–8. The last three enzymes in the pathway and ALAsynthase are located in the mitochondrion, whereas theother enzymes are cytosolic. Both erythroid and nonerythroid(“housekeeping”) forms of the first four enzymesare found. Heme biosynthesis occurs in mostmammalian cells with the exception of mature erythrocytes,which do not contain mitochondria. However,APIVPIIIIAIIAP

272 / CHAPTER 32APAPPAAPAPAPUroporphyrins were firstfound in the urine, but theyare not restricted to urine.P AUroporphyrin IP AUroporphyrin IIIMPMPPMMPMPMPCoproporphyrins were firstisolated from feces, but theyare also found in urine.P MP MCoproporphyrin ICoproporphyrin IIIFigure 32–3. Uroporphyrins and coproporphyrins. A (acetate); P (propionate); M(methyl) = ⎯CH 3 ; V (vinyl) = ⎯CH⎯CH 2 .approximately 85% of heme synthesis occurs in erythroidprecursor cells in the bone marrow and the majorityof the remainder in hepatocytes.The porphyrinogens described above are colorless,containing six extra hydrogen atoms as compared withthe corresponding colored porphyrins. These reducedporphyrins (the porphyrinogens) and not the correspondingporphyrins are the actual intermediates in thebiosynthesis of protoporphyrin and of heme.ALA Synthase Is the Key RegulatoryEnzyme in Hepatic Biosynthesis of HemeALA synthase occurs in both hepatic (ALAS1) and erythroid(ALAS2) forms. The rate-limiting reaction in thesynthesis of heme in liver is that catalyzed by ALAS1(Figure 32–5), a regulatory enzyme. It appears thatheme, probably acting through an aporepressor molecule,acts as a negative regulator of the synthesis ofALAS1. This repression-derepression mechanism is depicteddiagrammatically in Figure 32–9. Thus, the rateof synthesis of ALAS1 increases greatly in the absenceof heme and is diminished in its presence. The turnoverrate of ALAS1 in rat liver is normally rapid (half-lifeabout 1 hour), a common feature of an enzyme catalyzinga rate-limiting reaction. Heme also affects translationof the enzyme and its transfer from the cytosol tothe mitochondrion.Many drugs when administered to humans can resultin a marked increase in ALAS1. Most of thesedrugs are metabolized by a system in the liver that utilizesa specific hemoprotein, cytochrome P450 (seeChapter 53). During their metabolism, the utilizationof heme by cytochrome P450 is greatly increased,which in turn diminishes the intracellular heme concentration.This latter event effects a derepression ofALAS1 with a corresponding increased rate of hemesynthesis to meet the needs of the cells.MVMVMMFe 2+MMFe 2+PVFERROCHELATASEPVPMProtoporphyrin III (IX)(parent porphyrin of heme)Figure 32–4.Addition of iron to protoporphyrin to form heme.PMHeme(prosthetic group of hemoglobin)

PORPHYRINS & BILE PIGMENTS / 273COOHCOOHCOOHSuccinyl-CoA(“active”succinate)CH 2CH 2C OALASYNTHASECoA • SHCH 2CH 2COALASYNTHASECO 2CH 2CH 2COGlycineHS CoA+HC NH 2COOHPyridoxalphosphateHCNH 2COOHα-Amino-β-ketoadipateHCHNH 2δ-Aminolevulinate (ALA)COOHCOOHCOOHCH 2CH 2C OCH 2NH 2HOHNHCH 2CH 2CC H2H 2 OALADEHYDRATASECCCH 2NH 2COOH CH 2CH 2NHCH 2CCHTwo molecules ofδ-aminolevulinatePorphobilinogen(first precursor pyrrole)Figure 32–5. Biosynthesis of porphobilinogen. ALA synthase occurs in the mitochondria,whereas ALA dehydratase is present in the cytosol.Several factors affect drug-mediated derepression ofALAS1 in liver—eg, the administration of glucose canprevent it, as can the administration of hematin (an oxidizedform of heme).The importance of some of these regulatory mechanismsis further discussed below when the porphyriasare described.Regulation of the erythroid form of ALAS (ALAS2)differs from that of ALAS1. For instance, it is not inducedby the drugs that affect ALAS1, and it does notundergo feedback regulation by heme.PORPHYRINS ARE COLORED& FLUORESCEThe various porphyrinogens are colorless, whereas thevarious porphyrins are all colored. In the study of porphyrinsor porphyrin derivatives, the characteristic absorptionspectrum that each exhibits—in both the visibleand the ultraviolet regions of the spectrum—is of greatvalue. An example is the absorption curve for a solutionof porphyrin in 5% hydrochloric acid (Figure 32–10).Note particularly the sharp absorption band near 400nm. This is a distinguishing feature of the porphin ringand is characteristic of all porphyrins regardless of theside chains present. This band is termed the Soret bandafter its discoverer, the French physicist Charles Soret.When porphyrins dissolved in strong mineral acidsor in organic solvents are illuminated by ultravioletlight, they emit a strong red fluorescence. This fluorescenceis so characteristic that it is often used to detectsmall amounts of free porphyrins. The double bondsjoining the pyrrole rings in the porphyrins are responsiblefor the characteristic absorption and fluorescence ofthese compounds; these double bonds are absent in theporphyrinogens.An interesting application of the photodynamicproperties of porphyrins is their possible use in thetreatment of certain types of cancer, a procedure calledcancer phototherapy. Tumors often take up more porphyrinsthan do normal tissues. Thus, hematoporphyrinor other related compounds are administered toa patient with an appropriate tumor. The tumor is thenexposed to an argon laser, which excites the porphyrins,producing cytotoxic effects.Spectrophotometry Is Used to Testfor Porphyrins & Their PrecursorsCoproporphyrins and uroporphyrins are of clinical interestbecause they are excreted in increased amounts in

274 / CHAPTER 32AHOOCH 2 CH 2 CNH 24NH 3CCNHCOOHCH 2CH 2CCHFour molecules ofporphobilinogenPUROPORPHYRINOGEN ISYNTHASEpain and of a variety of neuropsychiatric findings); otherwise,patients will be subjected to inappropriate treatments.It has been speculated that King George III hada type of porphyria, which may account for his periodicconfinements in Windsor Castle and perhaps for someof his views regarding American colonists. Also, thephotosensitivity (favoring nocturnal activities) and severedisfigurement exhibited by some victims of congenitalerythropoietic porphyria have led to the suggestionthat these individuals may have been theprototypes of so-called werewolves. No evidence to supportthis notion has been adduced.ACCHydroxymethylbilane(linear tetrapyrrole)P A P A P AC H 2 C C C C H 2 CICCII IC C C C C CIINNHHCH 2 CH 2CCPSPONTANEOUSCYCLIZATIONHNIVCCACH 2CCPHNIIIType IuroporphyrinogenCCANNHHCH 2 CH 2CCAHNIVUROPORPHYRINOGEN IIISYNTHASECCPCH 2CCPHNIIIType IIIuroporphyrinogenFigure 32–6. Conversion of porphobilinogen to uroporphyrinogens.Uroporphyrinogen synthase I is alsocalled porphobilinogen (PBG) deaminase or hydroxymethylbilane(HMB) synthase.the porphyrias. These compounds, when present inurine or feces, can be separated from each other by extractionwith appropriate solvent mixtures. They canthen be identified and quantified using spectrophotometricmethods.ALA and PBG can also be measured in urine by appropriatecolorimetric tests.THE PORPHYRIAS ARE GENETICDISORDERS OF HEME METABOLISMThe porphyrias are a group of disorders due to abnormalitiesin the pathway of biosynthesis of heme; theycan be genetic or acquired. They are not prevalent, butit is important to consider them in certain circumstances(eg, in the differential diagnosis of abdominalPCCCCABiochemistry Underlies theCauses, Diagnoses, & Treatmentsof the PorphyriasSix major types of porphyria have been described, resultingfrom depressions in the activities of enzymes 3through 8 shown in Figure 32–9 (see also Table 32–2).Assay of the activity of one or more of these enzymesusing an appropriate source (eg, red blood cells) is thusimportant in making a definitive diagnosis in a suspectedcase of porphyria. Individuals with low activitiesof enzyme 1 (ALAS2) develop anemia, not porphyria(see Table 32–2). Patients with low activities of enzyme2 (ALA dehydratase) have been reported, but veryrarely; the resulting condition is called ALA dehydratase-deficientporphyria.In general, the porphyrias described are inherited inan autosomal dominant manner, with the exception ofcongenital erythropoietic porphyria, which is inheritedin a recessive mode. The precise abnormalities in thegenes directing synthesis of the enzymes involved inheme biosynthesis have been determined in some instances.Thus, the use of appropriate gene probes hasmade possible the prenatal diagnosis of some of theporphyrias.As is true of most inborn errors, the signs and symptomsof porphyria result from either a deficiency ofmetabolic products beyond the enzymatic block orfrom an accumulation of metabolites behind the block.If the enzyme lesion occurs early in the pathwayprior to the formation of porphyrinogens (eg, enzyme 3of Figure 32–9, which is affected in acute intermittentporphyria), ALA and PBG will accumulate in body tissuesand fluids (Figure 32–11). Clinically, patientscomplain of abdominal pain and neuropsychiatricsymptoms. The precise biochemical cause of thesesymptoms has not been determined but may relate toelevated levels of ALA or PBG or to a deficiency ofheme.On the other hand, enzyme blocks later in the pathwayresult in the accumulation of the porphyrinogens

PORPHYRINS & BILE PIGMENTS / 275APMPPIA4CO 2PIMIVIIIVIIAIIIPMIIIPP AUroporphyrinogen IA PUROPORPHYRINOGENDECARBOXYLASEP MCoproporphyrinogen IM PAIAMIMIVIIIVIIFigure 32–7. Decarboxylation of uroporphyrinogensto coproporphyrinogens in cytosol.(A, acetyl; M, methyl; P, propionyl.)P III PP AUroporphyrinogen III4CO 2P III PP MCoproporphyrinogen IIIPorphobilinogenUROPORPHYRINOGEN ISYNTHASEHydroxymethylbilane6HUROPORPHYRINOGEN IIISYNTHASESPONTANEOUS6HMITOCHONDRIA CYTOSOLUroporphyrinIIICoproporphyrinIII6HLightLight4CO 2COPROPORPHYRINOGENOXIDASEPROTOPORPHYRINOGENOXIDASEUroporphyrinogenIIICoproporphyrinogenIIIProtoporphyrinogen IIIProtoporphyrin IIIOr light in vitro6HUROPORPHYRINOGENDECARBOXYLASEUroporphyrinogenICoproporphyrinogenI4CO 2LightLight6HUroporphyrinICoproporphyrinIFERROCHELATASEFe 2+HemeFigure 32–8. Steps in the biosynthesis of the porphyrin derivatives from porphobilinogen. UroporphyrinogenI synthase is also called porphobilinogen deaminase or hydroxymethylbilane synthase.

276 / CHAPTER 32HemoproteinsProteinsHemeAporepressor8. FERROCHELATASEFe 2+Protoporphyrin III7. PROTOPORPHYRINOGENOXIDASEProtoporphyrinogen III6. COPROPORPHYRINOGENOXIDASECoproporphyrinogen III5. UROPORPHYRINOGENDECARBOXYLASEUroporphyrinogen III4. UROPORPHYRINOGEN IIISYNTHASEHydroxymethylbilane3. UROPORPHYRINOGEN ISYNTHASEPorphobilinogen2. ALADEHYDRATASEALA1. ALASYNTHASESuccinyl-CoA + GlycineFigure 32–9. Intermediates, enzymes, and regulation of heme synthesis.The enzyme numbers are those referred to in column 1 of Table32–2. Enzymes 1, 6, 7, and 8 are located in mitochondria, the others inthe cytosol. Mutations in the gene encoding enzyme 1 causes X-linkedsideroblastic anemia. Mutations in the genes encoding enzymes 2–8cause the porphyrias, though only a few cases due to deficiency of enzyme2 have been reported. Regulation of hepatic heme synthesis occursat ALA synthase (ALAS1) by a repression-derepression mechanismmediated by heme and its hypothetical aporepressor. Thedotted lines indicate the negative (− ) regulation by repression. Enzyme3 is also called porphobilinogen deaminase or hydroxymethylbilanesynthase.

PORPHYRINS & BILE PIGMENTS / 277Log absorbency54321Accumulation ofALA and PBG and/ordecrease in heme incells and body fluidsMutations in DNAAbnormalities of theenzymes of heme synthesisAccumulation ofporphyrinogens in skinand tissues300 400 500 600 700Wavelength (nm)Figure 32–10. Absorption spectrum of hematoporphyrin(0.01% solution in 5% HCl).Neuropsychiatric signsand symptomsSpontaneous oxidationof porphyrinogens toporphyrinsPhotosensitivityFigure 32–11. Biochemical causes of the majorsigns and symptoms of the porphyrias.Table 32–2. Summary of major findings in the porphyrias. 1Enzyme Involved 2 Type, Class, and MIM Number Major Signs and Symptoms Results of Laboratory Tests1. ALA synthase X-linked sideroblastic anemia 3 Anemia Red cell counts and hemoglobin(erythroid form) (erythropoietic) (MIM decreased201300)2. ALA dehydratase ALA dehydratase deficiency Abdominal pain, neuropsychiatric Urinary δ-aminolevulinic acid(hepatic) (MIM 125270)symptoms3. Uroporphyrinogen I Acute intermittent porphyria Abdominal pain, neuropsychiatric Urinary porphobilinogen positive,synthase 4 (hepatic) (MIM 176000) symptoms uroporphyrin positive4. Uroporphyrinogen III Congenital erythropoietic No photosensitivity Uroporphyrin positive, porphosynthase(erythropoietic) (MIM bilinogen negative263700)5. Uroporphyrinogen Porphyria cutanea tarda (he- Photosensitivity Uroporphyrin positive, porphodecarboxylasepatic) (MIM 176100) bilinogen negative6. Coproporphyrinogen Hereditary coproporphyria Photosensitivity, abdominal pain, Urinary porphobilinogen posioxidase(hepatic) (MIM 121300) neuropsychiatric symptoms tive, urinary uroporphyrinpositive, fecal protoporphyrinpositive7. Protoporphyrinogen Variegate porphyria (hepatic) Photosensitivity, abdominal pain, Urinary porphobilinogen posioxidase(MIM 176200) neuropsychiatric symptoms tive, fecal protoporphyrinpositive8. Ferrochelatase Protoporphyria (erythropoietic) Photosensitivity Fecal protoporphyrin posi-` (MIM 177000) tive, red cell protoporphyrinpositive1 Only the biochemical findings in the active stages of these diseases are listed. Certain biochemical abnormalities are detectable in the latentstages of some of the above conditions. Conditions 3, 5, and 8 are generally the most prevalent porphyrias.2 The numbering of the enzymes in this table corresponds to that used in Figure 32-9.3 X-linked sideroblastic anemia is not a porphyria but is included here because δ−aminolevulinic acid synthase is involved.4 This enzyme is also called porphobilinogen deaminase or hydroxymethylbilane synthase.

278 / CHAPTER 32indicated in Figures 32–9 and 32–11. Their oxidationproducts, the corresponding porphyrin derivatives,cause photosensitivity, a reaction to visible light ofabout 400 nm. The porphyrins, when exposed to lightof this wavelength, are thought to become “excited”and then react with molecular oxygen to form oxygenradicals. These latter species injure lysosomes and otherorganelles. Damaged lysosomes release their degradativeenzymes, causing variable degrees of skin damage, includingscarring.The porphyrias can be classified on the basis of theorgans or cells that are most affected. These are generallyorgans or cells in which synthesis of heme is particularlyactive. The bone marrow synthesizes considerablehemoglobin, and the liver is active in the synthesis ofanother hemoprotein, cytochrome P450. Thus, oneclassification of the porphyrias is to designate them aspredominantly either erythropoietic or hepatic; thetypes of porphyrias that fall into these two classes are socharacterized in Table 32–2. Porphyrias can also beclassified as acute or cutaneous on the basis of theirclinical features. Why do specific types of porphyria affectcertain organs more markedly than others? A partialanswer is that the levels of metabolites that causedamage (eg, ALA, PBG, specific porphyrins, or lack ofheme) can vary markedly in different organs or cells dependingupon the differing activities of their hemeformingenzymes.As described above, ALAS1 is the key regulatory enzymeof the heme biosynthetic pathway in liver. A largenumber of drugs (eg, barbiturates, griseofulvin) inducethe enzyme. Most of these drugs do so by inducing cytochromeP450 (see Chapter 53), which uses up hemeand thus derepresses (induces) ALAS1. In patients withporphyria, increased activities of ALAS1 result in increasedlevels of potentially harmful heme precursorsprior to the metabolic block. Thus, taking drugs thatcause induction of cytochrome P450 (so-called microsomalinducers) can precipitate attacks of porphyria.The diagnosis of a specific type of porphyria cangenerally be established by consideration of the clinicaland family history, the physical examination, and appropriatelaboratory tests. The major findings in the sixprincipal types of porphyria are listed in Table 32–2.High levels of lead can affect heme metabolism bycombining with SH groups in enzymes such as ferrochelataseand ALA dehydratase. This affects porphyrinmetabolism. Elevated levels of protoporphyrinare found in red blood cells, and elevated levels of ALAand of coproporphyrin are found in urine.It is hoped that treatment of the porphyrias at thegene level will become possible. In the meantime, treatmentis essentially symptomatic. It is important for patientsto avoid drugs that cause induction of cytochromeP450. Ingestion of large amounts of carbohydrates(glucose loading) or administration of hematin (ahydroxide of heme) may repress ALAS1, resulting in diminishedproduction of harmful heme precursors. Patientsexhibiting photosensitivity may benefit from administrationof β-carotene; this compound appears tolessen production of free radicals, thus diminishingphotosensitivity. Sunscreens that filter out visible lightcan also be helpful to such patients.CATABOLISM OF HEMEPRODUCES BILIRUBINUnder physiologic conditions in the human adult, 1–2× 10 8 erythrocytes are destroyed per hour. Thus, in 1day, a 70-kg human turns over approximately 6 g of hemoglobin.When hemoglobin is destroyed in the body,globin is degraded to its constituent amino acids,which are reused, and the iron of heme enters the ironpool, also for reuse. The iron-free porphyrin portion ofheme is also degraded, mainly in the reticuloendothelialcells of the liver, spleen, and bone marrow.The catabolism of heme from all of the heme proteinsappears to be carried out in the microsomal fractionsof cells by a complex enzyme system called hemeoxygenase. By the time the heme derived from hemeproteins reaches the oxygenase system, the iron has usuallybeen oxidized to the ferric form, constitutinghemin. The heme oxygenase system is substrate-inducible.As depicted in Figure 32–12, the hemin is reducedto heme with NADPH, and, with the aid ofmore NADPH, oxygen is added to the α-methenylbridge between pyrroles I and II of the porphyrin. Theferrous iron is again oxidized to the ferric form. Withthe further addition of oxygen, ferric ion is released,carbon monoxide is produced, and an equimolarquantity of biliverdin results from the splitting of thetetrapyrrole ring.In birds and amphibia, the green biliverdin IX is excreted;in mammals, a soluble enzyme called biliverdinreductase reduces the methenyl bridge between pyrroleIII and pyrrole IV to a methylene group to producebilirubin, a yellow pigment (Figure 32–12).It is estimated that 1 g of hemoglobin yields 35 mgof bilirubin. The daily bilirubin formation in humanadults is approximately 250–350 mg, deriving mainlyfrom hemoglobin but also from ineffective erythropoiesisand from various other heme proteins such ascytochrome P450.The chemical conversion of heme to bilirubin byreticuloendothelial cells can be observed in vivo as thepurple color of the heme in a hematoma is slowly convertedto the yellow pigment of bilirubin.

PORPHYRINS & BILE PIGMENTS / 279HemeOINαHeminHNIV N Fe 3+NIIPPNIIINADPHHNHHHNPPNADPMicrosomal heme oxygenase systemPNIV N Fe 2+PO 2INIIIαN IINADPHHemeHNONADPNADPHOHNIIBilirubinINNADPOHFe 3+ (reutilized)CO (exhaled)HNIIIPPIV N Fe 3+NIIO 2NIVPNPIIIHNOIBiliverdinFigure 32–12. Schematic representation of the microsomal heme oxygenase system. (Modified fromSchmid R, McDonough AF in: The Porphyrins. Dolphin D [editor]. Academic Press, 1978.)

280 / CHAPTER 32Bilirubin formed in peripheral tissues is transportedto the liver by plasma albumin. The further metabolismof bilirubin occurs primarily in the liver. It can be dividedinto three processes: (1) uptake of bilirubin byliver parenchymal cells, (2) conjugation of bilirubinwith glucuronate in the endoplasmic reticulum, and (3)secretion of conjugated bilirubin into the bile. Each ofthese processes will be considered separately.THE LIVER TAKES UP BILIRUBINBilirubin is only sparingly soluble in water, but its solubilityin plasma is increased by noncovalent binding toalbumin. Each molecule of albumin appears to haveone high-affinity site and one low-affinity site forbilirubin. In 100 mL of plasma, approximately 25 mgof bilirubin can be tightly bound to albumin at its highaffinitysite. Bilirubin in excess of this quantity can bebound only loosely and thus can easily be detached anddiffuse into tissues. A number of compounds such asantibiotics and other drugs compete with bilirubin forthe high-affinity binding site on albumin. Thus, thesecompounds can displace bilirubin from albumin andhave significant clinical effects.In the liver, the bilirubin is removed from albuminand taken up at the sinusoidal surface of the hepatocytesby a carrier-mediated saturable system. This facilitatedtransport system has a very large capacity, sothat even under pathologic conditions the system doesnot appear to be rate-limiting in the metabolism ofbilirubin.Since this facilitated transport system allows theequilibrium of bilirubin across the sinusoidal membraneof the hepatocyte, the net uptake of bilirubin willbe dependent upon the removal of bilirubin via subsequentmetabolic pathways.Once bilirubin enters the hepatocytes, it can bind tocertain cytosolic proteins, which help to keep it solubilizedprior to conjugation. Ligandin (a family of glutathioneS-transferases) and protein Y are the involvedproteins. They may also help to prevent efflux of bilirubinback into the blood stream.Conjugation of Bilirubin With GlucuronicAcid Occurs in the LiverBilirubin is nonpolar and would persist in cells (eg,bound to lipids) if not rendered water-soluble. Hepatocytesconvert bilirubin to a polar form, which is readilyexcreted in the bile, by adding glucuronic acid moleculesto it. This process is called conjugation and canemploy polar molecules other than glucuronic acid (eg,sulfate). Many steroid hormones and drugs are alsoconverted to water-soluble derivatives by conjugation inpreparation for excretion (see Chapter 53).The conjugation of bilirubin is catalyzed by a specificglucuronosyltransferase. The enzyme is mainlylocated in the endoplasmic reticulum, uses UDPglucuronicacid as the glucuronosyl donor, and is referredto as bilirubin-UGT. Bilirubin monoglucuronideis an intermediate and is subsequently converted to thediglucuronide (Figures 32–13 and 32–14). Most of thebilirubin excreted in the bile of mammals is in the formof bilirubin diglucuronide. However, when bilirubinconjugates exist abnormally in human plasma (eg, inobstructive jaundice), they are predominantly monoglucuronides.Bilirubin-UGT activity can be inducedby a number of clinically useful drugs, including phenobarbital.More information about glucuronosylationis presented below in the discussion of inherited disordersof bilirubin conjugation.Bilirubin Is Secreted Into BileSecretion of conjugated bilirubin into the bile occurs byan active transport mechanism, which is probably ratelimitingfor the entire process of hepatic bilirubin metabolism.The protein involved is MRP-2 (multidrugresistance-like protein 2), also called multispecific organicanion transporter (MOAT). It is located in theplasma membrane of the bile canalicular membraneand handles a number of organic anions. It is a memberof the family of ATP-binding cassette (ABC) transporters.The hepatic transport of conjugated bilirubininto the bile is inducible by those same drugs that arecapable of inducing the conjugation of bilirubin. Thus,the conjugation and excretion systems for bilirubin behaveas a coordinated functional unit.Figure 32–15 summarizes the three major processesinvolved in the transfer of bilirubin from blood to bile.Sites that are affected in a number of conditions causingjaundice (see below) are also indicated.– OOC(CH 2 O) 4 CMVMOO C C O C(CH 2 O) 4 COO –H 2 CH 2 CCH 2CH 2II III IV IO C C COFigure 32–13. Structure of bilirubin diglucuronide(conjugated, “direct-reacting” bilirubin). Glucuronicacid is attached via ester linkage to the two propionicacid groups of bilirubin to form an acylglucuronide.OMMV

PORPHYRINS & BILE PIGMENTS / 281UDP-GLUCOSEDEHYDROGENASEUDP-Glucose2NAD + 2NADH + 2H + UDP-Glucuronic acidFigure 32–14. Conjugation of bilirubinwith glucuronic acid. The glucuronate donor,UDP-glucuronic acid, is formed from UDPglucoseas depicted. The UDP-glucuronosyltransferaseis also called bilirubin-UGT.UDP-Glucuronic acid+BilirubinUDP-Glucuronic acid+Bilirubin monoglucuronideUDP-GLUCURONOSYL-TRANSFERASEUDP-GLUCURONOSYL-TRANSFERASEBilirubin monoglucuronide+UDPBilirubin diglucuronide+UDPBLOODHEPATOCYTEUDP-GlcUAUDP-GlcUABILE DUCTULEBilirubin • AlbuminBilirubin1. UPTAKEBilirubin diglucuronideBilirubin diglucuronide2. CONJUGATIONNeonatal jaundice“Toxic” jaundiceCrigler-Najjar syndromeGilbert syndrome3. SECRETIONDubin-Johnson syndromeFigure 32–15. Diagrammatic representation of thethree major processes (uptake, conjugation, and secretion)involved in the transfer of bilirubin from blood tobile. Certain proteins of hepatocytes, such as ligandin (afamily of glutathione S-transferase) and Y protein, bindintracellular bilirubin and may prevent its efflux into theblood stream. The process affected in a number of conditionscausing jaundice is also shown.Conjugated Bilirubin Is Reduced toUrobilinogen by Intestinal BacteriaAs the conjugated bilirubin reaches the terminal ileumand the large intestine, the glucuronides are removed byspecific bacterial enzymes (-glucuronidases), and thepigment is subsequently reduced by the fecal flora to agroup of colorless tetrapyrrolic compounds called urobilinogens(Figure 32–16). In the terminal ileum andlarge intestine, a small fraction of the urobilinogens is reabsorbedand reexcreted through the liver to constitutethe enterohepatic urobilinogen cycle. Under abnormalconditions, particularly when excessive bile pigment isformed or liver disease interferes with this intrahepaticcycle, urobilinogen may also be excreted in the urine.Normally, most of the colorless urobilinogensformed in the colon by the fecal flora are oxidized thereto urobilins (colored compounds) and are excreted inthe feces (Figure 32–16). Darkening of feces uponstanding in air is due to the oxidation of residual urobilinogensto urobilins.HYPERBILIRUBINEMIA CAUSESJAUNDICEWhen bilirubin in the blood exceeds 1 mg/dL (17.1µmol/L), hyperbilirubinemia exists. Hyperbilirubinemiamay be due to the production of more bilirubinthan the normal liver can excrete, or it may result fromthe failure of a damaged liver to excrete bilirubin producedin normal amounts. In the absence of hepaticdamage, obstruction of the excretory ducts of theliver—by preventing the excretion of bilirubin—willalso cause hyperbilirubinemia. In all these situations,bilirubin accumulates in the blood, and when it reachesa certain concentration (approximately 2–2.5 mg/dL),

282 / CHAPTER 32MH 2 CMINHEOHOHMMH 2 CMINHEHHOHOHHMHMH 2 CMINHEHHOHOHHMHNHHNNHHNNHNPHNH 2 C CH 2EPHNH 2 C CH 2EPHNHC CH 2EPMMesobilirubinogen(C 33 H 44 O 6 N 4 )PMStercobilinogen(L-Urobilinogen)PStercobilin(L-Urobilin)MFigure 32–16.Structure of some bile pigments.it diffuses into the tissues, which then become yellow.That condition is called jaundice or icterus.In clinical studies of jaundice, measurement ofbilirubin in the serum is of great value. A method forquantitatively assaying the bilirubin content of theserum was first devised by van den Bergh by applicationof Ehrlich’s test for bilirubin in urine. The Ehrlich reactionis based on the coupling of diazotized sulfanilicacid (Ehrlich’s diazo reagent) and bilirubin to producea reddish-purple azo compound. In the original procedureas described by Ehrlich, methanol was used toprovide a solution in which both bilirubin and thediazo regent were soluble. Van den Bergh inadvertentlyomitted the methanol on an occasion when assay of bilepigment in human bile was being attempted. To hissurprise, normal development of the color occurred “directly.”This form of bilirubin that would react withoutthe addition of methanol was thus termed “directreacting.”It was then found that this same direct reactionwould also occur in serum from cases of jaundicedue to biliary obstruction. However, it was still necessaryto add methanol to detect bilirubin in normalserum or that which was present in excess in serumfrom cases of hemolytic jaundice where no evidence ofobstruction was to be found. To that form of bilirubinwhich could be measured only after the addition ofmethanol, the term “indirect-reacting” was applied.It was subsequently discovered that the indirectbilirubin is “free” (unconjugated) bilirubin en route tothe liver from the reticuloendothelial tissues, where thebilirubin was originally produced by the breakdown ofheme porphyrins. Since this bilirubin is not water-soluble,it requires methanol to initiate coupling with thediazo reagent. In the liver, the free bilirubin becomesconjugated with glucuronic acid, and the conjugate,bilirubin glucuronide, can then be excreted into thebile. Furthermore, conjugated bilirubin, being watersoluble,can react directly with the diazo reagent, sothat the “direct bilirubin” of van den Bergh is actually abilirubin conjugate (bilirubin glucuronide).Depending on the type of bilirubin present inplasma—ie, unconjugated or conjugated—hyperbilirubinemiamay be classified as retention hyperbilirubinemia,due to overproduction, or regurgitation hyperbilirubinemia,due to reflux into the bloodstreambecause of biliary obstruction.Because of its hydrophobicity, only unconjugatedbilirubin can cross the blood-brain barrier into the centralnervous system; thus, encephalopathy due to hyperbilirubinemia(kernicterus) can occur only in connectionwith unconjugated bilirubin, as found in retentionhyperbilirubinemia. On the other hand, because of itswater-solubility, only conjugated bilirubin can appearin urine. Accordingly, choluric jaundice (choluria isthe presence of bile pigments in the urine) occurs onlyin regurgitation hyperbilirubinemia, and acholuricjaundice occurs only in the presence of an excess of unconjugatedbilirubin.Elevated Amounts of UnconjugatedBilirubin in Blood Occur in a Numberof ConditionsA. HEMOLYTIC ANEMIASHemolytic anemias are important causes of unconjugatedhyperbilirubinemia, though unconjugated hyperbilirubinemiais usually only slight (< 4 mg/dL; < 68.4µmol/L) even in the event of extensive hemolysis becauseof the healthy liver’s large capacity for handlingbilirubin.B. NEONATAL “PHYSIOLOGIC JAUNDICE”This transient condition is the most common cause ofunconjugated hyperbilirubinemia. It results from an ac-

PORPHYRINS & BILE PIGMENTS / 283celerated hemolysis around the time of birth and an immaturehepatic system for the uptake, conjugation, andsecretion of bilirubin. Not only is the bilirubin-UGTactivity reduced, but there probably is reduced synthesisof the substrate for that enzyme, UDP-glucuronic acid.Since the increased amount of bilirubin is unconjugated,it is capable of penetrating the blood-brain barrierwhen its concentration in plasma exceeds thatwhich can be tightly bound by albumin (20–25mg/dL). This can result in a hyperbilirubinemic toxicencephalopathy, or kernicterus, which can cause mentalretardation. Because of the recognized inducibilityof this bilirubin-metabolizing system, phenobarbitalhas been administered to jaundiced neonates and is effectivein this disorder. In addition, exposure to bluelight (phototherapy) promotes the hepatic excretion ofunconjugated bilirubin by converting some of thebilirubin to other derivatives such as maleimide fragmentsand geometric isomers that are excreted in thebile.C. CRIGLER-NAJJAR SYNDROME, TYPE I;CONGENITAL NONHEMOLYTIC JAUNDICEType I Crigler-Najjar syndrome is a rare autosomal recessivedisorder. It is characterized by severe congenitaljaundice (serum bilirubin usually exceeds 20 mg/dL)due to mutations in the gene encoding bilirubin-UGTactivity in hepatic tissues. The disease is often fatalwithin the first 15 months of life. Children with thiscondition have been treated with phototherapy, resultingin some reduction in plasma bilirubin levels. Phenobarbitalhas no effect on the formation of bilirubinglucuronides in patients with type I Crigler-Najjar syndrome.A liver transplant may be curative.D. CRIGLER-NAJJAR SYNDROME, TYPE IIThis rare inherited disorder also results from mutationsin the gene encoding bilirubin-UGT, but some activityof the enzyme is retained and the condition has a morebenign course than type I. Serum bilirubin concentrationsusually do not exceed 20 mg/dL. Patients withthis condition can respond to treatment with largedoses of phenobarbital.E. GILBERT SYNDROMEAgain, this is caused by mutations in the gene encodingbilirubin-UGT, but approximately 30% of the enzyme’sactivity is preserved and the condition is entirelyharmless.F. TOXIC HYPERBILIRUBINEMIAUnconjugated hyperbilirubinemia can result fromtoxin-induced liver dysfunction such as that caused bychloroform, arsphenamines, carbon tetrachloride, acetaminophen,hepatitis virus, cirrhosis, and Amanitamushroom poisoning. These acquired disorders are dueto hepatic parenchymal cell damage, which impairsconjugation.Obstruction in the Biliary Tree Is theCommonest Cause of ConjugatedHyperbilirubinemiaA. OBSTRUCTION OF THE BILIARY TREEConjugated hyperbilirubinemia commonly results fromblockage of the hepatic or common bile ducts, mostoften due to a gallstone or to cancer of the head of thepancreas. Because of the obstruction, bilirubin diglucuronidecannot be excreted. It thus regurgitates intothe hepatic veins and lymphatics, and conjugatedbilirubin appears in the blood and urine (choluric jaundice).The term cholestatic jaundice is used to include allcases of extrahepatic obstructive jaundice. It also coversthose cases of jaundice that exhibit conjugated hyperbilirubinemiadue to micro-obstruction of intrahepaticbiliary ductules by swollen, damaged hepatocytes (eg, asmay occur in infectious hepatitis).B. DUBIN-JOHNSON SYNDROMEThis benign autosomal recessive disorder consists ofconjugated hyperbilirubinemia in childhood or duringadult life. The hyperbilirubinemia is caused by mutationsin the gene encoding MRP-2 (see above), the proteininvolved in the secretion of conjugated bilirubininto bile. The centrilobular hepatocytes contain an abnormalblack pigment that may be derived from epinephrine.C. ROTOR SYNDROMEThis is a rare benign condition characterized by chronicconjugated hyperbilirubinemia and normal liver histology.Its precise cause has not been identified, but it isthought to be due to an abnormality in hepatic storage.Some Conjugated Bilirubin Can BindCovalently to AlbuminWhen levels of conjugated bilirubin remain high inplasma, a fraction can bind covalently to albumin (deltabilirubin). Because it is bound covalently to albumin,this fraction has a longer half-life in plasma than doesconventional conjugated bilirubin. Thus, it remains elevatedduring the recovery phase of obstructive jaundiceafter the remainder of the conjugated bilirubin has declinedto normal levels; this explains why some patientscontinue to appear jaundiced after conjugated bilirubinlevels have returned to normal.

284 / CHAPTER 32Table 32–3. Laboratory results in normal patients and patients with three different causes of jaundice.Condition Serum Bilirubin Urine Urobilinogen Urine Bilirubin Fecal UrobilinogenNormal Direct: 0.1–0.4 mg/dL 0–4 mg/24 h Absent 40–280 mg/24 hIndirect: 0.2–0.7 mg/dLHemolytic anemia ↑ Indirect Increased Absent IncreasedHepatitis ↑ Direct and indirect Decreased if micro- Present if micro- Decreasedobstruction is obstruction occurspresentObstructive jaundice 1 ↑ Direct Absent Present Trace to absent1 The commonest causes of obstructive (posthepatic) jaundice are cancer of the head of the pancreas and a gallstone lodged in the commonbile duct. The presence of bilirubin in the urine is sometimes referred to as choluria—therefore, hepatitis and obstruction of thecommon bile duct cause choluric jaundice, whereas the jaundice of hemolytic anemia is referred to as acholuric. The laboratory results inpatients with hepatitis are variable, depending on the extent of damage to parenchymal cells and the extent of micro-obstruction to bileductules. Serum levels of ALT and AST are usually markedly elevated in hepatitis, whereas serum levels of alkaline phosphatase are elevatedin obstructive liver disease.Urobilinogen & Bilirubin in UrineAre Clinical IndicatorsNormally, there are mere traces of urobilinogen in theurine. In complete obstruction of the bile duct, nourobilinogen is found in the urine, since bilirubin hasno access to the intestine, where it can be converted tourobilinogen. In this case, the presence of bilirubin(conjugated) in the urine without urobilinogen suggestsobstructive jaundice, either intrahepatic or posthepatic.In jaundice secondary to hemolysis, the increasedproduction of bilirubin leads to increased production ofurobilinogen, which appears in the urine in largeamounts. Bilirubin is not usually found in the urine inhemolytic jaundice (because unconjugated bilirubindoes not pass into the urine), so that the combinationof increased urobilinogen and absence of bilirubin issuggestive of hemolytic jaundice. Increased blood destructionfrom any cause brings about an increase inurine urobilinogen.Table 32–3 summarizes laboratory results obtainedon patients with three different causes of jaundice—hemolyticanemia (a prehepatic cause), hepatitis (a hepaticcause), and obstruction of the common bile duct (aposthepatic cause). Laboratory tests on blood (evaluationof the possibility of a hemolytic anemia and measurementof prothrombin time) and on serum (eg, electrophoresisof proteins; activities of the enzymes ALT,AST, and alkaline phosphatase) are also important inhelping to distinguish between prehepatic, hepatic, andposthepatic causes of jaundice.SUMMARY• Hemoproteins, such as hemoglobin and the cytochromes,contain heme. Heme is an iron-porphyrincompound (Fe 2+ -protoporphyrin IX) inwhich four pyrrole rings are joined by methenylbridges. The eight side groups (methyl, vinyl, andpropionyl substituents) on the four pyrrole rings ofheme are arranged in a specific sequence.• Biosynthesis of the heme ring occurs in mitochondriaand cytosol via eight enzymatic steps. It commenceswith formation of δ-aminolevulinate (ALA) fromsuccinyl-CoA and glycine in a reaction catalyzed byALA synthase, the regulatory enzyme of the pathway.• Genetically determined abnormalities of seven of theeight enzymes involved in heme biosynthesis result inthe inherited porphyrias. Red blood cells and liverare the major sites of metabolic expression of the porphyrias.Photosensitivity and neurologic problemsare common complaints. Intake of certain compounds(such as lead) can cause acquired porphyrias.Increased amounts of porphyrins or their precursorscan be detected in blood and urine, facilitating diagnosis.• Catabolism of the heme ring is initiated by the enzymeheme oxygenase, producing a linear tetrapyrrole.• Biliverdin is an early product of catabolism and onreduction yields bilirubin. The latter is transportedby albumin from peripheral tissues to the liver, whereit is taken up by hepatocytes. The iron of heme andthe amino acids of globin are conserved and reutilized.• In the liver, bilirubin is made water-soluble by conjugationwith two molecules of glucuronic acid and issecreted into the bile. The action of bacterial enzymesin the gut produces urobilinogen and urobilin,which are excreted in the feces and urine.• Jaundice is due to elevation of the level of bilirubinin the blood. The causes of jaundice can be classified

PORPHYRINS & BILE PIGMENTS / 285as prehepatic (eg, hemolytic anemias), hepatic (eg,hepatitis), and posthepatic (eg, obstruction of thecommon bile duct). Measurements of plasma totaland nonconjugated bilirubin, of urinary urobilinogenand bilirubin, and of certain serum enzymes aswell as inspection of stool samples help distinguishbetween these causes.REFERENCESAnderson KE et al: Disorders of heme biosynthesis: X-linked sideroblasticanemia and the porphyrias. In: The Metabolic andMolecular Bases of Inherited Disease, 8th ed. Scriver CR et al(editors). McGraw-Hill, 2001.Berk PD, Wolkoff AW: Bilirubin metabolism and the hyperbilirubinemias.In: Harrison’s Principles of Internal Medicine, 15thed. Braunwald E et al (editors). McGraw-Hill, 2001.Chowdhury JR et al: Hereditary jaundice and disorders of bilirubinmetabolism. In: The Metabolic and Molecular Bases of InheritedDisease, 8th ed. Scriver CR et al (editors). McGraw-Hill,2001.Desnick RJ: The porphyrias. In: Harrison’s Principles of InternalMedicine, 15th ed. Braunwald E et al (editors). McGraw-Hill,2001.Elder GH: Haem synthesis and the porphyrias. In: Scientific Foundationsof Biochemistry in Clinical Practice, 2nd ed. WilliamsDL, Marks V (editors). Butterworth-Heinemann, 1994.

SECTION IVStructure, Function, & Replicationof Informational MacromoleculesNucleotides 33Victor W. Rodwell, PhDBIOMEDICAL IMPORTANCENucleotides—the monomer units or building blocks ofnucleic acids—serve multiple additional functions. Theyform a part of many coenzymes and serve as donors ofphosphoryl groups (eg, ATP or GTP), of sugars (eg,UDP- or GDP-sugars), or of lipid (eg, CDP-acylglycerol).Regulatory nucleotides include the second messengerscAMP and cGMP, the control by ADP of oxidativephosphorylation, and allosteric regulation ofenzyme activity by ATP, AMP, and CTP. Syntheticpurine and pyrimidine analogs that contain halogens,thiols, or additional nitrogen are employed for chemotherapyof cancer and AIDS and as suppressors of theimmune response during organ transplantation.PURINES, PYRIMIDINES, NUCLEOSIDES,& NUCLEOTIDESPurines and pyrimidines are nitrogen-containing heterocycles,cyclic compounds whose rings contain bothcarbon and other elements (hetero atoms). Note thatthe smaller pyrimidine has the longer name and thelarger purine the shorter name and that their six-atomrings are numbered in opposite directions (Figure33–1). The planar character of purines and pyrimidinesfacilitates their close association, or “stacking,” whichstabilizes double-stranded DNA (Chapter 36). The oxoand amino groups of purines and pyrimidines exhibitketo-enol and amine-imine tautomerism (Figure 33–2),but physiologic conditions strongly favor the aminoand oxo forms.1N2CHH6 7C 5 N C 8CHCN 4 N93 HPurine3NHC2H4C 5CHN1CH6PyrimidineFigure 33–1. Purine and pyrimidine. The atoms arenumbered according to the international system.Nucleosides & NucleotidesNucleosides are derivatives of purines and pyrimidinesthat have a sugar linked to a ring nitrogen. Numeralswith a prime (eg, 2′ or 3′) distinguish atoms of thesugar from those of the heterocyclic base. The sugar inribonucleosides is D-ribose, and in deoxyribonucleosidesit is 2-deoxy-D-ribose. The sugar is linked to theheterocyclic base via a -N-glycosidic bond, almost alwaysto N-1 of a pyrimidine or to N-9 of a purine (Figure33–3).NH 2 NHO OHFigure 33–2. Tautomerism of the oxo and aminofunctional groups of purines and pyrimidines.286

NUCLEOTIDES / 287NH 2NH 2OONNNHNNHNN9NO1NH 2 NN9NO1NHOOHOOHOOHOOOHOHOHOHAdenosine Cytidine GuanosineUridineFigure 33–3.Ribonucleosides, drawn as the syn conformers.OHOHOHOHNNH 2NNNAdenineMononucleotides are nucleosides with a phosphorylgroup esterified to a hydroxyl group of the sugar. The3′- and 5′-nucleotides are nucleosides with a phosphorylgroup on the 3′- or 5′-hydroxyl group of the sugar,respectively. Since most nucleotides are 5′-, the prefix“5′-” is usually omitted when naming them. UMP anddAMP thus represent nucleotides with a phosphorylgroup on C-5 of the pentose. Additional phosphorylgroups linked by acid anhydride bonds to the phosphorylgroup of a mononucleotide form nucleosidediphosphates and triphosphates (Figure 33–4).Steric hindrance by the base restricts rotation aboutthe β-N-glycosidic bond of nucleosides and nucleotides.Both therefore exist as syn or anti conformers(Figure 33–5). While both conformers occur in nature,anti conformers predominate. Table 33–1 lists themajor purines and pyrimidines and their nucleosideand nucleotide derivatives. Single-letter abbreviationsare used to identify adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), whether free or presentin nucleosides or nucleotides. The prefix “d”(deoxy) indicates that the sugar is 2′-deoxy-D-ribose(eg, dGTP) (Figure 33–6).Nucleic Acids Also ContainAdditional BasesSmall quantities of additional purines and pyrimidinesoccur in DNA and RNAs. Examples include 5-methylcytosineof bacterial and human DNA, 5-hydroxymethylcytosineof bacterial and viral nucleic acids, andmono- and di-N-methylated adenine and guanine ofHOO –POOOPO –OOO –POCH 2OHO OHRiboseNNH 2NNNNNNH 2NNAdenosine 5′-monophosphate (AMP)HOOHOOAdenosine 5′-diphosphate (ADP)Adenosine 5′-triphosphate (ATP)Figure 33–4. ATP, its diphosphate, and itsmonophosphate.SynOHOHAntiFigure 33–5. The syn and anti conformers of adenosinediffer with respect to orientation about the N-glycosidicbond.OHOH

288 / CHAPTER 33Table 33–1. Bases, nucleosides, and nucleotides.NH 2NucleosideBase Base X = Ribose or Nucleotide, WhereFormula X = H Deoxyribose X = Ribose PhosphateNNNNAdenine Adenosine Adenosine monophosphateA A AMPXHH 2 NNONNNGuanine Guanosine Guanosine monophosphateG G GMPXNH 2NONCytosine Cytidine Cytidine monophosphateC C CMPXHNOONUracil Uridine Uridine monophosphateU U UMPXHNOONCH 3Thymine Thymidine Thymidine monophosphateT T TMPdXNNH 2NNNH 2NHNOHNOCH 3NNNNONONO– OOPO –OO– OOPO –OO– OOPO –OO– OOPO –OOHOHOHHOHOHOHHAMPdAMPUMPTMPFigure 33–6.AMP, dAMP, UMP, and TMP.

NUCLEOTIDES / 289ONNH 2CH 3NH5-MethylcytosineONNH 2NHCH 2 OH5-Hydroxymethylcytosine–OOPNH 2NNCH 2OONNOHNH 2 N NO CH 2O– O P ONNH 3 CNCH 3OCH 3OOHOOHNNHNN7Figure 33–9.cAMP, 3′,5′-cyclic AMP, and cGMP.NNHDimethylaminoadenineH 2 Nmammalian messenger RNAs (Figure 33–7). Theseatypical bases function in oligonucleotide recognitionand in regulating the half-lives of RNAs. Free nucleotidesinclude hypoxanthine, xanthine, and uric acid(see Figure 34–8), intermediates in the catabolism ofadenine and guanine. Methylated heterocyclic bases ofplants include the xanthine derivatives caffeine of coffee,theophylline of tea, and theobromine of cocoa (Figure33–8).Posttranslational modification of preformed polynucleotidescan generate additional bases such aspseudouridine, in which D-ribose is linked to C-5 ofuracil by a carbon-to-carbon bond rather than by aβ-N-glycosidic bond. The nucleotide pseudouridylicacid Ψ arises by rearrangement of UMP of a preformedtRNA. Similarly, methylation by S-adenosylmethionineof a UMP of preformed tRNA forms TMP (thymidinemonophosphate), which contains ribose rather than deoxyribose.N7-MethylguanineFigure 33–7. Four uncommon naturally occurringpyrimidines and purines.H 3 CNOCH 3NNNucleotides Serve DiversePhysiologic FunctionsNucleotides participate in reactions that fulfill physiologicfunctions as diverse as protein synthesis, nucleicacid synthesis, regulatory cascades, and signal transductionpathways.Nucleoside Triphosphates Have HighGroup Transfer PotentialAcid anhydrides, unlike phosphate esters, have highgroup transfer potential. ∆ 0 ′ for the hydrolysis of eachof the terminal phosphates of nucleoside triphosphatesis about −7 kcal/mol (−30 kJ/mol). The high grouptransfer potential of purine and pyrimidine nucleosidetriphosphates permits them to function as group transferreagents. Cleavage of an acid anhydride bond typicallyis coupled with a highly endergonic process suchas covalent bond synthesis—eg, polymerization of nucleosidetriphosphates to form a nucleic acid.In addition to their roles as precursors of nucleicacids, ATP, GTP, UTP, CTP, and their derivativeseach serve unique physiologic functions discussed inother chapters. Selected examples include the role ofATP as the principal biologic transducer of free energy;the second messenger cAMP (Figure 33–9); adenosine3′-phosphate-5′-phosphosulfate (Figure 33–10), thesulfate donor for sulfated proteoglycans (Chapter 48)and for sulfate conjugates of drugs; and the methylgroup donor S-adenosylmethionine (Figure 33–11).ONNCH 3Figure 33–8. Caffeine, a trimethylxanthine. The dimethylxanthinestheobromine and theophylline aresimilar but lack the methyl group at N-1 and at N-7, respectively.PAdenine Ribose P O SO2–3Figure 33–10. Adenosine 3′-phosphate-5′-phosphosulfate.

290 / CHAPTER 33NNH 2NNNTable 33–2. Many coenzymes and relatedcompounds are derivatives of adenosinemonophosphate.NH 2COO –CH CH 2+NH 3CH 2S +CH 3CH 2OHO OHROOPO –ONNCH 2n ONNAdenineFigure 33–11.MethionineAdenosineS-Adenosylmethionine.GTP serves as an allosteric regulator and as an energysource for protein synthesis, and cGMP (Figure 33–9)serves as a second messenger in response to nitric oxide(NO) during relaxation of smooth muscle (Chapter48). UDP-sugar derivatives participate in sugar epimerizationsand in biosynthesis of glycogen, glucosyl disaccharides,and the oligosaccharides of glycoproteins andproteoglycans (Chapters 47 and 48). UDP-glucuronicacid forms the urinary glucuronide conjugates of bilirubin(Chapter 32) and of drugs such as aspirin. CTPparticipates in biosynthesis of phosphoglycerides,sphingomyelin, and other substituted sphingosines(Chapter 24). Finally, many coenzymes incorporate nucleotidesas well as structures similar to purine andpyrimidine nucleotides (see Table 33–2).Nucleotides Are Polyfunctional AcidsNucleosides or free purine or pyrimidine bases are unchargedat physiologic pH. By contrast, the primaryphosphoryl groups (pK about 1.0) and secondary phosphorylgroups (pK about 6.2) of nucleotides ensure thatthey bear a negative charge at physiologic pH. Nucleotidescan, however, act as proton donors or acceptorsat pH values two or more units above or belowneutrality.Nucleotides Absorb Ultraviolet LightThe conjugated double bonds of purine and pyrimidinederivatives absorb ultraviolet light. The mutagenic effectof ultraviolet light results from its absorption bynucleotides in DNA with accompanying chemicalchanges. While spectra are pH-dependent, at pH 7.0 allthe common nucleotides absorb light at a wavelengthclose to 260 nm. The concentration of nucleotides andCoenzyme R R R nActive methionine Methionine* H H 0Amino acid adenylates Amino acid H H 1Active sulfate2−SO 3 H2−PO 3 13′,5′-Cyclic AMP H2−PO 3 1NAD*†H H 2NADP*†2−PO 3 H 2FAD†H H 2CoASH†H2−PO 3 2*Replaces phosphoryl group.† R is a B vitamin derivative.R'' OOR'D-Ribosenucleic acids thus often is expressed in terms of “absorbanceat 260 nm.”SYNTHETIC NUCLEOTIDE ANALOGSARE USED IN CHEMOTHERAPYSynthetic analogs of purines, pyrimidines, nucleosides,and nucleotides altered in either the heterocyclic ring orthe sugar moiety have numerous applications in clinicalmedicine. Their toxic effects reflect either inhibition ofenzymes essential for nucleic acid synthesis or their incorporationinto nucleic acids with resulting disruptionof base-pairing. Oncologists employ 5-fluoro- or 5-iodouracil, 3-deoxyuridine, 6-thioguanine and 6-mercaptopurine,5- or 6-azauridine, 5- or 6-azacytidine,and 8-azaguanine (Figure 33–12), which are incorporatedinto DNA prior to cell division. The purine analogallopurinol, used in treatment of hyperuricemia andgout, inhibits purine biosynthesis and xanthine oxidaseactivity. Cytarabine is used in chemotherapy of cancer.Finally, azathioprine, which is catabolized to 6-mercaptopurine,is employed during organ transplantation tosuppress immunologic rejection.

NUCLEOTIDES / 291OOHN5IHNONON6 NHOOHNO5FHOOHNON8 N2′HO H5-Iodo-2′-deoxyuridineONH5-FluorouracilHO OH6-AzauridineH 2 NN8-AzaguanineNHSHSHOHN6NNNH6-MercaptopurineFigure 33–12.H 2 NN6N6-ThioguanineNNHN 1Selected synthetic pyrimidine and purine analogs.263N54AlloburinolNHNNonhydrolyzable NucleosideTriphosphate Analogs Serve asResearch ToolsSynthetic nonhydrolyzable analogs of nucleosidetriphosphates (Figure 33–13) allow investigators to distinguishthe effects of nucleotides due to phosphoryltransfer from effects mediated by occupancy of allostericnucleotide-binding sites on regulated enzymes.POLYNUCLEOTIDESThe 5′-phosphoryl group of a mononucleotide can esterifya second ⎯OH group, forming a phosphodiester.Most commonly, this second ⎯OH group is the3′-OH of the pentose of a second nucleotide. Thisforms a dinucleotide in which the pentose moieties arelinked by a 3′ →5′ phosphodiester bond to form the“backbone” of RNA and DNA.While formation of a dinucleotide may be representedas the elimination of water between twomonomers, the reaction in fact strongly favors phosphodiesterhydrolysis. Phosphodiesterases rapidly catalyzethe hydrolysis of phosphodiester bonds whosespontaneous hydrolysis is an extremely slow process.Consequently, DNA persists for considerable periodsand has been detected even in fossils. RNAs are far lessstable than DNA since the 2′-hydroxyl group of RNA(absent from DNA) functions as a nucleophile duringhydrolysis of the 3′,5′-phosphodiester bond.Polynucleotides Are DirectionalMacromoleculesPhosphodiester bonds link the 3′- and 5′-carbons of adjacentmonomers. Each end of a nucleotide polymerthus is distinct. We therefore refer to the “5′- end” orthe “3′- end” of polynucleotides, the 5′- end being theone with a free or phosphorylated 5′-hydroxyl.Polynucleotides Have Primary StructureThe base sequence or primary structure of a polynucleotidecan be represented as shown below. The phosphodiesterbond is represented by P or p, bases by a singleletter, and pentoses by a vertical line.PA T C AP P POH

292 / CHAPTER 33Pu/Py R O P O P OO – O –OWhere all the phosphodiester bonds are 5′ →3′, amore compact notation is possible:pGpGpApTpCpAThis representation indicates that the 5′-hydroxyl—but not the 3′-hydroxyl—is phosphorylated.The most compact representation shows only thebase sequence with the 5′- end on the left and the 3′-end on the right. The phosphoryl groups are assumedbut not shown:GGATCAOParent nucleoside triphosphatePu/Py R O P O P CH 2O – O –OOOβ,γ-Methylene derivativeOPu/Py R O P O PO – O –β,γ-Imino derivativeHNOP O –O –OP O –O –OP O –O –Figure 33–13. Synthetic derivatives of nucleosidetriphosphates incapable of undergoing hydrolytic releaseof the terminal phosphoryl group. (Pu/Py, apurine or pyrimidine base; R, ribose or deoxyribose.)Shown are the parent (hydrolyzable) nucleosidetriphosphate (top) and the unhydrolyzable β-methylene(center) and γ-imino derivatives (bottom).SUMMARY• Under physiologic conditions, the amino and oxotautomers of purines, pyrimidines, and their derivativespredominate.• Nucleic acids contain, in addition to A, G, C, T, andU, traces of 5-methylcytosine, 5-hydroxymethylcytosine,pseudouridine (Ψ), or N-methylated bases.• Most nucleosides contain D-ribose or 2-deoxy-Driboselinked to N-1 of a pyrimidine or to N-9 of apurine by a β-glycosidic bond whose syn conformerspredominate.• A primed numeral locates the position of the phosphateon the sugars of mononucleotides (eg, 3′-GMP, 5′-dCMP). Additional phosphoryl groupslinked to the first by acid anhydride bonds form nucleosidediphosphates and triphosphates.• Nucleoside triphosphates have high group transferpotential and participate in covalent bond syntheses.The cyclic phosphodiesters cAMP and cGMP functionas intracellular second messengers.• Mononucleotides linked by 3′ →5′-phosphodiesterbonds form polynucleotides, directional macromoleculeswith distinct 3′- and 5′- ends. For pTpGpTp orTGCATCA, the 5′- end is at the left, and all phosphodiesterbonds are 3′→5′.• Synthetic analogs of purine and pyrimidine bases andtheir derivatives serve as anticancer drugs either byinhibiting an enzyme of nucleotide biosynthesis orby being incorporated into DNA or RNA.REFERENCESAdams RLP, Knowler JT, Leader DP: The Biochemistry of the NucleicAcids, 11th ed. Chapman & Hall, 1992.Blackburn GM, Gait MJ: Nucleic Acids in Chemistry & Biology. IRLPress, 1990.Bugg CE, Carson WM, Montgomery JA: Drugs by design. Sci Am1992;269(6):92.

Metabolism of Purine &Pyrimidine Nucleotides 34Victor W. Rodwell, PhDBIOMEDICAL IMPORTANCEThe biosynthesis of purines and pyrimidines is stringentlyregulated and coordinated by feedback mechanismsthat ensure their production in quantities and attimes appropriate to varying physiologic demand. Geneticdiseases of purine metabolism include gout,Lesch-Nyhan syndrome, adenosine deaminase deficiency,and purine nucleoside phosphorylase deficiency.By contrast, apart from the orotic acidurias, there arefew clinically significant disorders of pyrimidine catabolism.PURINES & PYRIMIDINES AREDIETARILY NONESSENTIALHuman tissues can synthesize purines and pyrimidinesfrom amphibolic intermediates. Ingested nucleic acidsand nucleotides, which therefore are dietarily nonessential,are degraded in the intestinal tract to mononucleotides,which may be absorbed or converted topurine and pyrimidine bases. The purine bases are thenoxidized to uric acid, which may be absorbed and excretedin the urine. While little or no dietary purine orpyrimidine is incorporated into tissue nucleic acids, injectedcompounds are incorporated. The incorporationof injected [ 3 H]thymidine into newly synthesized DNAthus is used to measure the rate of DNA synthesis.BIOSYNTHESIS OF PURINE NUCLEOTIDESPurine and pyrimidine nucleotides are synthesized invivo at rates consistent with physiologic need. Intracellularmechanisms sense and regulate the pool sizes ofnucleotide triphosphates (NTPs), which rise duringgrowth or tissue regeneration when cells are rapidly dividing.Early investigations of nucleotide biosynthesisemployed birds, and later ones used Escherichia coli.Isotopic precursors fed to pigeons established the sourceof each atom of a purine base (Figure 34–1) and initiatedstudy of the intermediates of purine biosynthesis.Three processes contribute to purine nucleotidebiosynthesis. These are, in order of decreasing importance:(1) synthesis from amphibolic intermediates293(synthesis de novo), (2) phosphoribosylation of purines,and (3) phosphorylation of purine nucleosides.INOSINE MONOPHOSPHATE (IMP)IS SYNTHESIZED FROM AMPHIBOLICINTERMEDIATESFigure 34–2 illustrates the intermediates and reactionsfor conversion of α-D-ribose 5-phosphate to inosinemonophosphate (IMP). Separate branches then lead toAMP and GMP (Figure 34–3). Subsequent phosphoryltransfer from ATP converts AMP and GMP to ADPand GDP. Conversion of GDP to GTP involves a secondphosphoryl transfer from ATP, whereas conversionof ADP to ATP is achieved primarily by oxidativephosphorylation (see Chapter 12).Multifunctional Catalysts Participate inPurine Nucleotide BiosynthesisIn prokaryotes, each reaction of Figure 34–2 is catalyzedby a different polypeptide. By contrast, in eukaryotes,the enzymes are polypeptides with multiplecatalytic activities whose adjacent catalytic sites facilitatechanneling of intermediates between sites. Threedistinct multifunctional enzymes catalyze reactions 3,4, and 6, reactions 7 and 8, and reactions 10 and 11 ofFigure 34–2.Antifolate Drugs or Glutamine AnalogsBlock Purine Nucleotide BiosynthesisThe carbons added in reactions 4 and 5 of Figure 34–2are contributed by derivatives of tetrahydrofolate.Purine deficiency states, which are rare in humans, generallyreflect a deficiency of folic acid. Compounds thatinhibit formation of tetrahydrofolates and thereforeblock purine synthesis have been used in cancerchemotherapy. Inhibitory compounds and the reactionsthey inhibit include azaserine (reaction 5, Figure 34–2),diazanorleucine (reaction 2), 6-mercaptopurine (reactions13 and 14), and mycophenolic acid (reaction 14).

294 / CHAPTER 34N 10 -FormyltetrahydrofolateAspartateRespiratory CO 2N1C6C 2 3NGlycineN78 C4 N 5 ,N 105 C C9NHAmide nitrogen of glutamine-MethenyltetrahydrofolateFigure 34–1. Sources of the nitrogen and carbonatoms of the purine ring. Atoms 4, 5, and 7 (shaded) derivefrom glycine.“SALVAGE REACTIONS” CONVERTPURINES & THEIR NUCLEOSIDES TOMONONUCLEOTIDESConversion of purines, their ribonucleosides, and theirdeoxyribonucleosides to mononucleotides involves socalled“salvage reactions” that require far less energythan de novo synthesis. The more important mechanisminvolves phosphoribosylation by PRPP (structureII, Figure 34–2) of a free purine (Pu) to form a purine5′-mononucleotide (Pu-RP).Pu + PR −PP → PRP + PP iTwo phosphoribosyl transferases then convert adenineto AMP and hypoxanthine and guanine to IMP orGMP (Figure 34–4). A second salvage mechanism involvesphosphoryl transfer from ATP to a purine ribonucleoside(PuR):PuR + ATP →PuR − P + ADPAdenosine kinase catalyzes phosphorylation of adenosineand deoxyadenosine to AMP and dAMP, and deoxycytidinekinase phosphorylates deoxycytidine and2′-deoxyguanosine to dCMP and dGMP.Liver, the major site of purine nucleotide biosynthesis,provides purines and purine nucleosides for salvageand utilization by tissues incapable of their biosynthesis.For example, human brain has a low level of PRPPamidotransferase (reaction 2, Figure 34–2) and hencedepends in part on exogenous purines. Erythrocytesand polymorphonuclear leukocytes cannot synthesize5-phosphoribosylamine (structure III, Figure 34–2)and therefore utilize exogenous purines to form nucleotides.AMP & GMP Feedback-Regulate PRPPGlutamyl AmidotransferaseSince biosynthesis of IMP consumes glycine, glutamine,tetrahydrofolate derivatives, aspartate, and ATP,it is advantageous to regulate purine biosynthesis. Themajor determinant of the rate of de novo purine nucleotidebiosynthesis is the concentration of PRPP,whose pool size depends on its rates of synthesis, utilization,and degradation. The rate of PRPP synthesisdepends on the availability of ribose 5-phosphate andon the activity of PRPP synthase, an enzyme sensitiveto feedback inhibition by AMP, ADP, GMP, andGDP.AMP & GMP Feedback-RegulateTheir Formation From IMPTwo mechanisms regulate conversion of IMP to GMPand AMP. AMP and GMP feedback-inhibit adenylosuccinatesynthase and IMP dehydrogenase (reactions12 and 14, Figure 34–3), respectively. Furthermore,conversion of IMP to adenylosuccinate en route toAMP requires GTP, and conversion of xanthinylate(XMP) to GMP requires ATP. This cross-regulationbetween the pathways of IMP metabolism thus servesto decrease synthesis of one purine nucleotide whenthere is a deficiency of the other nucleotide. AMP andGMP also inhibit hypoxanthine-guanine phosphoribosyltransferase,which converts hypoxanthine and guanineto IMP and GMP (Figure 34–4), and GMP feedback-inhibitsPRPP glutamyl amidotransferase (reaction2, Figure 34–2).REDUCTION OF RIBONUCLEOSIDEDIPHOSPHATES FORMSDEOXYRIBONUCLEOSIDEDIPHOSPHATESReduction of the 2′-hydroxyl of purine and pyrimidineribonucleotides, catalyzed by the ribonucleotide reductasecomplex (Figure 34–5), forms deoxyribonucleosidediphosphates (dNDPs). The enzyme complexis active only when cells are actively synthesizing DNA.Reduction requires thioredoxin, thioredoxin reductase,and NADPH. The immediate reductant, reducedthioredoxin, is produced by NADPH:thioredoxin reductase(Figure 34–5). Reduction of ribonucleosidediphosphates (NDPs) to deoxyribonucleoside diphosphates(dNDPs) is subject to complex regulatory controlsthat achieve balanced production of deoxyribonucleotidesfor synthesis of DNA (Figure 34–6).

PO5CH 2OHHOHHHOHOHα-D-Ribose 5-phosphate(I)ATP1Mg 2+PAMPPRPPSYNTHASEO5CH 2HO4 1H HH 3 2 O P O POH OHPRPP(II)P OGlutamine GlutamateH 2 O2PPiPRPP GLUTAMYLAMIDOTRANSFERASECH 2OHHOHHHOHGlycine7NH + 3H 2 C 5O C 49 O–P+NH 335-Phospho-β-D-ribosylamine(III)OOH 2CH 2 C 5O7NH + 3C 4NHMg 2 +H HATP ADP + PH HiOH OHGlycinamideribosyl-5-phosphate(IV)N 5 ,N 10 -Methenyl-H 4 folate H 4 folate4FORMYLTRANSFERASEHNH2C578 CH4C 9O NH OR-5- PFormylglycinamideribosyl-5-phosphate(V)Aspartate– OOC+HC NH 3CH 2VI SYNTHETASE5GlnATPMg 2+GluCOO –CHHC– OOCFumarate9– OOCHCH2 C– OOCOCN 6 5C1H3 4H2NCNNCHR-5- PAminoimidazole succinylcarboxamide ribosyl-5-phosphate(IX)H 2 O– OOC8IX SYNTHETASEOC– O 6 543H2NNNCHR-5- PAminoimidazolecarboxylate ribosyl-5-phosphate(VIII)ATP, Mg 2+CO H 2 O Ring closureN2H7HCN6H 72C58 CHCH4C 3 C 9VII CARBOXYLASE H 2 N N VII SYNTHETASEHN NH OR-5- PR-5- PAminoimidazoleFormylglycinamidineribosyl-5-phosphateribosyl-5-phosphate(VII)(VI)ADENYLOSUCCINASEH2 NOCH2NCCNCHNR-5- PN 10 -Formyl-H 4 folate H 4 folate10FORMYLTRANSFERASEOH 2 NCHOCNHCCNCHNR-5- PH 2 O11Ring closureIMP CYCLOHYDROLASEHNHCOCNCCNCHNR-5- PAminoimidazole carboxamideribosyl-5-phosphate(X)Formimidoimidazole carboxamideribosyl-5-phosphate(XI)Inosine monophosphate (IMP)(XII)Figure 34–2. Purine biosynthesis from ribose 5-phosphate and ATP. See text for explanations. (P , PO 3 2– or PO 2 – .)

296 / CHAPTER 34HNO–OOCNCHC COO – –H–OOC C C COOHH 2 2+NH H 2 ONH3N12 NGTP, Mg 2+–H H–OOC C C COO13 NNH 2NNInosine monophosphate(IMP)NN NADENYLOSUCCINASE N NADENYLOSUCCINATER-5- P SYNTHASER-5- R-5- PPAdenylosuccinate(AMPS)Adenosine monophosphate(AMP)+NADH 2 ONADH+ H+14IMP DEHYDROGENASEHNONO N NHR-5- PXanthosine monophosphate(XMP)Figure 34–3.Glutamine15 HNATPGlutamateTRANSAMIDINASEH 2 NConversion of IMP to AMP and GMP.ONNNR-5- PGuanosine monophosphate(GMP)BIOSYNTHESIS OF PYRIMIDINENUCLEOTIDESFigure 34–7 summarizes the roles of the intermediatesand enzymes of pyrimidine nucleotide biosynthesis.The catalyst for the initial reaction is cytosolic carbamoylphosphate synthase II, a different enzyme from the mitochondrialcarbamoyl phosphate synthase I of urea synthesis(Figure 29–9). Compartmentation thus providestwo independent pools of carbamoyl phosphate. PRPP,an early participant in purine nucleotide synthesis (Figure34–2), is a much later participant in pyrimidinebiosynthesis.Multifunctional ProteinsCatalyze the Early Reactionsof Pyrimidine BiosynthesisFive of the first six enzyme activities of pyrimidinebiosynthesis reside on multifunctional polypeptides.One such polypeptide catalyzes the first three reactionsof Figure 34–2 and ensures efficient channeling of carbamoylphosphate to pyrimidine biosynthesis. A secondbifunctional enzyme catalyzes reactions 5 and 6.THE DEOXYRIBONUCLEOSIDES OFURACIL & CYTOSINE ARE SALVAGEDWhile mammalian cells reutilize few free pyrimidines,“salvage reactions” convert the ribonucleosides uridineand cytidine and the deoxyribonucleosides thymidineand deoxycytidine to their respective nucleotides. ATPdependentphosphoryltransferases (kinases) catalyze thephosphorylation of the nucleoside diphosphates 2′-deoxycytidine,2′-deoxyguanosine, and 2′-deoxyadenosineto their corresponding nucleoside triphosphates. In addition,orotate phosphoribosyltransferase (reaction 5,Figure 34–7), an enzyme of pyrimidine nucleotide synthesis,salvages orotic acid by converting it to orotidinemonophosphate (OMP).Methotrexate Blocks Reductionof DihydrofolateReaction 12 of Figure 34–7 is the only reaction of pyrimidinenucleotide biosynthesis that requires a tetrahydrofolatederivative. The methylene group of N 5 ,N 10 -methylene-tetrahydrofolateis reduced to the methyl group thatis transferred, and tetrahydrofolate is oxidized to dihydro-

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES / 297NHNNH 2NONHNAdenineNPRPPPRPPPP iPADENINEPHOSPHORIBOSYLTRANSFERASEPP iONHNNH 2NH 2COHHOHOAMPNNHHOHNRibonucleosidediphosphateReducedthioredoxinRIBONUCLEOTIDEREDUCTASETHIOREDOXINREDUCTASE2′-DeoxyribonucleosidediphosphateOxidizedthioredoxinNADP + NADPH + H +Figure 34–5. Reduction of ribonucleoside diphosphatesto 2′-deoxyribonucleoside diphosphates.H 2 NN NHHypoxanthineHNONGuanineHYPOXANTHINE-GUANINEPHOSPHORIBOSYLTRANSFERASENNHPRPPPOH 2 NPP iP ONH 2CHNHHNOH OHIMPONH 2CHHOOHHNNHHOH OHGMPFigure 34–4. Phosphoribosylation of adenine, hypoxanthine,and guanine to form AMP, IMP, and GMP,respectively.folate. For further pyrimidine synthesis to occur, dihydrofolatemust be reduced back to tetrahydrofolate, a reactioncatalyzed by dihydrofolate reductase. Dividing cells,which must generate TMP and dihydrofolate, thus are especiallysensitive to inhibitors of dihydrofolate reductasesuch as the anticancer drug methotrexate.Certain Pyrimidine Analogs AreSubstrates for Enzymes of PyrimidineNucleotide BiosynthesisOrotate phosphoribosyltransferase (reaction 5, Figure34–7) converts the drug allopurinol (Figure 33–12) toa nucleotide in which the ribosyl phosphate is attachedto N-1 of the pyrimidine ring. The anticancer drug5-fluorouracil (Figure 33–12) is also phosphoribosylatedby orotate phosphoribosyl transferase.REGULATION OF PYRIMIDINENUCLEOTIDE BIOSYNTHESISGene Expression & Enzyme ActivityBoth Are RegulatedThe activities of the first and second enzymes of pyrimidinenucleotide biosynthesis are controlled by allostericCDPUDPGDPADP– – –– – ––++++2′dCDPATP2′dUDP2′dGDP2′dADP2′dCTP2′dTTP2′dGTP2′dATPFigure 34–6. Regulation of the reduction of purineand pyrimidine ribonucleotides to their respective2′-deoxyribonucleotides. Solid lines represent chemicalflow. Broken lines show negative (– ) or positive (+ )feedback regulation.

298 / CHAPTER 34CO 2 + Glutamine + ATPCARBAMOYLPHOSPHATESYNTHASE II+ H 3N 32COO1PCarbamoylphosphate(CAP)O–O C 45 CH+216 C H+ H3 NCOO –AsparticacidASPARTATETRANSCAR-BAMOYLASE2O– O C 4H 52 N CH321 62C CHO NH COO – 3P iCarbamoylaspartic acid(CAA)DIHYDRO-OROTASEH 2 ONAD +OCHN CH 2O C CHNCOOH–DIHYDROOROTATEDEHYDROGENASENADH + H + 4Dihydrooroticacid (DHOA)OCO 26OPP iPRPPO4HN 3 5HN5HNO216NR-5- PUMPOROTIDYLIC ACIDDECARBOXYLASEO N COO –R-5- POMPOROTATEPHOSPHORIBOSYL-TRANSFERASEONHOrotic acid(OA)COO –ATP7ADPATPADPATPCTPSYNTHASEUDPUTP89NH2NADPH + H + NADP +10dUDP (deoxyuridine diphosphate)H 2 ORIBONUCLEOTIDEREDUCTASE11GlutamineTHYMIDYLATESYNTHASEdUMPO12P iN5 ,N 10 -Methylene H 4 folateH 2 folateNHNCH 3O NO NR-5- P - P - PdR-5-PCTPTMPFigure 34–7. The biosynthetic pathway for pyrimidine nucleotides.

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES / 299regulation. Carbamoyl phosphate synthase II (reaction1, Figure 34–7) is inhibited by UTP and purine nucleotidesbut activated by PRPP. Aspartate transcarbamoylase(reaction 2, Figure 34–7) is inhibited byCTP but activated by ATP. In addition, the first threeand the last two enzymes of the pathway are regulatedby coordinate repression and derepression.Purine & Pyrimidine NucleotideBiosynthesis Are Coordinately RegulatedPurine and pyrimidine biosynthesis parallel one anothermole for mole, suggesting coordinated control oftheir biosynthesis. Several sites of cross-regulation characterizepurine and pyrimidine nucleotide biosynthesis.The PRPP synthase reaction (reaction 1, Figure 34–2),which forms a precursor essential for both processes, isfeedback-inhibited by both purine and pyrimidine nucleotides.HUMANS CATABOLIZE PURINESTO URIC ACIDHumans convert adenosine and guanosine to uric acid(Figure 34–8). Adenosine is first converted to inosineby adenosine deaminase. In mammals other thanhigher primates, uricase converts uric acid to the watersolubleproduct allantoin. However, since humans lackuricase, the end product of purine catabolism in humansis uric acid.GOUT IS A METABOLIC DISORDEROF PURINE CATABOLISMVarious genetic defects in PRPP synthetase (reaction 1,Figure 34–2) present clinically as gout. Each defect—eg, an elevated V max , increased affinity for ribose 5-phosphate, or resistance to feedback inhibition—resultsin overproduction and overexcretion of purine catabolites.When serum urate levels exceed the solubilitylimit, sodium urate crystalizes in soft tissues and jointsand causes an inflammatory reaction, gouty arthritis.However, most cases of gout reflect abnormalities inrenal handling of uric acid.NNH 2NHO H2CONNOHH HH HOHHO H2 AdenosineH 2 ONH + 4OHNNN NOH HH HOH OHInosineHNP iONN NHHypoxanthineH 2 NHOHNRibose 1-phosphateH 2 NHNH 2 O + O 2H O2 O 2NHNO NH NHXanthineH 2 O + O 2ONH2 COONNH HH HOH OHGuanosineNP iN NHGuanineHN 3Figure 34–8. Formation of uric acid from purine nucleosidesby way of the purine bases hypoxanthine, xanthine, and guanine.Purine deoxyribonucleosides are degraded by the samecatabolic pathway and enzymes, all of which exist in the mucosaof the mammalian gastrointestinal tract.H 2 O 2OHNHN179O 3NHNHUric acidO

300 / CHAPTER 34OTHER DISORDERS OFPURINE CATABOLISMWhile purine deficiency states are rare in human subjects,there are numerous genetic disorders of purine catabolism.Hyperuricemias may be differentiated basedon whether patients excrete normal or excessive quantitiesof total urates. Some hyperuricemias reflect specificenzyme defects. Others are secondary to diseases suchas cancer or psoriasis that enhance tissue turnover.Lesch-Nyhan SyndromeLesch-Nyhan syndrome, an overproduction hyperuricemiacharacterized by frequent episodes of uric acidlithiasis and a bizarre syndrome of self-mutilation, reflectsa defect in hypoxanthine-guanine phosphoribosyltransferase, an enzyme of purine salvage (Figure34–4). The accompanying rise in intracellular PRPP resultsin purine overproduction. Mutations that decreaseor abolish hypoxanthine-guanine phosphoribosyltransferaseactivity include deletions, frameshift mutations,base substitutions, and aberrant mRNA splicing.Von Gierke’s DiseasePurine overproduction and hyperuricemia in vonGierke’s disease (glucose-6-phosphatase deficiency)occurs secondary to enhanced generation of the PRPPprecursor ribose 5-phosphate. An associated lactic acidosiselevates the renal threshold for urate, elevatingtotal body urates.HypouricemiaHypouricemia and increased excretion of hypoxanthineand xanthine are associated with xanthine oxidase deficiencydue to a genetic defect or to severe liver damage.Patients with a severe enzyme deficiency may exhibitxanthinuria and xanthine lithiasis.Adenosine Deaminase & PurineNucleoside Phosphorylase DeficiencyAdenosine deaminase deficiency is associated with animmunodeficiency disease in which both thymusderivedlymphocytes (T cells) and bone marrow-derivedlymphocytes (B cells) are sparse and dysfunctional.Purine nucleoside phosphorylase deficiency isassociated with a severe deficiency of T cells but apparentlynormal B cell function. Immune dysfunctions appearto result from accumulation of dGTP and dATP,which inhibit ribonucleotide reductase and thereby depletecells of DNA precursors.CATABOLISM OF PYRIMIDINESPRODUCES WATER-SOLUBLEMETABOLITESUnlike the end products of purine catabolism, thoseof pyrimidine catabolism are highly water-soluble:CO 2 , NH 3 , β-alanine, and β-aminoisobutyrate (Figure34–9). Excretion of β-aminoisobutyrate increases inleukemia and severe x-ray radiation exposure due to increaseddestruction of DNA. However, many personsof Chinese or Japanese ancestry routinely excreteβ-aminoisobutyrate. Humans probably transaminateβ-aminoisobutyrate to methylmalonate semialdehyde,which then forms succinyl-CoA (Figure 19–2).Pseudouridine Is Excreted UnchangedSince no human enzyme catalyzes hydrolysis or phosphorolysisof pseudouridine, this unusual nucleoside isexcreted unchanged in the urine of normal subjects.OVERPRODUCTION OF PYRIMIDINECATABOLITES IS ONLY RARELYASSOCIATED WITH CLINICALLYSIGNIFICANT ABNORMALITIESSince the end products of pyrimidine catabolism arehighly water-soluble, pyrimidine overproduction resultsin few clinical signs or symptoms. In hyperuricemia associatedwith severe overproduction of PRPP, there isoverproduction of pyrimidine nucleotides and increasedexcretion of β-alanine. Since N 5 ,N 10 -methylene-tetrahydrofolateis required for thymidylate synthesis,disorders of folate and vitamin B 12 metabolismresult in deficiencies of TMP.Orotic AciduriasThe orotic aciduria that accompanies Reye’s syndromeprobably is a consequence of the inability of severelydamaged mitochondria to utilize carbamoyl phosphate,which then becomes available for cytosolic overproductionof orotic acid. Type I orotic aciduria reflects a deficiencyof both orotate phosphoribosyltransferase andorotidylate decarboxylase (reactions 5 and 6, Figure34–7); the rarer type II orotic aciduria is due to a deficiencyonly of orotidylate decarboxylase (reaction 6,Figure 34–7).Deficiency of a Urea Cycle Enzyme Resultsin Excretion of Pyrimidine PrecursorsIncreased excretion of orotic acid, uracil, and uridineaccompanies a deficiency in liver mitochondrial ornithinetranscarbamoylase (reaction 2, Figure 29–9).

METABOLISM OF PURINE & PYRIMIDINE NUCLEOTIDES / 301CH 3NO NHCytosine1 /2 O 2NH3OOHN CH 3HNO NO NHHThymineUracilNADPH + H +NADP +OOHNCH 3HNHHHHHO N HO N HHHDihydrouracilDihydrothymineH 2 OH 2 OCOO −COO −H CH CHH C 32 N 22 NHC CH 2C CH 2ONONHHβ-Ureidopropionate(N-carbamoyl-β-alanine)β-Ureidoisobutyrate(N-carbamoyl-β-aminoisobutyrate)CO 2 + NH 3H 3 N + CH 2 CH 2 COO − H 3 N + CH 2 CH COO −β-AlanineNH 2β -AminoisobutyrateFigure 34–9.Catabolism of pyrimidines.Excess carbamoyl phosphate exits to the cytosol, whereit stimulates pyrimidine nucleotide biosynthesis. Theresulting mild orotic aciduria is increased by highnitrogenfoods.Drugs May Precipitate Orotic AciduriaAllopurinol (Figure 33–12), an alternative substrate fororotate phosphoribosyltransferase (reaction 5, Figure34–7), competes with orotic acid. The resulting nucleotideproduct also inhibits orotidylate decarboxylase(reaction 6, Figure 34–7), resulting in orotic aciduriaand orotidinuria. 6-Azauridine, following conversionto 6-azauridylate, also competitively inhibits orotidylatedecarboxylase (reaction 6, Figure 34–7), enhancing excretionof orotic acid and orotidine.SUMMARY• Ingested nucleic acids are degraded to purines andpyrimidines. New purines and pyrimidines areformed from amphibolic intermediates and thus aredietarily nonessential.• Several reactions of IMP biosynthesis require folatederivatives and glutamine. Consequently, antifolatedrugs and glutamine analogs inhibit purine biosynthesis.• Oxidation and amination of IMP forms AMP andGMP, and subsequent phosphoryl transfer fromATP forms ADP and GDP. Further phosphoryltransfer from ATP to GDP forms GTP. ADP is convertedto ATP by oxidative phosphorylation. Reductionof NDPs forms dNDPs.• Hepatic purine nucleotide biosynthesis is stringentlyregulated by the pool size of PRPP and by feedbackinhibition of PRPP-glutamyl amidotransferase byAMP and GMP.• Coordinated regulation of purine and pyrimidinenucleotide biosynthesis ensures their presence in proportionsappropriate for nucleic acid biosynthesisand other metabolic needs.• Humans catabolize purines to uric acid (pK a 5.8),present as the relatively insoluble acid at acidic pH oras its more soluble sodium urate salt at a pH nearneutrality. Urate crystals are diagnostic of gout.Other disorders of purine catabolism include Lesch-Nyhan syndrome, von Gierke’s disease, and hypouricemias.• Since pyrimidine catabolites are water-soluble, theiroverproduction does not result in clinical abnormalities.Excretion of pyrimidine precursors can, however,result from a deficiency of ornithine transcarbamoylasebecause excess carbamoyl phosphate isavailable for pyrimidine biosynthesis.

302 / CHAPTER 34REFERENCESBenkovic SJ: The transformylase enzymes in de novo purinebiosynthesis. Trends Biochem Sci 1994;9:320.Brooks EM et al: Molecular description of three macro-deletionsand an Alu-Alu recombination-mediated duplication in theHPRT gene in four patients with Lesch-Nyhan disease.Mutat Res 2001;476:43.Curto R, Voit EO, Cascante M: Analysis of abnormalities in purinemetabolism leading to gout and to neurological dysfunctionsin man. Biochem J 1998;329:477.Harris MD, Siegel LB, Alloway JA: Gout and hyperuricemia. AmFamily Physician 1999;59:925.Lipkowitz MS et al: Functional reconstitution, membrane targeting,genomic structure, and chromosomal localization of ahuman urate transporter. J Clin Invest 2001;107:1103.Martinez J et al: Human genetic disorders, a phylogenetic perspective.J Mol Biol 2001;308:587.Puig JG et al: Gout: new questions for an ancient disease. Adv ExpMed Biol 1998;431:1.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.Tvrdik T et al: Molecular characterization of two deletion eventsinvolving Alu-sequences, one novel base substitution and twotentative hotspot mutations in the hypoxanthine phosphoribosyltransferasegene in five patients with Lesch-Nyhansyndrome.Hum Genet 1998;103:311.Zalkin H, Dixon JE: De novo purine nucleotide synthesis. ProgNucleic Acid Res Mol Biol 1992;42:259.

Nucleic Acid Structure & Function 35Daryl K. Granner, MDBIOMEDICAL IMPORTANCEThe discovery that genetic information is coded alongthe length of a polymeric molecule composed of onlyfour types of monomeric units was one of the major scientificachievements of the twentieth century. Thispolymeric molecule, DNA, is the chemical basis ofheredity and is organized into genes, the fundamentalunits of genetic information. The basic informationpathway—ie, DNA directs the synthesis of RNA,which in turn directs protein synthesis—has been elucidated.Genes do not function autonomously; theirreplication and function are controlled by various geneproducts, often in collaboration with components ofvarious signal transduction pathways. Knowledge of thestructure and function of nucleic acids is essential inunderstanding genetics and many aspects of pathophysiologyas well as the genetic basis of disease.DNA CONTAINS THEGENETIC INFORMATIONThe demonstration that DNA contained the genetic informationwas first made in 1944 in a series of experimentsby Avery, MacLeod, and McCarty. They showedthat the genetic determination of the character (type) ofthe capsule of a specific pneumococcus could be transmittedto another of a different capsular type by introducingpurified DNA from the former coccus into thelatter. These authors referred to the agent (later shownto be DNA) accomplishing the change as “transformingfactor.” Subsequently, this type of genetic manipulationhas become commonplace. Similar experiments haverecently been performed utilizing yeast, cultured mammaliancells, and insect and mammalian embryos as recipientsand cloned DNA as the donor of genetic information.DNA Contains Four DeoxynucleotidesThe chemical nature of the monomeric deoxynucleotideunits of DNA—deoxyadenylate, deoxyguanylate,deoxycytidylate, and thymidylate—is described inChapter 33. These monomeric units of DNA are heldin polymeric form by 3′,5′-phosphodiester bridges constitutinga single strand, as depicted in Figure 35–1.The informational content of DNA (the genetic code)resides in the sequence in which these monomers—purine and pyrimidine deoxyribonucleotides—are ordered.The polymer as depicted possesses a polarity;one end has a 5′-hydroxyl or phosphate terminal whilethe other has a 3′-phosphate or hydroxyl terminal. Theimportance of this polarity will become evident. Sincethe genetic information resides in the order of themonomeric units within the polymers, there must exista mechanism of reproducing or replicating this specificinformation with a high degree of fidelity. That requirement,together with x-ray diffraction data fromthe DNA molecule and the observation of Chargaffthat in DNA molecules the concentration of deoxyadenosine(A) nucleotides equals that of thymidine(T) nucleotides (A = T), while the concentration of deoxyguanosine(G) nucleotides equals that of deoxycytidine(C) nucleotides (G = C), led Watson, Crick, andWilkins to propose in the early 1950s a model of a double-strandedDNA molecule. The model they proposedis depicted in Figure 35–2. The two strands of thisdouble-stranded helix are held in register by hydrogenbonds between the purine and pyrimidine bases of therespective linear molecules. The pairings between thepurine and pyrimidine nucleotides on the oppositestrands are very specific and are dependent upon hydrogenbonding of A with T and G with C (Figure 35–3).This common form of DNA is said to be righthandedbecause as one looks down the double helix thebase residues form a spiral in a clockwise direction. Inthe double-stranded molecule, restrictions imposed bythe rotation about the phosphodiester bond, the favoredanti configuration of the glycosidic bond (Figure33–8), and the predominant tautomers (see Figure33–3) of the four bases (A, G, T, and C) allow A to paironly with T and G only with C, as depicted in Figure35–3. This base-pairing restriction explains the earlierobservation that in a double-stranded DNA moleculethe content of A equals that of T and the content of Gequals that of C. The two strands of the double-helicalmolecule, each of which possesses a polarity, are antiparallel;ie, one strand runs in the 5′ to 3′ directionand the other in the 3′ to 5′ direction. This is analogousto two parallel streets, each running one way but carryingtraffic in opposite directions. In the doublestrandedDNA molecules, the genetic information re-303

304 / CHAPTER 35O5′CH 2NNGNNHNH 2NH 2POHHOOHHHPOCH 2HHOOHHCNHPNOOOH 3 CNHTOCH 2 NOH HCH 2H HOHOPHHO3′HHNNHPNH 2NANOFigure 35–1. A segment of one strand of a DNA molecule in which the purine and pyrimidine bases guanine(G), cytosine (C), thymine (T), and adenine (A) are held together by a phosphodiester backbone between 2′-deoxyribosylmoieties attached to the nucleobases by an N-glycosidic bond. Note that the backbone has a polarity(ie, a direction). Convention dictates that a single-stranded DNA sequence is written in the 5′ to 3′ direction (ie,pGpCpTpA, where G, C, T, and A represent the four bases and p represents the interconnecting phosphates).sides in the sequence of nucleotides on one strand, thetemplate strand. This is the strand of DNA that iscopied during nucleic acid synthesis. It is sometimes referredto as the noncoding strand. The opposite strandis considered the coding strand because it matches theRNA transcript that encodes the protein.The two strands, in which opposing bases are heldtogether by hydrogen bonds, wind around a central axisin the form of a double helix. Double-stranded DNAexists in at least six forms (A–E and Z). The B form isusually found under physiologic conditions (low salt,high degree of hydration). A single turn of B-DNAabout the axis of the molecule contains ten base pairs.The distance spanned by one turn of B-DNA is 3.4nm. The width (helical diameter) of the double helix inB-DNA is 2 nm.As depicted in Figure 35–3, three hydrogen bondshold the deoxyguanosine nucleotide to the deoxycytidinenucleotide, whereas the other pair, the A–T pair, isheld together by two hydrogen bonds. Thus, the G–Cbonds are much more resistant to denaturation, or“melting,” than A–T-rich regions.The Denaturation (Melting) of DNAIs Used to Analyze Its StructureThe double-stranded structure of DNA can be separatedinto two component strands (melted) in solutionby increasing the temperature or decreasing the saltconcentration. Not only do the two stacks of bases pullapart but the bases themselves unstack while still connectedin the polymer by the phosphodiester backbone.Concomitant with this denaturation of the DNA moleculeis an increase in the optical absorbance of thepurine and pyrimidine bases—a phenomenon referredto as hyperchromicity of denaturation. Because of the

NUCLEIC ACID STRUCTURE & FUNCTION / 305CH 3ONONHHNNHNMinor grooveMajor grooveS A T SPPS T A SPPS C G SPPS G C So34 AThymidineHNNAdenosineHNNONHNONCytosine H NNNo20 AFigure 35–2. A diagrammatic representation of theWatson and Crick model of the double-helical structureof the B form of DNA. The horizontal arrow indicatesthe width of the double helix (20 Å), and the verticalarrow indicates the distance spanned by one completeturn of the double helix (34 Å). One turn of B-DNA includesten base pairs (bp), so the rise is 3.4 Å per bp.The central axis of the double helix is indicated by thevertical rod. The short arrows designate the polarity ofthe antiparallel strands. The major and minor groovesare depicted. (A, adenine; C, cytosine; G, guanine;T, thymine; P, phosphate; S, sugar [deoxyribose].)stacking of the bases and the hydrogen bonding betweenthe stacks, the double-stranded DNA moleculeexhibits properties of a rigid rod and in solution is a viscousmaterial that loses its viscosity upon denaturation.The strands of a given molecule of DNA separateover a temperature range. The midpoint is called themelting temperature, or T m . The T m is influenced bythe base composition of the DNA and by the salt concentrationof the solution. DNA rich in G–C pairs,which have three hydrogen bonds, melts at a higher temperaturethan that rich in A–T pairs, which have two hydrogenbonds. A tenfold increase of monovalent cationconcentration increases the T m by 16.6 °C. Formamide,which is commonly used in recombinant DNA experiments,destabilizes hydrogen bonding between bases,thereby lowering the T m . This allows the strands of DNAHGuanosineFigure 35–3. Base pairing between deoxyadenosineand thymidine involves the formation of two hydrogenbonds. Three such bonds form between deoxycytidineand deoxyguanosine. The broken lines represent hydrogenbonds.or DNA-RNA hybrids to be separated at much lowertemperatures and minimizes the phosphodiester bondbreakage that occurs at high temperatures.Renaturation of DNA RequiresBase Pair MatchingSeparated strands of DNA will renature or reassociatewhen appropriate physiologic temperature and salt conditionsare achieved. The rate of reassociation dependsupon the concentration of the complementary strands.Reassociation of the two complementary DNA strandsof a chromosome after DNA replication is a physiologicexample of renaturation (see below). At a given temperatureand salt concentration, a particular nucleic acidstrand will associate tightly only with a complementarystrand. Hybrid molecules will also form under appropriateconditions. For example, DNA will form a hybridwith a complementary DNA (cDNA) or with acognate messenger RNA (mRNA; see below). Whencombined with gel electrophoresis techniques that separatehybrid molecules by size and radioactive labeling toprovide a detectable signal, the resulting analytic techniquesare called Southern (DNA/cDNA) and Northernblotting (DNA/RNA), respectively. These proce-

306 / CHAPTER 35dures allow for very specific identification of hybridsfrom mixtures of DNA or RNA (see Chapter 40).There Are Grooves in the DNA MoleculeCareful examination of the model depicted in Figure35–2 reveals a major groove and a minor groove windingalong the molecule parallel to the phosphodiesterbackbones. In these grooves, proteins can interact specificallywith exposed atoms of the nucleotides (usually byH bonding) and thereby recognize and bind to specificnucleotide sequences without disrupting the base pairingof the double-helical DNA molecule. As discussed inChapters 37 and 39, regulatory proteins control the expressionof specific genes via such interactions.DNA Exists in Relaxed& Supercoiled FormsIn some organisms such as bacteria, bacteriophages, andmany DNA-containing animal viruses, the ends of theDNA molecules are joined to create a closed circle withno covalently free ends. This of course does not destroythe polarity of the molecules, but it eliminates all free 3′and 5′ hydroxyl and phosphoryl groups. Closed circlesexist in relaxed or supercoiled forms. Supercoils are introducedwhen a closed circle is twisted around its own axisor when a linear piece of duplex DNA, whose ends arefixed, is twisted. This energy-requiring process puts themolecule under stress, and the greater the number of supercoils,the greater the stress or torsion (test this bytwisting a rubber band). Negative supercoils are formedwhen the molecule is twisted in the direction oppositefrom the clockwise turns of the right-handed doublehelix found in B-DNA. Such DNA is said to be underwound.The energy required to achieve this state is, in asense, stored in the supercoils. The transition to anotherform that requires energy is thereby facilitated by the underwinding.One such transition is strand separation,which is a prerequisite for DNA replication and transcription.Supercoiled DNA is therefore a preferred formin biologic systems. Enzymes that catalyze topologicchanges of DNA are called topoisomerases. Topoisomerasescan relax or insert supercoils. The best-characterizedexample is bacterial gyrase, which induces negativesupercoiling in DNA using ATP as energy source. Homologsof this enzyme exist in all organisms and are importanttargets for cancer chemotherapy.DNA PROVIDES A TEMPLATE FORREPLICATION & TRANSCRIPTIONThe genetic information stored in the nucleotide sequenceof DNA serves two purposes. It is the source ofinformation for the synthesis of all protein molecules ofthe cell and organism, and it provides the informationinherited by daughter cells or offspring. Both of thesefunctions require that the DNA molecule serve as atemplate—in the first case for the transcription of theinformation into RNA and in the second case for thereplication of the information into daughter DNA molecules.The complementarity of the Watson and Crick double-strandedmodel of DNA strongly suggests thatreplication of the DNA molecule occurs in a semiconservativemanner. Thus, when each strand of the double-strandedparental DNA molecule separates from itscomplement during replication, each serves as a templateon which a new complementary strand is synthesized(Figure 35–4). The two newly formed doublestrandeddaughter DNA molecules, each containingone strand (but complementary rather than identical)from the parent double-stranded DNA molecule, arethen sorted between the two daughter cells (Figure35–5). Each daughter cell contains DNA moleculeswith information identical to that which the parentpossessed; yet in each daughter cell the DNA moleculeof the parent cell has been only semiconserved.THE CHEMICAL NATURE OF RNA DIFFERSFROM THAT OF DNARibonucleic acid (RNA) is a polymer of purine andpyrimidine ribonucleotides linked together by 3′,5′-phosphodiester bridges analogous to those in DNA(Figure 35–6). Although sharing many features withDNA, RNA possesses several specific differences:(1) In RNA, the sugar moiety to which the phosphatesand purine and pyrimidine bases are attached isribose rather than the 2′-deoxyribose of DNA.(2) The pyrimidine components of RNA differ fromthose of DNA. Although RNA contains the ribonucleotidesof adenine, guanine, and cytosine, it does notpossess thymine except in the rare case mentionedbelow. Instead of thymine, RNA contains the ribonucleotideof uracil.(3) RNA exists as a single strand, whereas DNA existsas a double-stranded helical molecule. However,given the proper complementary base sequence withopposite polarity, the single strand of RNA—asdemonstrated in Figure 35–7—is capable of foldingback on itself like a hairpin and thus acquiring doublestrandedcharacteristics.(4) Since the RNA molecule is a single strand complementaryto only one of the two strands of a gene, itsguanine content does not necessarily equal its cytosinecontent, nor does its adenine content necessarily equalits uracil content.

NUCLEIC ACID STRUCTURE & FUNCTION / 307G COLDOLD5′ G C 3′CGGCOriginalparent moleculeATAATGGCCAATFirst-generationdaughter moleculesTAGCTG3′A5′TACCGCGACTACTSecond-generationdaughter moleculesATATTGAACGT3′ T A 5′ 3′ T A 5′OLDNEW NEWOLDT AT AFigure 35–4. The double-stranded structure of DNAand the template function of each old strand (darkshading) on which a new (light shading) complementarystrand is synthesized.(5) RNA can be hydrolyzed by alkali to 2′,3′ cyclicdiesters of the mononucleotides, compounds that cannotbe formed from alkali-treated DNA because of theabsence of a 2′-hydroxyl group. The alkali lability ofRNA is useful both diagnostically and analytically.Information within the single strand of RNA is containedin its sequence (“primary structure”) of purineand pyrimidine nucleotides within the polymer. Thesequence is complementary to the template strand ofthe gene from which it was transcribed. Because of thisTGAACGTFigure 35–5. DNA replication is semiconservative.During a round of replication, each of the two strandsof DNA is used as a template for synthesis of a new,complementary strand.complementarity, an RNA molecule can bind specificallyvia the base-pairing rules to its template DNAstrand; it will not bind (“hybridize”) with the other(coding) strand of its gene. The sequence of the RNAmolecule (except for U replacing T) is the same as thatof the coding strand of the gene (Figure 35–8).Nearly All of the Several Species of RNAAre Involved in Some Aspect of ProteinSynthesisThose cytoplasmic RNA molecules that serve as templatesfor protein synthesis (ie, that transfer genetic informationfrom DNA to the protein-synthesizing machinery)are designated messenger RNAs, or mRNAs.Many other cytoplasmic RNA molecules (ribosomalRNAs; rRNAs) have structural roles wherein they con-

308 / CHAPTER 35O5′CH 2NNGNNHNH 2NH 2POHHOOHHOHPOCH 2HHOOHHOCNHPNOOCH 2HHOOHHOOUNHPNHOOCH 2HH3′OHHONNHPNH 2NANOFigure 35–6. A segment of a ribonucleic acid (RNA) molecule in which the purine and pyrimidine bases—guanine (G), cytosine (C), uracil (U), and adenine (A)—are held together by phosphodiester bonds between ribosylmoieties attached to the nucleobases by N-glycosidic bonds. Note that the polymer has a polarity as indicatedby the labeled 3′- and 5′-attached phosphates.tribute to the formation and function of ribosomes (theorganellar machinery for protein synthesis) or serve asadapter molecules (transfer RNAs; tRNAs) for thetranslation of RNA information into specific sequencesof polymerized amino acids.Some RNA molecules have intrinsic catalytic activity.The activity of these ribozymes often involves thecleavage of a nucleic acid. An example is the role ofRNA in catalyzing the processing of the primary transcriptof a gene into mature messenger RNA.Much of the RNA synthesized from DNA templatesin eukaryotic cells, including mammalian cells, is degradedwithin the nucleus, and it never serves as either astructural or an informational entity within the cellularcytoplasm.In all eukaryotic cells there are small nuclear RNA(snRNA) species that are not directly involved in proteinsynthesis but play pivotal roles in RNA processing.These relatively small molecules vary in size from 90 toabout 300 nucleotides (Table 35–1).The genetic material for some animal and plantviruses is RNA rather than DNA. Although some RNAviruses never have their information transcribed into aDNA molecule, many animal RNA viruses—specifically,the retroviruses (the HIV virus, for example)—aretranscribed by an RNA-dependent DNA polymerase,the so-called reverse transcriptase, to produce a double-strandedDNA copy of their RNA genome. Inmany cases, the resulting double-stranded DNA transcriptis integrated into the host genome and subsequentlyserves as a template for gene expression andfrom which new viral RNA genomes can be transcribed.RNA Is Organized in SeveralUnique StructuresIn all prokaryotic and eukaryotic organisms, three mainclasses of RNA molecules exist: messenger RNA(mRNA), transfer RNA (tRNA), and ribosomal RNA

NUCLEIC ACID STRUCTURE & FUNCTION / 3095′CCGAAAUUCGUUUUCGGGCUUUGGCCAACAGCStemLoopFigure 35–7. Diagrammatic representation of thesecondary structure of a single-stranded RNA moleculein which a stem loop, or “hairpin,” has been formed andis dependent upon the intramolecular base pairing.Note that A forms hydrogen bonds with U in RNA.(rRNA). Each differs from the others by size, function,and general stability.A. MESSENGER RNA (MRNA)This class is the most heterogeneous in size and stability.All members of the class function as messengersconveying the information in a gene to the proteinsynthesizingmachinery, where each serves as a templateon which a specific sequence of amino acids is polymerizedto form a specific protein molecule, the ultimategene product (Figure 35–9).3′Table 35–1. Some of the species of small stableRNAs found in mammalian cells.Length MoleculesName (nucleotides) per Cell LocalizationU1 165 1 × 10 6 Nucleoplasm/hnRNAU2 188 5 × 10 5 NucleoplasmU3 216 3 × 10 5 NucleolusU4 139 1 × 10 5 NucleoplasmU5 118 2 × 10 5 NucleoplasmU6 106 3 × 10 5 Perichromatin granules4.5S 91–95 3 x 10 5 Nucleus and cytoplasm7S 280 5 × 10 5 Nucleus and cytoplasm7-2 290 1 × 10 5 Nucleus and cytoplasm7-3 300 2 × 10 5 NucleusMessenger RNAs, particularly in eukaryotes, havesome unique chemical characteristics. The 5′ terminalof mRNA is “capped” by a 7-methylguanosine triphosphatethat is linked to an adjacent 2′-O-methyl ribonucleosideat its 5′-hydroxyl through the three phosphates(Figure 35–10). The mRNA molecules frequently containinternal 6-methyladenylates and other 2′-O-ribosemethylated nucleotides. The cap is involved in therecognition of mRNA by the translating machinery,and it probably helps stabilize the mRNA by preventingthe attack of 5′-exonucleases. The protein-synthesizingmachinery begins translating the mRNA into proteinsbeginning downstream of the 5′ or capped terminal.The other end of most mRNA molecules, the 3′-hydroxylterminal, has an attached polymer of adenylateresidues 20–250 nucleotides in length. The specificfunction of the poly(A) “tail” at the 3′-hydroxyl terminalof mRNAs is not fully understood, but it seems thatit maintains the intracellular stability of the specificmRNA by preventing the attack of 3′-exonucleases.Some mRNAs, including those for some histones, donot contain poly(A). The poly(A) tail, because it willform a base pair with oligodeoxythymidine polymersattached to a solid substrate like cellulose, can be usedto separate mRNA from other species of RNA, includingmRNA molecules that lack this tail.DNA strands:CodingTemplateRNAtranscript5′ — TGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATG— 3′3′ — ACCTTAACACTCGCCTATTGTTAAAGTGTGTCCTTTGTCGATACTGGTAC— 5′5′ pAUUGUGAGCGGAUAACAAUUUCACACAGGAAACAGCUAUGACCAUG 3′Figure 35–8. The relationship between the sequences of an RNA transcript and its gene, in which the codingand template strands are shown with their polarities. The RNA transcript with a 5′ to 3′ polarity is complementaryto the template strand with its 3′ to 5′ polarity. Note that the sequence in the RNA transcript and itspolarity is the same as that in the coding strand, except that the U of the transcript replaces the T of the gene.

310 / CHAPTER 355′3′DNA3′5′mRNA5′ 3′5′RibosomeProtein synthesis on mRNA template3′CompletedproteinmoleculeFigure 35–9. The expression of genetic informationin DNA into the form of an mRNAtranscript. This is subsequently translated byribosomes into a specific protein molecule.In mammalian cells, including cells of humans, themRNA molecules present in the cytoplasm are not theRNA products immediately synthesized from the DNAtemplate but must be formed by processing from a precursormolecule before entering the cytoplasm. Thus,in mammalian nuclei, the immediate products of genetranscription constitute a fourth class of RNA molecules.These nuclear RNA molecules are very heterogeneousin size and are quite large. The heterogeneousnuclear RNA (hnRNA) molecules may have a molecularweight in excess of 10 7 , whereas the molecularweight of mRNA molecules is generally less than 2 ×10 6 . As discussed in Chapter 37, hnRNA molecules areprocessed to generate the mRNA molecules which thenenter the cytoplasm to serve as templates for proteinsynthesis.B. TRANSFER RNA (TRNA)tRNA molecules vary in length from 74 to 95 nucleotides.They also are generated by nuclear processingof a precursor molecule (Chapter 37). The tRNA moleculesserve as adapters for the translation of the informationin the sequence of nucleotides of the mRNAinto specific amino acids. There are at least 20 speciesof tRNA molecules in every cell, at least one (and oftenseveral) corresponding to each of the 20 amino acids requiredfor protein synthesis. Although each specifictRNA differs from the others in its sequence of nucleotides,the tRNA molecules as a class have many featuresin common. The primary structure—ie, the nucleotidesequence—of all tRNA molecules allowsextensive folding and intrastrand complementarity togenerate a secondary structure that appears like acloverleaf (Figure 35–11).All tRNA molecules contain four main arms. Theacceptor arm terminates in the nucleotides CpCpAOH.These three nucleotides are added posttranscriptionally.The tRNA-appropriate amino acid is attached tothe 3′-OH group of the A moiety of the acceptor arm.The D, TC, and extra arms help define a specifictRNA.Although tRNAs are quite stable in prokaryotes, theyare somewhat less stable in eukaryotes. The opposite istrue for mRNAs, which are quite unstable in prokaryotesbut generally stable in eukaryotic organisms.C. RIBOSOMAL RNA (RRNA)A ribosome is a cytoplasmic nucleoprotein structurethat acts as the machinery for the synthesis of proteinsfrom the mRNA templates. On the ribosomes, themRNA and tRNA molecules interact to translate into aspecific protein molecule information transcribed fromthe gene. In active protein synthesis, many ribosomesare associated with an mRNA molecule in an assemblycalled the polysome.The components of the mammalian ribosome,which has a molecular weight of about 4.2 × 10 6 and asedimentation velocity of 80S (Svedberg units), areshown in Table 35–2. The mammalian ribosome containstwo major nucleoprotein subunits—a larger onewith a molecular weight of 2.8 × 10 6 (60S) and asmaller subunit with a molecular weight of 1.4 × 10 6(40S). The 60S subunit contains a 5S ribosomal RNA(rRNA), a 5.8S rRNA, and a 28S rRNA; there are alsoprobably more than 50 specific polypeptides. The 40Ssubunit is smaller and contains a single 18S rRNA andapproximately 30 distinct polypeptide chains. All of theribosomal RNA molecules except the 5S rRNA areprocessed from a single 45S precursor RNA molecule inthe nucleolus (Chapter 37). 5S rRNA is independentlytranscribed. The highly methylated ribosomal RNAmolecules are packaged in the nucleolus with the specificribosomal proteins. In the cytoplasm, the ribosomesremain quite stable and capable of many translationcycles. The functions of the ribosomal RNAmolecules in the ribosomal particle are not fully understood,but they are necessary for ribosomal assemblyand seem to play key roles in the binding of mRNA to

NUCLEIC ACID STRUCTURE & FUNCTION / 311OHCOHCHCHHCHOH 2 NHNNONNCH 3CAPH 2 C 5′OO –POOOPO –OO –5′CH 2OOPHCH3′ CONNCHH2′CNH 2NNOOCH 3NHmRNAOOPO –O5′CH 2HCHOHNCHO3′ CCOHFigure 35–10. The cap structure attached to the 5′ terminal of most eukaryotic messengerRNA molecules. A 7-methylguanosine triphosphate (black) is attached at the 5′ terminalof the mRNA (shown in blue), which usually contains a 2′-O-methylpurine nucleotide.These modifications (the cap and methyl group) are added after the mRNA is transcribedfrom DNA.OOPO –ribosomes and its translation. Recent studies suggestthat an rRNA component performs the peptidyl transferaseactivity and thus is an enzyme (a ribozyme).D. SMALL STABLE RNAA large number of discrete, highly conserved, and smallstable RNA species are found in eukaryotic cells. Themajority of these molecules are complexed with proteinsto form ribonucleoproteins and are distributed inthe nucleus, in the cytoplasm, or in both. They range insize from 90 to 300 nucleotides and are present in100,000–1,000,000 copies per cell.Small nuclear RNAs (snRNAs), a subset of theseRNAs, are significantly involved in mRNA processingand gene regulation. Of the several snRNAs, U1, U2,U4, U5, and U6 are involved in intron removal and theprocessing of hnRNA into mRNA (Chapter 37). TheU7 snRNA may be involved in production of the correct3′ ends of histone mRNA—which lacks a poly(A)tail. The U4 and U6 snRNAs may also be required forpoly(A) processing.

312 / CHAPTER 35Region of hydrogenbonding betweenbase pairsD arm5′PU3′AcceptorarmaaACCAnticodon armGTExtra armTψC armψAlkylated purineFigure 35–11. Typical aminoacyl tRNA in which theamino acid (aa) is attached to the 3′ CCA terminal. Theanticodon, TΨC, and dihydrouracil (D) arms are indicated,as are the positions of the intramolecular hydrogenbonding between these base pairs. (From WatsonJD: Molecular Biology of the Gene, 3rd ed. Copyright ©1976, 1970, 1965, by W.A. Benjamin, Inc., Menlo Park, California.)CGSPECIFIC NUCLEASES DIGESTNUCLEIC ACIDSEnzymes capable of degrading nucleic acids have beenrecognized for many years. These nucleases can be classifiedin several ways. Those which exhibit specificityfor deoxyribonucleic acid are referred to as deoxyribonucleases.Those which specifically hydrolyze ribonucleicacids are ribonucleases. Within both ofthese classes are enzymes capable of cleaving internalphosphodiester bonds to produce either 3′-hydroxyland 5′-phosphoryl terminals or 5′-hydroxyl and 3′-phosphoryl terminals. These are referred to as endonucleases.Some are capable of hydrolyzing both strandsof a double-stranded molecule, whereas others canonly cleave single strands of nucleic acids. Some nucleasescan hydrolyze only unpaired single strands, whileothers are capable of hydrolyzing single strands participatingin the formation of a double-stranded molecule.There exist classes of endonucleases that recognize specificsequences in DNA; the majority of these are therestriction endonucleases, which have in recent yearsbecome important tools in molecular genetics and medicalsciences. A list of some currently recognized restrictionendonucleases is presented in Table 40–2.Some nucleases are capable of hydrolyzing a nucleotideonly when it is present at a terminal of a molecule;these are referred to as exonucleases. Exonucleasesact in one direction (3′ →5′ or 5′ →3′) only. Inbacteria, a 3′ →5′ exonuclease is an integral part of theDNA replication machinery and there serves to edit—or proofread—the most recently added deoxynucleotidefor base-pairing errors.Table 35–2. Components of mammalian ribosomes. 1Mass Protein RNAComponent (mw) Number Mass Size Mass Bases40S subunit 1.4 × 10 6 ~35 7 × 10 5 18S 7 × 10 5 190060S subunit 2.8 × 10 6 ~50 1 × 10 6 5S 35,000 1205.8S 45,000 16028S 1.6 × 10 6 47001 The ribosomal subunits are defined according to their sedimentation velocityin Svedberg units (40S or 60S). This table illustrates the total mass(MW) of each. The number of unique proteins and their total mass (MW) andthe RNA components of each subunit in size (Svedberg units), mass, andnumber of bases are listed.

NUCLEIC ACID STRUCTURE & FUNCTION / 313SUMMARY• DNA consists of four bases—A, G, C, and T—which are held in linear array by phosphodiesterbonds through the 3′ and 5′ positions of adjacent deoxyribosemoieties.• DNA is organized into two strands by the pairing ofbases A to T and G to C on complementary strands.These strands form a double helix around a centralaxis.• The 3 × 10 9 base pairs of DNA in humans are organizedinto the haploid complement of 23 chromosomes.The exact sequence of these 3 billion nucleotidesdefines the uniqueness of each individual.• DNA provides a template for its own replication andthus maintenance of the genotype and for the transcriptionof the 30,000–50,000 genes into a varietyof RNA molecules.• RNA exists in several different single-stranded structures,most of which are involved in protein synthesis.The linear array of nucleotides in RNA consistsof A, G, C, and U, and the sugar moiety is ribose.• The major forms of RNA include messenger RNA(mRNA), ribosomal RNA (rRNA), and transferRNA (tRNA). Certain RNA molecules act as catalysts(ribozymes).REFERENCESGreen R, Noller HF: Ribosomes and translation. Annu Rev Biochem1997;66:689.Guthrie C, Patterson B: Spliceosomal snRNAs. Ann Rev Genet1988;22:387.Hunt T: DNA Makes RNA Makes Protein. Elsevier, 1983.Watson JD, Crick FHC: Molecular structure of nucleic acids. Nature1953;171:737.Watson JD: The Double Helix. Atheneum, 1968.Watson JD et al: Molecular Biology of the Gene, 5th ed. Benjamin-Cummings, 2000.

DNA Organization, Replication,& Repair 36Daryl K. Granner, MD, & P. Anthony Weil, PhDBIOMEDICAL IMPORTANCE*The genetic information in the DNA of a chromosomecan be transmitted by exact replication or it can be exchangedby a number of processes, including crossingover, recombination, transposition, and conversion.These provide a means of ensuring adaptability and diversityfor the organism but, when these processes goawry, can also result in disease. A number of enzymesystems are involved in DNA replication, alteration,and repair. Mutations are due to a change in the basesequence of DNA and may result from the faulty replication,movement, or repair of DNA and occur with afrequency of about one in every 10 6 cell divisions. Abnormalitiesin gene products (either in protein functionor amount) can be the result of mutations that occur incoding or regulatory-region DNA. A mutation in agerm cell will be transmitted to offspring (so-called verticaltransmission of hereditary disease). A number offactors, including viruses, chemicals, ultraviolet light,and ionizing radiation, increase the rate of mutation.Mutations often affect somatic cells and so are passedon to successive generations of cells, but only within anorganism. It is becoming apparent that a number ofdiseases—and perhaps most cancers—are due to thecombined effects of vertical transmission of mutationsas well as horizontal transmission of induced mutations.CHROMATIN IS THE CHROMOSOMALMATERIAL EXTRACTED FROM NUCLEIOF CELLS OF EUKARYOTIC ORGANISMSChromatin consists of very long double-stranded DNAmolecules and a nearly equal mass of rather small basicproteins termed histones as well as a smaller amount ofnonhistone proteins (most of which are acidic and*So far as is possible, the discussion in this chapter and in Chapters37, 38, and 39 will pertain to mammalian organisms, which are, ofcourse, among the higher eukaryotes. At times it will be necessaryto refer to observations in prokaryotic organisms such as bacteriaand viruses, but in such cases the information will be of a kind thatcan be extrapolated to mammalian organisms.314larger than histones) and a small quantity of RNA. Thenonhistone proteins include enzymes involved in DNAreplication, such as DNA topoisomerases. Also includedare proteins involved in transcription, such asthe RNA polymerase complex. The double-strandedDNA helix in each chromosome has a length that isthousands of times the diameter of the cell nucleus.One purpose of the molecules that comprise chromatin,particularly the histones, is to condense theDNA. Electron microscopic studies of chromatin havedemonstrated dense spherical particles called nucleosomes,which are approximately 10 nm in diameterand connected by DNA filaments (Figure 36–1). Nucleosomesare composed of DNA wound around a collectionof histone molecules.Histones Are the Most AbundantChromatin ProteinsThe histones are a small family of closely related basicproteins. H1 histones are the ones least tightly boundto chromatin Figure 36–1) and are, therefore, easily removedwith a salt solution, after which chromatin becomessoluble. The organizational unit of this solublechromatin is the nucleosome. Nucleosomes containfour classes of histones: H2A, H2B, H3, and H4. Thestructures of all four histones—H2A, H2B, H3, andH4, the so-called core histones forming the nucleosome—havebeen highly conserved between species.This extreme conservation implies that the function ofhistones is identical in all eukaryotes and that the entiremolecule is involved quite specifically in carrying outthis function. The carboxyl terminal two-thirds of themolecules have a typical random amino acid composition,while their amino terminal thirds are particularlyrich in basic amino acids. These four core histones aresubject to at least five types of covalent modification:acetylation, methylation, phosphorylation, ADPribosylation,and covalent linkage (H2A only) to ubiquitin.These histone modifications probably play animportant role in chromatin structure and function asillustrated in Table 36–1.The histones interact with each other in very specificways. H3 and H4 form a tetramer containing two mol-

DNA ORGANIZATION, REPLICATION, & REPAIR / 315Figure 36–1. Electron micrograph of nucleosomesattached by strands of nucleic acid. (The bar represents2.5 µm.) (Reproduced, with permission, from Oudet P,Gross-Bellard M, Chambon P: Electron microscopic andbiochemical evidence that chromatin structure is a repeatingunit. Cell 1975;4:281.)ecules of each (H3/H4) 2 , while H2A and H2B formdimers (H2A-H2B). Under physiologic conditions,these histone oligomers associate to form the histone octamerof the composition (H3/H4) 2 -(H2A-H2B) 2 .The Nucleosome Contains Histone & DNAWhen the histone octamer is mixed with purified, double-strandedDNA, the same x-ray diffraction pattern isformed as that observed in freshly isolated chromatin.Electron microscopic studies confirm the existence ofreconstituted nucleosomes. Furthermore, the reconstitutionof nucleosomes from DNA and histones H2A,H2B, H3, and H4 is independent of the organismal orcellular origin of the various components. The histoneH1 and the nonhistone proteins are not necessary forthe reconstitution of the nucleosome core.Table 36–1. Possible roles of modified histones.1. Acetylation of histones H3 and H4 is associated with the activationor inactivation of gene transcription (Chapter 37).2. Acetylation of core histones is associated with chromosomalassembly during DNA replication.3. Phosphorylation of histone H1 is associated with the condensationof chromosomes during the replication cycle.4. ADP-ribosylation of histones is associated with DNA repair.5. Methylation of histones is correlated with activation andrepression of gene transcription.In the nucleosome, the DNA is supercoiled in a lefthandedhelix over the surface of the disk-shaped histoneoctamer (Figure 36–2). The majority of core histoneproteins interact with the DNA on the inside of the supercoilwithout protruding, though the amino terminaltails of all the histones probably protrude outside of thisstructure and are available for regulatory covalent modifications(see Table 36–1).The (H3/H4) 2 tetramer itself can confer nucleosome-likeproperties on DNA and thus has a centralrole in the formation of the nucleosome. The additionof two H2A-H2B dimers stabilizes the primary particleand firmly binds two additional half-turns of DNA previouslybound only loosely to the (H3/H4) 2 . Thus,1.75 superhelical turns of DNA are wrapped aroundthe surface of the histone octamer, protecting 146 basepairs of DNA and forming the nucleosome core particle(Figure 36–2). The core particles are separated by anabout 30-bp linker region of DNA. Most of the DNAis in a repeating series of these structures, giving the socalled“beads-on-a-string” appearance when examinedby electron microscopy (see Figure 36–1).The assembly of nucleosomes is mediated by one ofseveral chromatin assembly factors facilitated by histonechaperones, proteins such as the anionic nuclear proteinnucleoplasmin. As the nucleosome is assembled, histonesare released from the histone chaperones. Nucleosomesappear to exhibit preference for certain regions onspecific DNA molecules, but the basis for this nonrandomdistribution, termed phasing, is not completelyHistoneH1Histone octamerDNAFigure 36–2. Model for the structure of the nucleosome,in which DNA is wrapped around the surface of aflat protein cylinder consisting of two each of histonesH2A, H2B, H3, and H4 that form the histone octamer.The 146 base pairs of DNA, consisting of 1.75 superhelicalturns, are in contact with the histone octamer. Thisprotects the DNA from digestion by a nuclease. The positionof histone H1, when it is present, is indicated bythe dashed outline at the bottom of the figure.

316 / CHAPTER 36understood. It is probably related to the relative physicalflexibility of certain nucleotide sequences that are able toaccommodate the regions of kinking within the supercoilas well as the presence of other DNA-bound factorsthat limit the sites of nucleosome deposition.The super-packing of nucleosomes in nuclei is seeminglydependent upon the interaction of the H1 histoneswith adjacent nucleosomes.HIGHER-ORDER STRUCTURES PROVIDEFOR THE COMPACTION OF CHROMATINElectron microscopy of chromatin reveals two higherorders of structure—the 10-nm fibril and the 30-nmchromatin fiber—beyond that of the nucleosome itself.The disk-like nucleosome structure has a 10-nm diameterand a height of 5 nm. The 10-nm fibril consists ofnucleosomes arranged with their edges separated by asmall distance (30 bp of DNA) with their flat faces parallelwith the fibril axis (Figure 36–3). The 10-nm fibrilis probably further supercoiled with six or seven nucleosomesper turn to form the 30-nm chromatin fiber(Figure 36–3). Each turn of the supercoil is relativelyflat, and the faces of the nucleosomes of successiveturns would be nearly parallel to each other. H1 histonesappear to stabilize the 30-nm fiber, but their positionand that of the variable length spacer DNA are notclear. It is probable that nucleosomes can form a varietyof packed structures. In order to form a mitotic chromosome,the 30-nm fiber must be compacted in lengthanother 100-fold (see below).In interphase chromosomes, chromatin fibers appearto be organized into 30,000–100,000 bp loops ordomains anchored in a scaffolding (or supporting matrix)within the nucleus. Within these domains, someDNA sequences may be located nonrandomly. It hasbeen suggested that each looped domain of chromatincorresponds to one or more separate genetic functions,containing both coding and noncoding regions of thecognate gene or genes.SOME REGIONS OF CHROMATIN ARE“ACTIVE” & OTHERS ARE “INACTIVE”Generally, every cell of an individual metazoan organismcontains the same genetic information. Thus, the differencesbetween cell types within an organism must be explainedby differential expression of the common geneticinformation. Chromatin containing active genes (ie,transcriptionally active chromatin) has been shown todiffer in several ways from that of inactive regions. Thenucleosome structure of active chromatin appears to bealtered, sometimes quite extensively, in highly active regions.DNA in active chromatin contains large regions(about 100,000 bases long) that are sensitive to digestionby a nuclease such as DNase I. DNase I makes single-strandcuts in any segment of DNA (no sequencespecificity). It will digest DNA not protected by proteininto its component deoxynucleotides. The sensitivity toDNase I of chromatin regions being actively transcribedreflects only a potential for transcription rather thantranscription itself and in several systems can be correlatedwith a relative lack of 5-methyldeoxycytidine in theDNA and particular histone covalent modifications(phosphorylation, acetylation, etc; see Table 36–1).Within the large regions of active chromatin thereexist shorter stretches of 100–300 nucleotides that exhibitan even greater (another tenfold) sensitivity toDNase I. These hypersensitive sites probably resultfrom a structural conformation that favors access of thenuclease to the DNA. These regions are often locatedimmediately upstream from the active gene and are thelocation of interrupted nucleosomal structure caused bythe binding of nonhistone regulatory transcription factorproteins. (See Chapters 37 and 39.) In many cases, itseems that if a gene is capable of being transcribed, itvery often has a DNase-hypersensitive site(s) in the chromatinimmediately upstream. As noted above, nonhistoneregulatory proteins involved in transcription controland those involved in maintaining access to the templatestrand lead to the formation of hypersensitive sites. Hypersensitivesites often provide the first clue about thepresence and location of a transcription control element.Transcriptionally inactive chromatin is denselypacked during interphase as observed by electron microscopicstudies and is referred to as heterochromatin;transcriptionally active chromatin stains lessdensely and is referred to as euchromatin. Generally,euchromatin is replicated earlier than heterochromatinin the mammalian cell cycle (see below).There are two types of heterochromatin: constitutiveand facultative. Constitutive heterochromatin is alwayscondensed and thus inactive. It is found in theregions near the chromosomal centromere and at chromosomalends (telomeres). Facultative heterochromatinis at times condensed, but at other times it is activelytranscribed and, thus, uncondensed and appearsas euchromatin. Of the two members of the X chromosomepair in mammalian females, one X chromosome isalmost completely inactive transcriptionally and is heterochromatic.However, the heterochromatic X chromosomedecondenses during gametogenesis and becomestranscriptionally active during early embryogenesis—thus, it is facultative heterochromatin.Certain cells of insects, eg, Chironomus, containgiant chromosomes that have been replicated for tencycles without separation of daughter chromatids.These copies of DNA line up side by side in precise registerand produce a banded chromosome containing regionsof condensed chromatin and lighter bands of

Metaphasechromosome1400 nmCondensedloops700 nmNuclear-scaffoldassociatedformChromosomescaffoldNon-condensedloops300 nm30-nmchromatin fibrilcomposed ofnucleosomes30 nm“Beadson-a-string”10-nmchromatinfibrilH1OctOctH1H1Oct10 nmNakeddouble-helicalDNA2 nmFigure 36–3. Shown is the extent of DNA packaging in metaphase chromosomes (top) to noted duplex DNA (bottom).Chromosomal DNA is packaged and organized at several levels as shown (see Table 36–2). Each phase of condensationor compaction and organization (bottom to top) decreases overall DNA accessibility to an extent that theDNA sequences in metaphase chromosomes are almost totally transcriptionally inert. In toto, these five levels ofDNA compaction result in nearly a 10 4 -fold linear decrease in end-to-end DNA length. Complete condensation anddecondensation of the linear DNA in chromosomes occur in the space of hours during the normal replicative cellcycle (see Figure 36–20).317

318 / CHAPTER 36more extended chromatin. Transcriptionally active regionsof these polytene chromosomes are especiallydecondensed into “puffs” that can be shown to containthe enzymes responsible for transcription and to be thesites of RNA synthesis (Figure 36–4).A5CBR3BBR3Figure 36–4. Illustration of the tight correlation betweenthe presence of RNA polymerase II and RNA synthesis.A number of genes are activated when Chironomustentans larvae are subjected to heat shock (39 °Cfor 30 minutes). A: Distribution of RNA polymerase II(also called type B) in isolated chromosome IV from thesalivary gland (at arrows). The enzyme was detected byimmunofluorescence using an antibody directedagainst the polymerase. The 5C and BR3 are specificbands of chromosome IV, and the arrows indicate puffs.B: Autoradiogram of a chromosome IV that was incubatedin 3 H-uridine to label the RNA. Note the correspondenceof the immunofluorescence and presenceof the radioactive RNA (black dots). Bar = 7 µm. (Reproduced,with permission, from Sass H: RNA polymerase B inpolytene chromosomes. Cell 1982;28:274. Copyright ©1982 by the Massachusetts Institute of Technology.)5CDNA IS ORGANIZEDINTO CHROMOSOMESAt metaphase, mammalian chromosomes possess atwofold symmetry, with the identical duplicated sisterchromatids connected at a centromere, the relative positionof which is characteristic for a given chromosome(Figure 36–5). The centromere is an adenine-thymine(A–T) rich region ranging in size from 10 2 (brewers’yeast) to 10 6 (mammals) base pairs. It binds several proteinswith high affinity. This complex, called the kinetochore,provides the anchor for the mitotic spindle. Itthus is an essential structure for chromosomal segregationduring mitosis.The ends of each chromosome contain structurescalled telomeres. Telomeres consist of short, repeatTG-rich sequences. Human telomeres have a variablenumber of repeats of the sequence 5′-TTAGGG-3′,which can extend for several kilobases. Telomerase, amultisubunit RNA-containing complex related to viralRNA-dependent DNA polymerases (reverse transcriptases),is the enzyme responsible for telomere synthesisand thus for maintaining the length of the telomere.Since telomere shortening has been associated withboth malignant transformation and aging, telomerasehas become an attractive target for cancer chemotherapyand drug development. Each sister chromatid containsone double-stranded DNA molecule. During interphase,the packing of the DNA molecule is less densethan it is in the condensed chromosome duringmetaphase. Metaphase chromosomes are nearly completelytranscriptionally inactive.The human haploid genome consists of about3 × 10 9 bp and about 1.7 × 10 7 nucleosomes. Thus, eachof the 23 chromatids in the human haploid genomewould contain on the average 1.3 × 10 8 nucleotides inone double-stranded DNA molecule. The length ofeach DNA molecule must be compressed about 8000-fold to generate the structure of a condensed metaphasechromosome! In metaphase chromosomes, the 30-nmchromatin fibers are also folded into a series of loopeddomains, the proximal portions of which are anchoredto a nonhistone proteinaceous scaffolding within thenucleus (Figure 36–3). The packing ratios of each ofthe orders of DNA structure are summarized in Table36–2.The packaging of nucleoproteins within chromatidsis not random, as evidenced by the characteristic patternsobserved when chromosomes are stained with specificdyes such as quinacrine or Giemsa’s stain (Figure36–6).From individual to individual within a singlespecies, the pattern of staining (banding) of the entirechromosome complement is highly reproducible; nonetheless,it differs significantly from other species, eventhose closely related. Thus, the packaging of the nucleoproteinsin chromosomes of higher eukaryotes must insome way be dependent upon species-specific characteristicsof the DNA molecules.A combination of specialized staining techniquesand high-resolution microscopy has allowed geneticists

DNA ORGANIZATION, REPLICATION, & REPAIR / 319Sister chromatid No. 2Sister chromatid No. 1CentromereFigure 36–5. The two sister chromatids ofhuman chromosome 12 (× 27,850). The locationof the A+T-rich centromeric region connectingsister chromatids is indicated, as are two of thefour telomeres residing at the very ends of thechromatids that are attached one to the other atthe centromere. (Modified and reproduced, withpermission, from DuPraw EJ: DNA and Chromosomes.Holt, Rinehart, and Winston, 1970.)Telomeres(TTAGG) nto quite precisely map thousands of genes to specific regionsof mouse and human chromosomes. With the recentelucidation of the human and mouse genome sequences,it has become clear that many of these visualmapping methods were remarkably accurate.Coding Regions Are Often Interruptedby Intervening SequencesThe protein coding regions of DNA, the transcriptsof which ultimately appear in the cytoplasm as singlemRNA molecules, are usually interrupted in the eukaryoticgenome by large intervening sequences ofTable 36–2. The packing ratios of each of theorders of DNA structure.Chromatin FormPacking RatioNaked double-helical DNA ~1.010-nm fibril of nucleosomes 7–1025- to 30-nm chromatin fiber of superheli- 40–60cal nucleosomesCondensed metaphase chromosome of 8000loopsnonprotein coding DNA. Accordingly, the primarytranscripts of DNA (mRNA precursors, originallytermed hnRNA because this species of RNA was quiteheterogeneous in size [length] and mostly restricted tothe nucleus), contain noncoding intervening sequencesof RNA that must be removed in a process which alsojoins together the appropriate coding segments to formthe mature mRNA. Most coding sequences for a singlemRNA are interrupted in the genome (and thus in theprimary transcript) by at least one—and in some casesas many as 50—noncoding intervening sequences (introns).In most cases, the introns are much longer thanthe continuous coding regions (exons). The processingof the primary transcript, which involves removal of intronsand splicing of adjacent exons, is described in detailin Chapter 37.The function of the intervening sequences, or introns,is not clear. They may serve to separate functionaldomains (exons) of coding information in a formthat permits genetic rearrangement by recombinationto occur more rapidly than if all coding regions for agiven genetic function were contiguous. Such an enhancedrate of genetic rearrangement of functional domainsmight allow more rapid evolution of biologicfunction. The relationships among chromosomalDNA, gene clusters on the chromosome, the exonintronstructure of genes, and the final mRNA productare illustrated in Figure 36–7.

320 / CHAPTER 361 2 3 4 56 7 8 9 10 111213 14 15 16 171819 20 21 22 XYFigure 36–6. A human karyotype (of a man with a normal 46,XY constitution), inwhich the metaphase chromosomes have been stained by the Giemsa method andaligned according to the Paris Convention. (Courtesy of H Lawce and F Conte.)MUCH OF THE MAMMALIAN GENOMEIS REDUNDANT & MUCH ISNOT TRANSCRIBEDThe haploid genome of each human cell consists of3 × 10 9 base pairs of DNA subdivided into 23 chromosomes.The entire haploid genome contains sufficientDNA to code for nearly 1.5 million average-sizedgenes. However, studies of mutation rates and of thecomplexities of the genomes of higher organismsstrongly suggest that humans have < 100,000 proteinsencoded by the ~1.1% of the human genome that iscomposed of exonic DNA. This implies that most ofthe DNA is noncoding—ie, its information is nevertranslated into an amino acid sequence of a proteinmolecule. Certainly, some of the excess DNA sequencesserve to regulate the expression of genes during development,differentiation, and adaptation to the environment.Some excess clearly makes up the intervening sequencesor introns (24% of the total human genome)that split the coding regions of genes, but much of theexcess appears to be composed of many families of repeatedsequences for which no functions have beenclearly defined. A summary of the salient features of thehuman genome is presented in Chapter 40.The DNA in a eukaryotic genome can be dividedinto different “sequence classes.” These are uniquesequence,or nonrepetitive, DNA and repetitivesequenceDNA. In the haploid genome, unique-sequenceDNA generally includes the single copy genesthat code for proteins. The repetitive DNA in the haploidgenome includes sequences that vary in copy numberfrom two to as many as 10 7 copies per cell.More Than Half the DNA in EukaryoticOrganisms Is in Unique orNonrepetitive SequencesThis estimation (and the distribution of repetitivesequenceDNA) is based on a variety of DNA-RNA hybridizationtechniques and, more recently, on directDNA sequencing. Similar techniques are used to estimatethe number of active genes in a population ofunique-sequence DNA. In brewers’ yeast (Saccharomycescerevisiae, a lower eukaryote), about two thirdsof its 6200 genes are expressed. In typical tissues in ahigher eukaryote (eg, mammalian liver and kidney), between10,000 and 15,000 genes are expressed. Differentcombinations of genes are expressed in each tissue,

DNA ORGANIZATION, REPLICATION, & REPAIR / 321Chromosome(1–2 × 10 3 genes)1.5 × 10 8 bpGene cluster(~20 genes)1.5 × 10 6 bpGene2 × 10 4 bpPrimary transcript8 × 10 3 ntmRNA2 × 10 3 ntFigure 36–7. The relationship between chromosomal DNA and mRNA. The humanhaploid DNA complement of 3 × 10 9 base pairs (bp) is distributed between 23 chromosomes.Genes are clustered on these chromosomes. An average gene is 2 × 10 4 bp inlength, including the regulatory region (hatched area), which is usually located at the 5′end of the gene. The regulatory region is shown here as being adjacent to the transcriptioninitiation site (arrow). Most eukaryotic genes have alternating exons and introns. Inthis example, there are nine exons (dark blue areas) and eight introns (light blue areas).The introns are removed from the primary transcript by the processing reaction, and theexons are ligated together in sequence to form the mature mRNA. (nt, nucleotides.)of course, and how this is accomplished is one of themajor unanswered questions in biology.In Human DNA, at Least 30% of theGenome Consists of Repetitive SequencesRepetitive-sequence DNA can be broadly classified asmoderately repetitive or as highly repetitive. The highlyrepetitive sequences consist of 5–500 base pair lengthsrepeated many times in tandem. These sequences areusually clustered in centromeres and telomeres of thechromosome and are present in about 1–10 millioncopies per haploid genome. These sequences are transcriptionallyinactive and may play a structural role inthe chromosome (see Chapter 40).The moderately repetitive sequences, which are definedas being present in numbers of less than 10 6copies per haploid genome, are not clustered but are interspersedwith unique sequences. In many cases, theselong interspersed repeats are transcribed by RNA polymeraseII and contain caps indistinguishable from thoseon mRNA.Depending on their length, moderately repetitivesequences are classified as long interspersed repeatsequences (LINEs) or short interspersed repeatsequences (SINEs). Both types appear to beretroposons, ie, they arose from movement from onelocation to another (transposition) through an RNAintermediate by the action of reverse transcriptase thattranscribes an RNA template into DNA. Mammaliangenomes contain 20–50 thousand copies of the 6–7 kbLINEs. These represent species-specific families of repeatelements. SINEs are shorter (70–300 bp), andthere may be more than 100,000 copies per genome.Of the SINEs in the human genome, one family, theAlu family, is present in about 500,000 copies per haploidgenome and accounts for at least 5–6% of thehuman genome. Members of the human Alu familyand their closely related analogs in other animals aretranscribed as integral components of hnRNA or as discreteRNA molecules, including the well-studied 4.5SRNA and 7S RNA. These particular family membersare highly conserved within a species as well as betweenmammalian species. Components of the short inter-

322 / CHAPTER 36spersed repeats, including the members of the Alu family,may be mobile elements, capable of jumping intoand out of various sites within the genome (see below).This can have disastrous results, as exemplified by theinsertion of Alu sequences into a gene, which, when somutated, causes neurofibromatosis.Microsatellite Repeat SequencesOne category of repeat sequences exists as both dispersedand grouped tandem arrays. The sequences consistof 2–6 bp repeated up to 50 times. These microsatellitesequences most commonly are found asdinucleotide repeats of AC on one strand and TG onthe opposite strand, but several other forms occur, includingCG, AT, and CA. The AC repeat sequences areestimated to occur at 50,000–100,000 locations in thegenome. At any locus, the number of these repeats mayvary on the two chromosomes, thus providing heterozygosityof the number of copies of a particular microsatellitenumber in an individual. This is a heritabletrait, and, because of their number and the ease of detectingthem using the polymerase chain reaction(PCR) (Chapter 40), AC repeats are very useful in constructinggenetic linkage maps. Most genes are associatedwith one or more microsatellite markers, so the relativeposition of genes on chromosomes can beassessed, as can the association of a gene with a disease.Using PCR, a large number of family members can berapidly screened for a certain microsatellite polymorphism.The association of a specific polymorphismwith a gene in affected family members—and the lackof this association in unaffected members—may be thefirst clue about the genetic basis of a disease.Trinucleotide sequences that increase in number(microsatellite instability) can cause disease. The unstablep(CGG) n repeat sequence is associated with thefragile X syndrome. Other trinucleotide repeats thatundergo dynamic mutation (usually an increase) areassociated with Huntington’s chorea (CAG), myotonicdystrophy (CTG), spinobulbar muscular atrophy (CAG),and Kennedy’s disease (CAG).ONE PERCENT OF CELLULAR DNAIS IN MITOCHONDRIAThe majority of the peptides in mitochondria (about54 out of 67) are coded by nuclear genes. The rest arecoded by genes found in mitochondrial (mt) DNA.Human mitochondria contain two to ten copies of asmall circular double-stranded DNA molecule thatmakes up approximately 1% of total cellular DNA.This mtDNA codes for mt ribosomal and transferRNAs and for 13 proteins that play key roles in the respiratorychain. The linearized structural map of thehuman mitochondrial genes is shown in Figure 36–8.Some of the features of mtDNA are shown in Table36–3.An important feature of human mitochondrialmtDNA is that—because all mitochondria are contributedby the ovum during zygote formation—it istransmitted by maternal nonmendelian inheritance.kb2 4 6 8 10 12 14 16OHPH1PH2ATPase8 6ND3ND4LOH12S 16S ND1 ND2 CO1 CO2 CO3 ND4 ND5 CYT BPLOLFigure 36–8. Maps of human mitochondrial genes. The maps represent the heavy (upper strand) and light(lower map) strands of linearized mitochondrial (mt) DNA, showing the genes for the subunits of NADHcoenzymeQ oxidoreductase (ND1 through ND6), cytochrome c oxidase (CO1 through CO3), cytochrome b(CYT B), and ATP synthase (ATPase 8 and 6) and for the 12S and 16S ribosomal mt rRNAs. The transfer RNAs are denotedby small open boxes. The origin of heavy-strand (OH) and light-strand (OL) replication and the promotersfor the initiation of heavy-strand (PH1 and PH2) and light-strand (PL) transcription are indicated by arrows.(Reproduced, with permission, from Moraes CT et al: Mitochondrial DNA deletions in progressive external ophthalmoplegiaand Kearns-Sayre syndrome. N Engl J Med 1989;320:1293.)ND6

DNA ORGANIZATION, REPLICATION, & REPAIR / 323Table 36–3. Some major features of the structureand function of human mitochondrial DNA. 1• Is circular, double-stranded, and composed of heavy (H)and a light (L) chains or strands.• Contains 16,569 bp.• Encodes 13 protein subunits of the respiratory chain (of atotal of about 67):Seven subunits of NADH dehydrogenase (complex I)Cytochrome b of complex IIIThree subunits of cytochrome oxidase (complex IV)Two subunits of ATP synthase• Encodes large (16s) and small (12s) mt ribosomal RNAs.• Encodes 22 mt tRNA molecules.• Genetic code differs slightly from the standard code:UGA (standard stop codon) is read as Trp.AGA and AGG (standard codons for Arg) are read as stopcodons.• Contains very few untranslated sequences.• High mutation rate (five to ten times that of nuclear DNA).• Comparisons of mtDNA sequences provide evidence aboutevolutionary origins of primates and other species.1 Adapted from Harding AE: Neurological disease and mitochondrialgenes. Trends Neurol Sci 1991;14:132.crossing over occurs as shown in Figure 36–9. Thisusually results in an equal and reciprocal exchange ofgenetic information between homologous chromosomes.If the homologous chromosomes possess differentalleles of the same genes, the crossover may producenoticeable and heritable genetic linkage differences. Inthe rare case where the alignment of homologous chromosomesis not exact, the crossing over or recombinationevent may result in an unequal exchange of information.One chromosome may receive less geneticmaterial and thus a deletion, while the other partner ofthe chromosome pair receives more genetic materialand thus an insertion or duplication (Figure 36–9).Unequal crossing over does occur in humans, as evidencedby the existence of hemoglobins designatedLepore and anti-Lepore (Figure 36–10). The fartherapart two sequences are on an individual chromosome,the greater the likelihood of a crossover recombinationThus, in diseases resulting from mutations of mtDNA,an affected mother would in theory pass the disease toall of her children but only her daughters would transmitthe trait. However, in some cases, deletions inmtDNA occur during oogenesis and thus are not inheritedfrom the mother. A number of diseases have nowbeen shown to be due to mutations of mtDNA. Theseinclude a variety of myopathies, neurologic disorders,and some cases of diabetes mellitus.GENETIC MATERIAL CAN BE ALTERED& REARRANGEDAn alteration in the sequence of purine and pyrimidinebases in a gene due to a change—a removal or an insertion—ofone or more bases may result in an alteredgene product. Such alteration in the genetic material resultsin a mutation whose consequences are discussedin detail in Chapter 38.Chromosomal Recombination Is One Wayof Rearranging Genetic MaterialGenetic information can be exchanged between similaror homologous chromosomes. The exchange or recombinationevent occurs primarily during meiosis inmammalian cells and requires alignment of homologousmetaphase chromosomes, an alignment that almostalways occurs with great exactness. A process ofFigure 36–9. The process of crossing-over betweenhomologous metaphase chromosomes to generate recombinantchromosomes. See also Figure 36–12.

324 / CHAPTER 36Gγ Aγ δ βδ βGγ Aγ δ βGγAγAnti-LeporeGγ Aγ δβLeporeFigure 36–10. The process of unequal crossoverin the region of the mammalian genomethat harbors the structural genes encoding hemoglobinsand the generation of the unequalrecombinant products hemoglobin delta-betaLepore and beta-delta anti-Lepore. The examplesgiven show the locations of the crossoverregions between amino acid residues. (Redrawnand reproduced, with permission, from Clegg JB,Weatherall DJ: β 0 Thalassemia: Time for a reappraisal?Lancet 1974;2:133.)event. This is the basis for genetic mapping methods.Unequal crossover affects tandem arrays of repeatedDNAs whether they are related globin genes, as in Figure36–10, or more abundant repetitive DNA. Unequalcrossover through slippage in the pairing can resultin expansion or contraction in the copy number ofthe repeat family and may contribute to the expansionand fixation of variant members throughout the array.Chromosomal Integration OccursWith Some VirusesSome bacterial viruses (bacteriophages) are capable ofrecombining with the DNA of a bacterial host in such away that the genetic information of the bacteriophage isincorporated in a linear fashion into the genetic informationof the host. This integration, which is a form ofrecombination, occurs by the mechanism illustrated inFigure 36–11. The backbone of the circular bacteriophagegenome is broken, as is that of the DNA moleculeof the host; the appropriate ends are resealed withthe proper polarity. The bacteriophage DNA is figurativelystraightened out (“linearized”) as it is integratedinto the bacterial DNA molecule—frequently a closedcircle as well. The site at which the bacteriophagegenome integrates or recombines with the bacterialgenome is chosen by one of two mechanisms. If thebacteriophage contains a DNA sequence homologousto a sequence in the host DNA molecule, then a recombinationevent analogous to that occurring between homologouschromosomes can occur. However, somebacteriophages synthesize proteins that bind specificsites on bacterial chromosomes to a nonhomologoussite characteristic of the bacteriophage DNA molecule.Integration occurs at the site and is said to be “sitespecific.”Many animal viruses, particularly the oncogenicviruses—either directly or, in the case of RNA virusessuch as HIV that causes AIDS, their DNA transcriptsgenerated by the action of the viral RNA-dependentDNA polymerase, or reverse transcriptase—can be integratedinto chromosomes of the mammalian cell. Theintegration of the animal virus DNA into the animalgenome generally is not “site-specific” but does displaysite preferences.Transposition Can ProduceProcessed GenesIn eukaryotic cells, small DNA elements that clearly arenot viruses are capable of transposing themselves in and111 2BCAACBB1 2BFigure 36–11. The integration of a circular genomefrom a virus (with genes A, B, and C) into the DNA moleculeof a host (with genes 1 and 2) and the consequentordering of the genes.ACCA22

DNA ORGANIZATION, REPLICATION, & REPAIR / 325out of the host genome in ways that affect the functionof neighboring DNA sequences. These mobile elements,sometimes called “jumping DNA,” can carryflanking regions of DNA and, therefore, profoundly affectevolution. As mentioned above, the Alu family ofmoderately repeated DNA sequences has structuralcharacteristics similar to the termini of retroviruses,which would account for the ability of the latter tomove into and out of the mammalian genome.Direct evidence for the transposition of other smallDNA elements into the human genome has been providedby the discovery of “processed genes” for immunoglobulinmolecules, α-globin molecules, and severalothers. These processed genes consist of DNAsequences identical or nearly identical to those of themessenger RNA for the appropriate gene product. Thatis, the 5′ nontranscribed region, the coding regionwithout intron representation, and the 3′ poly(A) tailare all present contiguously. This particular DNA sequencearrangement must have resulted from the reversetranscription of an appropriately processed messengerRNA molecule from which the intron regionshad been removed and the poly(A) tail added. The onlyrecognized mechanism this reverse transcript couldhave used to integrate into the genome would havebeen a transposition event. In fact, these “processedgenes” have short terminal repeats at each end, as doknown transposed sequences in lower organisms. In theabsence of their transcription and thus genetic selectionfor function, many of the processed genes have beenrandomly altered through evolution so that they nowcontain nonsense codons which preclude their ability toencode a functional, intact protein (see Chapter 38).Thus, they are referred to as “pseudogenes.”Gene Conversion ProducesRearrangementsBesides unequal crossover and transposition, a thirdmechanism can effect rapid changes in the genetic material.Similar sequences on homologous or nonhomologouschromosomes may occasionally pair up and eliminateany mismatched sequences between them. Thismay lead to the accidental fixation of one variant or anotherthroughout a family of repeated sequences andthereby homogenize the sequences of the members ofrepetitive DNA families. This latter process is referredto as gene conversion.Sister Chromatids ExchangeIn diploid eukaryotic organisms such as humans, aftercells progress through the S phase they contain atetraploid content of DNA. This is in the form of sisterchromatids of chromosome pairs. Each of these sisterchromatids contains identical genetic information sinceeach is a product of the semiconservative replication ofthe original parent DNA molecule of that chromosome.Crossing over occurs between these genetically identicalsister chromatids. Of course, these sister chromatid exchanges(Figure 36–12) have no genetic consequence aslong as the exchange is the result of an equal crossover.Immunoglobulin Genes RearrangeIn mammalian cells, some interesting gene rearrangementsoccur normally during development and differentiation.For example, in mice the V Land C Lgenes for asingle immunoglobulin molecule (see Chapter 39) arewidely separated in the germ line DNA. In the DNA of adifferentiated immunoglobulin-producing (plasma) cell,the same V Land C Lgenes have been moved physicallycloser together in the genome and into the same transcriptionunit. However, even then, this rearrangementof DNA during differentiation does not bring the V LandC Lgenes into contiguity in the DNA. Instead, the DNAFigure 36–12. Sister chromatid exchanges betweenhuman chromosomes. These are detectable by Giemsastaining of the chromosomes of cells replicated for twocycles in the presence of bromodeoxyuridine. The arrowsindicate some regions of exchange. (Courtesy ofSWolff and J Bodycote.)

326 / CHAPTER 36contains an interspersed or interruption sequence ofabout 1200 base pairs at or near the junction of the Vand C regions. The interspersed sequence is transcribedinto RNA along with the V Land C Lgenes, and the interspersedinformation is removed from the RNA during itsnuclear processing (Chapters 37 and 39).DNA SYNTHESIS & REPLICATIONARE RIGIDLY CONTROLLEDThe primary function of DNA replication is understoodto be the provision of progeny with the geneticinformation possessed by the parent. Thus, the replicationof DNA must be complete and carried out in sucha way as to maintain genetic stability within the organismand the species. The process of DNA replication iscomplex and involves many cellular functions and severalverification procedures to ensure fidelity in replication.About 30 proteins are involved in the replicationof the E coli chromosome, and this process is almostcertainly more complex in eukaryotic organisms. Thefirst enzymologic observations on DNA replicationwere made in E coli by Kornberg, who described in thatorganism the existence of an enzyme now called DNApolymerase I. This enzyme has multiple catalytic activities,a complex structure, and a requirement for thetriphosphates of the four deoxyribonucleosides of adenine,guanine, cytosine, and thymine. The polymerizationreaction catalyzed by DNA polymerase I of E colihas served as a prototype for all DNA polymerases ofboth prokaryotes and eukaryotes, even though it is nowrecognized that the major role of this polymerase is tocomplete replication on the lagging strand.In all cells, replication can occur only from a singlestrandedDNA (ssDNA) template. Mechanisms mustexist to target the site of initiation of replication and tounwind the double-stranded DNA (dsDNA) in that region.The replication complex must then form. Afterreplication is complete in an area, the parent and daughterstrands must re-form dsDNA. In eukaryotic cells, an additionalstep must occur. The dsDNA must precisely reformthe chromatin structure, including nucleosomes,that existed prior to the onset of replication. Although thisentire process is not well understood in eukaryotic cells,replication has been quite precisely described in prokaryoticcells, and the general principles are thought to be thesame in both. The major steps are listed in Table 36–4, illustratedin Figure 36–13, and discussed, in sequence,below. A number of proteins, most with specific enzymaticaction, are involved in this process (Table 36–5).The Origin of ReplicationAt the origin of replication (ori), there is an associationof sequence-specific dsDNA-binding proteins withTable 36–4. Steps involved in DNA replicationin eukaryotes.1. Identification of the origins of replication.2. Unwinding (denaturation) of dsDNA to provide an ssDNAtemplate.3. Formation of the replication fork.4. Initiation of DNA synthesis and elongation.5. Formation of replication bubbles with ligation of the newlysynthesized DNA segments.6. Reconstitution of chromatin structure.a series of direct repeat DNA sequences. In bacteriophageλ, the oriλ is bound by the λ-encoded O proteinto four adjacent sites. In E coli, the oriC is bound bythe protein dnaA. In both cases, a complex is formedconsisting of 150–250 bp of DNA and multimers ofthe DNA-binding protein. This leads to the local denaturationand unwinding of an adjacent A+T-rich regionof DNA. Functionally similar autonomouslyreplicating sequences (ARS) have been identified inyeast cells. The ARS contains a somewhat degenerate11-bp sequence called the origin replication element(ORE). The ORE binds a set of proteins, analogous tothe dnaA protein of E coli, which is collectively calledthe origin recognition complex (ORC). The ORE islocated adjacent to an approximately 80-bp A+T-richsequence that is easy to unwind. This is called the DNAunwinding element (DUE). The DUE is the origin ofreplication in yeast.Consensus sequences similar to ori or ARS in structureor function have not been precisely defined inmammalian cells, though several of the proteins thatparticipate in ori recognition and function have beenidentified and appear quite similar to their yeast counterpartsin both amino acid sequence and function.Unwinding of DNAThe interaction of proteins with ori defines the start siteof replication and provides a short region of ssDNA essentialfor initiation of synthesis of the nascent DNAstrand. This process requires the formation of a numberof protein-protein and protein-DNA interactions. Acritical step is provided by a DNA helicase that allowsfor processive unwinding of DNA. In uninfected E coli,this function is provided by a complex of dnaB helicaseand the dnaC protein. Single-stranded DNA-bindingproteins (SSBs) stabilize this complex. In λ phageinfectedE coli, the phage protein P binds to dnaB andthe P/dnaB complex binds to oriλ by interacting withthe O protein. dnaB is not an active helicase when in theP/dnaB/O complex. Three E coli heat shock proteins(dnaK, dnaJ, and GrpE) cooperate to remove the P

DNA ORGANIZATION, REPLICATION, & REPAIR / 327OriA + T - richregionOri-bindingprotein( )A + T regionDenaturationBindingof SSB ( )Leadingstrand3′5′Binding offactors,formation ofreplication fork,initiation ofreplication3′PolymeraseHelicasePrimase5′3′SSB5′Laggingstrand3′Replication fork5′= Ori–binding protein= Polymerase= Nascent DNA= RNA primer= Helicase= Primase= SSBFigure 36–13. Steps involved in DNA replication. This figure describes DNA replication in an E coli cell, but thegeneral steps are similar in eukaryotes. A specific interaction of a protein (the O protein) to the origin of replication(ori) results in local unwinding of DNA at an adjacent A+T-rich region. The DNA in this area is maintained in thesingle-strand conformation (ssDNA) by single-strand-binding proteins (SSBs). This allows a variety of proteins, includinghelicase, primase, and DNA polymerase, to bind and to initiate DNA synthesis. The replication fork proceeds asDNA synthesis occurs continuously (long arrow) on the leading strand and discontinuously (short arrows) on the laggingstrand. The nascent DNA is always synthesized in the 5′ to 3′ direction, as DNA polymerases can add a nucleotideonly to the 3′ end of a DNA strand.protein and activate the dnaB helicase. In cooperationwith SSB, this leads to DNA unwinding and activereplication. In this way, the replication of the λ phage isaccomplished at the expense of replication of the hostE coli cell.Formation of the Replication ForkA replication fork consists of four components thatform in the following sequence: (1) the DNA helicaseunwinds a short segment of the parental duplex DNA;(2) a primase initiates synthesis of an RNA moleculethat is essential for priming DNA synthesis; (3) theDNA polymerase initiates nascent, daughter strandsynthesis; and (4) SSBs bind to ssDNA and preventpremature reannealing of ssDNA to dsDNA. These reactionsare illustrated in Figure 36–13.The polymerase III holoenzyme (the dnaE geneproduct in E coli) binds to template DNA as part of amultiprotein complex that consists of several polymeraseaccessory factors (β, γ, δ, δ′, and τ). DNA polymerasesonly synthesize DNA in the 5′ to 3′ direction,

328 / CHAPTER 36Table 36–5. Classes of proteins involvedin replication.ProteinDNA polymerasesHelicasesTopoisomerasesDNA primaseFunctionDeoxynucleotide polymerizationProcessive unwinding of DNARelieve torsional strain that resultsfrom helicase-induced unwindingInitiates synthesis of RNA primersSingle-strand binding Prevent premature reannealing ofproteinsdsDNADNA ligaseSeals the single strand nick betweenthe nascent chain and Okazaki fragmentson lagging strandand only one of the several different types of polymerasesis involved at the replication fork. Because theDNA strands are antiparallel (Chapter 35), the polymerasefunctions asymmetrically. On the leading (forward)strand, the DNA is synthesized continuously.On the lagging (retrograde) strand, the DNA is synthesizedin short (1–5 kb; see Figure 36–16) fragments,the so-called Okazaki fragments. Several Okazaki fragments(up to 250) must be synthesized, in sequence, foreach replication fork. To ensure that this happens, thehelicase acts on the lagging strand to unwind dsDNA ina 5′ to 3′ direction. The helicase associates with the primaseto afford the latter proper access to the template.This allows the RNA primer to be made and, in turn,the polymerase to begin replicating the DNA. This isan important reaction sequence since DNA polymerasescannot initiate DNA synthesis de novo. Themobile complex between helicase and primase has beencalled a primosome. As the synthesis of an Okazakifragment is completed and the polymerase is released, anew primer has been synthesized. The same polymerasemolecule remains associated with the replication forkand proceeds to synthesize the next Okazaki fragment.The DNA Polymerase ComplexA number of different DNA polymerase molecules engagein DNA replication. These share three importantproperties: (1) chain elongation, (2) processivity, and(3) proofreading. Chain elongation accounts for therate (in nucleotides per second) at which polymerizationoccurs. Processivity is an expression of the numberof nucleotides added to the nascent chain before thepolymerase disengages from the template. The proofreadingfunction identifies copying errors and correctsthem. In E coli, polymerase III (pol III) functions at thereplication fork. Of all polymerases, it catalyzes thehighest rate of chain elongation and is the most processive.It is capable of polymerizing 0.5 Mb of DNA duringone cycle on the leading strand. Pol III is a large(> 1 MDa), ten-subunit protein complex in E coli. Thetwo identical β subunits of pol III encircle the DNAtemplate in a sliding “clamp,” which accounts for thestability of the complex and for the high degree of processivitythe enzyme exhibits.Polymerase II (pol II) is mostly involved in proofreadingand DNA repair. Polymerase I (pol I) completeschain synthesis between Okazaki fragments onthe lagging strand. Eukaryotic cells have counterpartsfor each of these enzymes plus some additional ones. Acomparison is shown in Table 36–6.In mammalian cells, the polymerase is capable ofpolymerizing about 100 nucleotides per second, a rateat least tenfold slower than the rate of polymerization ofdeoxynucleotides by the bacterial DNA polymerasecomplex. This reduced rate may result from interferenceby nucleosomes. It is not known how the replicationcomplex negotiates nucleosomes.Initiation & Elongation of DNA SynthesisThe initiation of DNA synthesis (Figure 36–14) requirespriming by a short length of RNA, about10–200 nucleotides long. This priming process involvesthe nucleophilic attack by the 3′-hydroxyl group of theRNA primer on the α phosphate of the first enteringdeoxynucleoside triphosphate (N in Figure 36–14)with the splitting off of pyrophosphate. The 3′-hydroxylgroup of the recently attached deoxyribonucleosidemonophosphate is then free to carry out anucleophilic attack on the next entering deoxyribonucleosidetriphosphate (N + 1 in Figure 36–14), again atits α phosphate moiety, with the splitting off of pyrophosphate.Of course, selection of the proper deoxyribonucleotidewhose terminal 3′-hydroxyl group isto be attacked is dependent upon proper base pairingTable 36–6. A comparison of prokaryotic andeukaryotic DNA polymerases.E coli Mammalian FunctionI α Gap filling and synthesis of laggingstrandII ε DNA proofreading and repairβγDNA repairMitochondrial DNA synthesisIII δ Processive, leading strand synthesis

OHHHHHOOPCX 1X 2X 3X 4X4COHHOHHRNA primerOHOOPCHHOHHOHOOPCHHOHHOHFirst entering dNTPO –POOOO –POOO –OHPOO –CHHOHOHHNHOPOCHHOHHNOHOOPCHHOHHSecond entering dNTPO –POHOHN+1CO O OPO O O –PH HH HO O – OH HO –Figure 36–14. The initiation of DNA synthesis upon a primer of RNA and the subsequentattachment of the second deoxyribonucleoside triphosphate.329

330 / CHAPTER 36with the other strand of the DNA molecule accordingto the rules proposed originally by Watson and Crick(Figure 36–15). When an adenine deoxyribonucleosidemonophosphoryl moiety is in the template position, athymidine triphosphate will enter and its α phosphatewill be attacked by the 3′-hydroxyl group of thedeoxyribonucleoside monophosphoryl most recentlyadded to the polymer. By this stepwise process, thetemplate dictates which deoxyribonucleoside triphosphateis complementary and by hydrogen bondingholds it in place while the 3′-hydroxyl group of thegrowing strand attacks and incorporates the new nucleotideinto the polymer. These segments of DNAattached to an RNA initiator component are theOkazaki fragments (Figure 36–16). In mammals, aftermany Okazaki fragments are generated, the replicationcomplex begins to remove the RNA primers, to fill inthe gaps left by their removal with the proper basepaireddeoxynucleotide, and then to seal the fragmentsof newly synthesized DNA by enzymes referred to asDNA ligases.Replication Exhibits PolarityAs has already been noted, DNA molecules are doublestrandedand the two strands are antiparallel, ie, runningin opposite directions. The replication of DNA inprokaryotes and eukaryotes occurs on both strands simultaneously.However, an enzyme capable of polymerizingDNA in the 3′ to 5′ direction does not exist inany organism, so that both of the newly replicatedDNA strands cannot grow in the same direction simultaneously.Nevertheless, the same enzyme does replicateboth strands at the same time. The single enzyme replicatesone strand (“leading strand”) in a continuousmanner in the 5′ to 3′ direction, with the same overallforward direction. It replicates the other strand (“laggingstrand”) discontinuously while polymerizing the3′TPCA5′AGOHGURNA primerOHOHCOHUOHTGAACADNA templateTTTAGACGrowing DNA polymerCGAGTP P POHOH3′TGAAEntering TTPC5′Figure 36–15. The RNA-primed synthesis of DNA demonstrating the template function of thecomplementary strand of parental DNA.

DNA ORGANIZATION, REPLICATION, & REPAIR / 3313′DNA template5′5′ 3′Newly synthesizedRNA10 bp10 bpDNA strandprimer100 bpOkazaki fragmentsFigure 36–16. The discontinuous polymerization of deoxyribonucleotides on the laggingstrand; formation of Okazaki fragments during lagging strand DNA synthesis is illustrated.Okazaki fragments are 100–250 nt long in eukaryotes, 1000–2000 bp in prokaryotes.nucleotides in short spurts of 150–250 nucleotides,again in the 5′ to 3′ direction, but at the same time itfaces toward the back end of the preceding RNAprimer rather than toward the unreplicated portion.This process of semidiscontinuous DNA synthesis isshown diagrammatically in Figures 36–13 and 36–16.In the mammalian nuclear genome, most of theRNA primers are eventually removed as part of thereplication process, whereas after replication of the mitochondrialgenome the small piece of RNA remains asan integral part of the closed circular DNA structure.Formation of Replication BubblesReplication proceeds from a single ori in the circularbacterial chromosome, composed of roughly 6 × 10 6 bpof DNA. This process is completed in about 30 minutes,a replication rate of 3 × 10 5 bp/min. The entiremammalian genome replicates in approximately 9hours, the average period required for formation of atetraploid genome from a diploid genome in a replicatingcell. If a mammalian genome (3 × 10 9 bp) replicatedat the same rate as bacteria (ie, 3 × 10 5 bp/min)from but a single ori, replication would take over 150hours! Metazoan organisms get around this problemusing two strategies. First, replication is bidirectional.Second, replication proceeds from multiple origins ineach chromosome (a total of as many as 100 in humans).Thus, replication occurs in both directionsalong all of the chromosomes, and both strands arereplicated simultaneously. This replication process generates“replication bubbles” (Figure 36–17).The multiple sites that serve as origins for DNAreplication in eukaryotes are poorly defined except in afew animal viruses and in yeast. However, it is clear thatinitiation is regulated both spatially and temporally,since clusters of adjacent sites initiate replication synchronously.There are suggestions that functional domainsof chromatin replicate as intact units, implyingthat the origins of replication are specifically locatedwith respect to transcription units.During the replication of DNA, there must be a separationof the two strands to allow each to serve as atemplate by hydrogen bonding its nucleotide bases tothe incoming deoxynucleoside triphosphate. The separationof the DNA double helix is promoted by SSBs, specificprotein molecules that stabilize the single-strandedstructure as the replication fork progresses. These stabi-Origin of replication“Replication bubble”3′5′5′3′Unwinding proteinsat replication forksDirectionsof replicationFigure 36–17. The generation of “replication bubbles” during the process of DNA synthesis. The bidirectionalreplication and the proposed positions of unwinding proteins at the replication forks are depicted.

332 / CHAPTER 36lizing proteins bind cooperatively and stoichiometricallyto the single strands without interfering with the abilitiesof the nucleotides to serve as templates (Figure36–13). In addition to separating the two strands of thedouble helix, there must be an unwinding of the molecule(once every 10 nucleotide pairs) to allow strand separation.This must happen in segments, given the timeduring which DNA replication occurs. There are multiple“swivels” interspersed in the DNA molecules of allorganisms. The swivel function is provided by specificenzymes that introduce “nicks” in one strand of theunwinding double helix, thereby allowing the unwindingprocess to proceed. The nicks are quickly resealedwithout requiring energy input, because of the formationof a high-energy covalent bond between the nickedphosphodiester backbone and the nicking-sealing enzyme.The nicking-resealing enzymes are called DNAtopoisomerases. This process is depicted diagrammaticallyin Figure 36–18 and there compared with theATP-dependent resealing carried out by the DNA ligases.Topoisomerases are also capable of unwinding supercoiledDNA. Supercoiled DNA is a higher-orderedstructure occurring in circular DNA molecules wrappedaround a core, as depicted in Figure 36–19.There exists in one species of animal viruses (retroviruses)a class of enzymes capable of synthesizing a sin-Step 1DNA topoisomerase I = EDNA ligase = EE + ATP E P R A(AMP-Enzyme)5′P -EO 3′H3′ 5′Enzyme (E) -generatedsingle-strand nick5′P3′ 5′O 3′HSingle-strand nickpresentStep 2EEP R A5′P -E5′PP R AOHFormation of highenergybondOHStep 3EP R A(AMP)Nick repairedNick repairedFigure 36–18. Comparison of two types of nick-sealing reactions on DNA. Theseries of reactions at left is catalyzed by DNA topoisomerase I, that at right byDNA ligase; P = phosphate, R = ribose, A = ademine. (Slightly modified and reproduced,with permission, from Lehninger AL: Biochemistry, 2nd ed. Worth, 1975.)

DNA ORGANIZATION, REPLICATION, & REPAIR / 333preexisting and newly assembled histone octamers arerandomly distributed to each arm of the replicationfork.Figure 36–19. Supercoiling of DNA. A left-handedtoroidal (solenoidal) supercoil, at left, will convert to aright-handed interwound supercoil, at right, when thecylindric core is removed. Such a transition is analogousto that which occurs when nucleosomes are disruptedby the high salt extraction of histones from chromatin.gle-stranded and then a double-stranded DNA moleculefrom a single-stranded RNA template. This polymerase,RNA-dependent DNA polymerase, or “reversetranscriptase,” first synthesizes a DNA-RNA hybridmolecule utilizing the RNA genome as a template. Aspecific nuclease, RNase H, degrades the RNA strand,and the remaining DNA strand in turn serves as a templateto form a double-stranded DNA molecule containingthe information originally present in the RNAgenome of the animal virus.Reconstitution of Chromatin StructureThere is evidence that nuclear organization and chromatinstructure are involved in determining the regulationand initiation of DNA synthesis. As notedabove, the rate of polymerization in eukaryotic cells,which have chromatin and nucleosomes, is tenfoldslower than that in prokaryotic cells, which havenaked DNA. It is also clear that chromatin structuremust be re-formed after replication. Newly replicatedDNA is rapidly assembled into nucleosomes, and theDNA Synthesis Occurs Duringthe S Phase of the Cell CycleIn animal cells, including human cells, the replicationof the DNA genome occurs only at a specified timeduring the life span of the cell. This period is referred toas the synthetic or S phase. This is usually temporallyseparated from the mitotic phase by nonsynthetic periodsreferred to as gap 1 (G1) and gap 2 (G2), occurringbefore and after the S phase, respectively (Figure36–20). Among other things, the cell prepares for DNAsynthesis in G1 and for mitosis in G2. The cell regulatesits DNA synthesis grossly by allowing it to occuronly at specific times and mostly in cells preparing todivide by a mitotic process.It appears that all eukaryotic cells have gene productsthat govern the transition from one phase of thecell cycle to another. The cyclins are a family of proteinswhose concentration increases and decreasesthroughout the cell cycle—thus their name. The cyclinsturn on, at the appropriate time, different cyclindependentprotein kinases (CDKs) that phosphorylatesubstrates essential for progression through the cellcycle (Figure 36–21). For example, cyclin D levels risein late G1 phase and allow progression beyond the start(yeast) or restriction point (mammals), the point beyondwhich cells irrevocably proceed into the S orDNA synthesis phase.The D cyclins activate CDK4 and CDK6. Thesetwo kinases are also synthesized during G1 in cells undergoingactive division. The D cyclins and CDK4 andCDK6 are nuclear proteins that assemble as a complexin late G1 phase. The complex is an active serinethreonineprotein kinase. One substrate for this kinaseis the retinoblastoma (Rb) protein. Rb is a cell cycleregulator because it binds to and inactivates a transcriptionfactor (E2F) necessary for the transcription of certaingenes (histone genes, DNA replication proteins,etc) needed for progression from G1 to S phase. Thephosphorylation of Rb by CDK4 or CDK6 results inthe release of E2F from Rb-mediated transcription repression—thus,gene activation ensues and cell cycleprogression takes place.Other cyclins and CDKs are involved in differentaspects of cell cycle progression (Table 36–7). Cyclin Eand CDK2 form a complex in late G1. Cyclin E israpidly degraded, and the released CDK2 then forms acomplex with cyclin A. This sequence is necessary forthe initiation of DNA synthesis in S phase. A complexbetween cyclin B and CDK1 is rate-limiting for theG2/M transition in eukaryotic cells.

334 / CHAPTER 36Improper spindledetectedDamaged DNAdetectedG 2SIncompletereplicationdetectedMGlDamaged DNAdetectedFigure 36–20. Mammalian cell cycle and cellcycle checkpoints. DNA, chromosome, and chromosomesegregation integrity is continuouslymonitored throughout the cell cycle. If DNA damageis detected in either the G1 or the G2 phase ofthe cell cycle, if the genome is incompletely replicated,or if normal chromosome segregation machineryis incomplete (ie, a defective spindle), cellswill not progress through the phase of the cycle inwhich defects are detected. In some cases, if thedamage cannot be repaired, such cells undergoprogrammed cell death (apoptosis).Many of the cancer-causing viruses (oncoviruses)and cancer-inducing genes (oncogenes) are capable ofalleviating or disrupting the apparent restriction thatnormally controls the entry of mammalian cells fromG1 into the S phase. From the foregoing, one mighthave surmised that excessive production of a cyclin—orproduction at an inappropriate time—might result inabnormal or unrestrained cell division. In this context itis noteworthy that the bcl oncogene associated with Bcell lymphoma appears to be the cyclin D1 gene. Similarly,the oncoproteins (or transforming proteins) pro-duced by several DNA viruses target the Rb transcriptionrepressor for inactivation, inducing cell division inappropriately.During the S phase, mammalian cells containgreater quantities of DNA polymerase than during thenonsynthetic phases of the cell cycle. Furthermore,those enzymes responsible for formation of the substratesfor DNA synthesis—ie, deoxyribonucleosidetriphosphates—are also increased in activity, and theiractivity will diminish following the synthetic phaseuntil the reappearance of the signal for renewed DNACdk1-cyclin BCdk1-cyclin AG 2 MG 1Cdk4-cyclin DCdk6-cyclin DSRestrictionpointCdk2-cyclin ACdk2-cyclin EFigure 36–21. Schematic illustration of thepoints during the mammalian cell cycle duringwhich the indicated cyclins and cyclin-dependentkinases are activated. The thickness of the variouscolored lines is indicative of the extent of activity.

DNA ORGANIZATION, REPLICATION, & REPAIR / 335Table 36–7. Cyclins and cyclin-dependent kinasesinvolved in cell cycle progression.Cyclin Kinase FunctionD CDK4, CDK6 Progression past restriction point atG1/S boundaryE, A CDK2 Initiation of DNA synthesis in early SphaseB CDK1 Transition from G2 to Msynthesis. During the S phase, the nuclear DNA iscompletely replicated once and only once. It seemsthat once chromatin has been replicated, it is marked soas to prevent its further replication until it again passesthrough mitosis. The molecular mechanisms for thisphenomenon have yet to be elucidated.In general, a given pair of chromosomes will replicatesimultaneously and within a fixed portion of the Sphase upon every replication. On a chromosome, clustersof replication units replicate coordinately. The natureof the signals that regulate DNA synthesis at theselevels is unknown, but the regulation does appear to bean intrinsic property of each individual chromosome.Enzymes Repair Damaged DNAThe maintenance of the integrity of the information inDNA molecules is of utmost importance to the survivalof a particular organism as well as to survival of thespecies. Thus, it can be concluded that surviving specieshave evolved mechanisms for repairing DNA damageoccurring as a result of either replication errors or environmentalinsults.As described in Chapter 35, the major responsibilityfor the fidelity of replication resides in the specific pairingof nucleotide bases. Proper pairing is dependentupon the presence of the favored tautomers of thepurine and pyrimidine nucleotides, but the equilibriumwhereby one tautomer is more stable than another isonly about 10 4 or 10 5 in favor of that with the greaterstability. Although this is not favorable enough to ensurethe high fidelity that is necessary, favoring of thepreferred tautomers—and thus of the proper base pairing—couldbe ensured by monitoring the base pairingtwice. Such double monitoring does appear to occur inboth bacterial and mammalian systems: once at thetime of insertion of the deoxyribonucleoside triphosphates,and later by a follow-up energy-requiring mechanismthat removes all improper bases which may occurin the newly formed strand. This “proofreading” preventstautomer-induced misincorporation from occurringmore frequently than once every 10 8 –10 10 basepairs of DNA synthesized. The mechanisms responsiblefor this monitoring mechanism in E coli include the 3′to 5′ exonuclease activities of one of the subunits of thepol III complex and of the pol I molecule. The analogousmammalian enzymes (δ and α) do not seem topossess such a nuclease proofreading function. Otherenzymes provide this repair function.Replication errors, even with a very efficient repairsystem, lead to the accumulation of mutations. Ahuman has 10 14 nucleated cells each with 3 × 10 9 basepairs of DNA. If about 10 16 cell divisions occur in alifetime and 10 −10 mutations per base pair per cell generationescape repair, there may eventually be as manyas one mutation per 10 6 bp in the genome. Fortunately,most of these will probably occur in DNA that does notencode proteins or will not affect the function of encodedproteins and so are of no consequence. In addition,spontaneous and chemically induced damage toDNA must be repaired.Damage to DNA by environmental, physical, andchemical agents may be classified into four types(Table 36–8). Abnormal regions of DNA, either fromcopying errors or DNA damage, are replaced by fourmechanisms: (1) mismatch repair, (2) base excisionrepair,(3) nucleotide excision-repair, and (4) doublestrandbreak repair (Table 36–9). These mechanismsexploit the redundancy of information inherent in thedouble helical DNA structure. The defective region inone strand can be returned to its original form by relyingon the complementary information stored in theunaffected strand.Table 36–8. Types of damage to DNA.I. Single-base alterationA. DepurinationB. Deamination of cytosine to uracilC. Deamination of adenine to hypoxanthineD. Alkylation of baseE. Insertion or deletion of nucleotideF. Base-analog incorporationII.Two-base alterationA. UV light–induced thymine-thymine (pyrimidine) dimerB. Bifunctional alkylating agent cross-linkageIII. Chain breaksA. Ionizing radiationB. Radioactive disintegration of backbone elementC. Oxidative free radical formationIV. Cross-linkageA. Between bases in same or opposite strandsB. Between DNA and protein molecules (eg, histones)

336 / CHAPTER 36Table 36–9. Mechanism of DNA repairMechanism Problem SolutionMismatch Copying errors (single Methyl-directedrepair base or two- to five- strand cutting, exobaseunpaired loops) nuclease digestion,and replacementBase Spontaneous, chem- Base removal by N-excision- ical, or radiation dam- glycosylase, abasicrepair age to a single base sugar removal, replacementNucleotide Spontaneous, chem- Removal of an apexcision-ical, or radiation dam- proximately 30-repair age to a DNA segment nucleotide oligomerand replacementDouble- Ionizing radiation, Synapsis, unwindstrandchemotherapy, ing, alignment,break repair oxidative free ligationradicals3′5′3′5′3′5′CH 3CH 3CH 3 CH 3CH 3 CH 35′3′SINGLE-SITE STRAND CUTBY GATC ENDONUCLEASE5′3′DEFECT REMOVEDBY EXONUCLEASE5′3′DEFECT REPAIREDBY POLYMERASECH 3 CH 33′ 5′Mismatch RepairMismatch repair corrects errors made when DNA iscopied. For example, a C could be inserted opposite anA, or the polymerase could slip or stutter and insert twoto five extra unpaired bases. Specific proteins scan thenewly synthesized DNA, using adenine methylationwithin a GATC sequence as the point of reference (Figure36–22). The template strand is methylated, and thenewly synthesized strand is not. This difference allowsthe repair enzymes to identify the strand that containsthe errant nucleotide which requires replacement. If amismatch or small loop is found, a GATC endonucleasecuts the strand bearing the mutation at a site correspondingto the GATC. An exonuclease then digeststhis strand from the GATC through the mutation, thusremoving the faulty DNA. This can occur from eitherend if the defect is bracketed by two GATC sites. Thisdefect is then filled in by normal cellular enzymes accordingto base pairing rules. In E coli, three proteins(Mut S, Mut C, and Mut H) are required for recognitionof the mutation and nicking of the strand. Othercellular enzymes, including ligase, polymerase, andSSBs, remove and replace the strand. The process issomewhat more complicated in mammalian cells, asabout six proteins are involved in the first steps.Faulty mismatch repair has been linked to hereditarynonpolyposis colon cancer (HNPCC), one of themost common inherited cancers. Genetic studies linkedHNPCC in some families to a region of chromosome2. The gene located, designated hMSH2, was subsequentlyshown to encode the human analog of the3′5′CH 3 CH 3RELIGATEDBY LIGASEFigure 36–22. Mismatch repair of DNA. This mechanismcorrects a single mismatch base pair (eg, C to Arather than T to A) or a short region of unpaired DNA.The defective region is recognized by an endonucleasethat makes a single-strand cut at an adjacent methylatedGATC sequence. The DNA strand is removedthrough the mutation, replaced, and religated.E coli MutS protein that is involved in mismatch repair(see above). Mutations of hMSH2 account for 50–60%of HNPCC cases. Another gene, hMLH1, is associatedwith most of the other cases. hMLH1 is the human analogof the bacterial mismatch repair gene MutL. Howdoes faulty mismatch repair result in colon cancer? Thehuman genes were localized because microsatellite instabilitywas detected. That is, the cancer cells had a microsatelliteof a length different from that found in thenormal cells of the individual. It appears that the affectedcells, which harbor a mutated hMSH2 orhMLH1 mismatch repair enzyme, are unable to removesmall loops of unpaired DNA, and the microsatellitethus increases in size. Ultimately, microsatellite DNAexpansion must affect either the expression or the functionof a protein critical in surveillance of the cell cyclein these colon cells.5′3′

DNA ORGANIZATION, REPLICATION, & REPAIR / 337Base Excision-RepairThe depurination of DNA, which happens spontaneouslyowing to the thermal lability of the purine N-glycosidic bond, occurs at a rate of 5000–10,000/cell/dat 37 °C. Specific enzymes recognize a depurinated siteand replace the appropriate purine directly, without interruptionof the phosphodiester backbone.Cytosine, adenine, and guanine bases in DNA spontaneouslyform uracil, hypoxanthine, or xanthine, respectively.Since none of these normally exist in DNA,it is not surprising that specific N-glycosylases can recognizethese abnormal bases and remove the base itselffrom the DNA. This removal marks the site of the defectand allows an apurinic or apyrimidinic endonucleaseto excise the abasic sugar. The proper base isthen replaced by a repair DNA polymerase, and a ligasereturns the DNA to its original state (Figure 36–23).This series of events is called base excision-repair. By asimilar series of steps involving initially the recognitionof the defect, alkylated bases and base analogs can be removedfrom DNA and the DNA returned to its originalinformational content. This mechanism is suitablefor replacement of a single base but is not effective atreplacing regions of damaged DNA.Nucleotide Excision-RepairThis mechanism is used to replace regions of damagedDNA up to 30 bases in length. Common examples ofDNA damage include ultraviolet (UV) light, which inducesthe formation of cyclobutane pyrimidine-pyrimidinedimers, and smoking, which causes formation ofbenzo[a]pyrene-guanine adducts. Ionizing radiation,cancer chemotherapeutic agents, and a variety of chemicalsfound in the environment cause base modification,strand breaks, cross-linkage between bases on oppositestrands or between DNA and protein, and numerousother defects. These are repaired by a process called nucleotideexcision-repair (Figure 36–24). This complexprocess, which involves more gene products than the twoother types of repair, essentially involves the hydrolysis oftwo phosphodiester bonds on the strand containing thedefect. A special excision nuclease (exinuclease), consistingof at least three subunits in E coli and 16 polypeptidesin humans, accomplishes this task. In eukaryoticcells the enzymes cut between the third to fifth phosphodiesterbond 3′ from the lesion, and on the 5′ side the cutis somewhere between the twenty-first and twenty-fifthbonds. Thus, a fragment of DNA 27–29 nucleotideslong is excised. After the strand is removed it is replaced,again by exact base pairing, through the action of yet anotherpolymerase (δ/ε in humans), and the ends arejoined to the existing strands by DNA ligase.Xeroderma pigmentosum (XP) is an autosomal recessivegenetic disease. The clinical syndrome includes3′5′A T C G G C T C A T C C G A TT A G C C G A G T A G G C T A5′3′Heat energyA T C G G C T U A T C C G A TT A G C C G A G T A G G C T AAUT C G G C T*URACIL DNA GLYCOSYLASEA T C C G A TT A G C C G A G T A G G C T AAT C G G CNUCLEASEST C C G A TT A G C C G A G T A G G C T AA T C G G C T C A T C C G A TT A G C C G A G T A G G C T ADNA POLYMERASE + DNA LIGASEFigure 36–23. Base excision-repair of DNA. The enzymeuracil DNA glycosylase removes the uracil createdby spontaneous deamination of cytosine in the DNA. Anendonuclease cuts the backbone near the defect; then,after an endonuclease removes a few bases, the defectis filled in by the action of a repair polymerase and thestrand is rejoined by a ligase. (Courtesy of B Alberts.)marked sensitivity to sunlight (ultraviolet) with subsequentformation of multiple skin cancers and prematuredeath. The risk of developing skin cancer is increased1000- to 2000-fold. The inherited defect seemsto involve the repair of damaged DNA, particularlythymine dimers. Cells cultured from patients with xerodermapigmentosum exhibit low activity for the nucleotideexcision-repair process. Seven complementationgroups have been identified using hybrid cellanalyses, so at least seven gene products (XPA–XPG)are involved. Two of these (XPA and XPC) are involvedin recognition and excision. XPB and XPD arehelicases and, interestingly, are subunits of the transcriptionfactor TFIIH (see Chapter 37).Double-Strand Break RepairThe repair of double-strand breaks is part of the physiologicprocess of immunoglobulin gene rearrangement. It

338 / CHAPTER 363′5′3′5′3′5′3′5′5′3′RECOGNITION AND UNWINDING5′3′OLIGONUCLEOTIDE EXCISIONBY CUTTING AT TWO SITES5′3′DEGRADATION OF MUTATED DNARESYNTHESIS AND RELIGATIONFigure 36–24. Nucleotide excision-repair. Thismechanism is employed to correct larger defects inDNA and generally involves more proteins than eithermismatch or base excision-repair. After defect recognition(indicated by XXXX) and unwinding of the DNA encompassingthe defect, an excision nuclease (exinuclease)cuts the DNA upstream and downstream of thedefective region. This gap is then filled in by a polymerase(δ/ε in humans) and religated.5′3′is also an important mechanism for repairing damagedDNA, such as occurs as a result of ionizing radiation oroxidative free radical generation. Some chemotherapeuticagents destroy cells by causing ds breaks or preventingtheir repair.Two proteins are initially involved in the nonhomologousrejoining of a ds break. Ku, a heterodimer of70 kDa and 86 kDa subunits, binds to free DNA endsand has latent ATP-dependent helicase activity. TheDNA-bound Ku heterodimer recruits a unique proteinkinase, DNA-dependent protein kinase (DNA-PK).DNA-PK has a binding site for DNA free ends and anotherfor dsDNA just inside these ends. It therefore allowsfor the approximation of the two separated ends.The free end DNA-Ku-DNA-PK complex activates thekinase activity in the latter. DNA-PK reciprocally phosphorylatesKu and the other DNA-PK molecule, on theopposing strand, in trans. DNA-PK then dissociatesfrom the DNA and Ku, resulting in activation of theKu helicase. This results in unwinding of the two ends.The unwound, approximated DNA forms base pairs;the extra nucleotide tails are removed by an exonuclease;and the gaps are filled and closed by DNA ligase.This repair mechanism is illustrated in Figure 36–25.Some Repair Enzymes Are MultifunctionalSomewhat surprising is the recent observation thatDNA repair proteins can serve other purposes. For example,some repair enzymes are also found as componentsof the large TFIIH complex that plays a centralrole in gene transcription (Chapter 37). Another componentof TFIIH is involved in cell cycle regulation.Thus, three critical cellular processes may be linkedthrough use of common proteins. There is also goodevidence that some repair enzymes are involved in generearrangements that occur normally.In patients with ataxia-telangiectasia, an autosomalrecessive disease in humans resulting in the developmentof cerebellar ataxia and lymphoreticular neoplasms,there appears to exist an increased sensitivity to damageby x-ray. Patients with Fanconi’s anemia, an autosomalrecessive anemia characterized also by an increased frequencyof cancer and by chromosomal instability, probablyhave defective repair of cross-linking damage.Ku and DNA-PK bindApproximationUnwindingAlignment and base pairingLigationPPFigure 36–25. Double-strand break repair of DNA.The proteins Ku and DNA-dependent protein kinasecombine to approximate the two strands and unwindthem. The aligned fragments form base pairs; the extraends are removed, probably by a DNA-PK-associatedendo- or exonuclease, and the gaps are filled in; andcontinuity is restored by ligation.

DNA ORGANIZATION, REPLICATION, & REPAIR / 339All three of these clinical syndromes are associatedwith an increased frequency of cancer. It is likely thatother human diseases resulting from disordered DNArepair capabilities will be found in the future.DNA & Chromosome Integrity IsMonitored Throughout the Cell CycleGiven the importance of normal DNA and chromosomefunction to survival, it is not surprising that eukaryoticcells have developed elaborate mechanisms to monitorthe integrity of the genetic material. As detailed above, anumber of complex multi-subunit enzyme systems haveevolved to repair damaged DNA at the nucleotide sequencelevel. Similarly, DNA mishaps at the chromosomelevel are also monitored and repaired. As shown inFigure 36–20, DNA integrity and chromosomal integrityare continuously monitored throughout the cellcycle. The four specific steps at which this monitoringoccurs have been termed checkpoint controls. If problemsare detected at any of these checkpoints, progressionthrough the cycle is interrupted and transit through thecell cycle is halted until the damage is repaired. The molecularmechanisms underlying detection of DNA damageduring the G1 and G2 phases of the cycle are understoodbetter than those operative during S and M phases.The tumor suppressor p53, a protein of MW 53kDa, plays a key role in both G1 and G2 checkpoint control.Normally a very unstable protein, p53 is a DNAbinding transcription factor, one of a family of relatedproteins, that is somehow stabilized in response to DNAdamage, perhaps by direct p53-DNA interactions. Increasedlevels of p53 activate transcription of an ensembleof genes that collectively serve to delay transit through thecycle. One of these induced proteins, p21 CIP , is a potentCDK-cyclin inhibitor (CKI) that is capable of efficientlyinhibiting the action of all CDKs. Clearly, inhibition ofCDKs will halt progression through the cell cycle (seeFigures 36–19 and 36–20). If DNA damage is too extensiveto repair, the affected cells undergo apoptosis (programmedcell death) in a p53-dependent fashion. In thiscase, p53 induces the activation of a collection of genesthat induce apoptosis. Cells lacking functional p53 fail toundergo apoptosis in response to high levels of radiationor DNA-active chemotherapeutic agents. It may come asno surprise, then, that p53 is one of the most frequentlymutated genes in human cancers. Additional research intothe mechanisms of checkpoint control will prove invaluablefor the development of effective anticancer therapeuticoptions.SUMMARY• DNA in eukaryotic cells is associated with a varietyof proteins, resulting in a structure called chromatin.• Much of the DNA is associated with histone proteinsto form a structure called the nucleosome. Nucleosomesare composed of an octamer of histones and150 bp of DNA.• Nucleosomes and higher-order structures formedfrom them serve to compact the DNA.• As much as 90% of DNA may be transcriptionallyinactive as a result of being nuclease-resistant, highlycompacted, and nucleosome-associated.• DNA in transcriptionally active regions is sensitive tonuclease attack; some regions are exceptionally sensitiveand are often found to contain transcriptioncontrol sites.• Transcriptionally active DNA (the genes) is oftenclustered in regions of each chromosome. Withinthese regions, genes may be separated by inactiveDNA in nucleosomal structures. The transcriptionunit—that portion of a gene that is copied by RNApolymerase—consists of coding regions of DNA(exons) interrupted by intervening sequences of noncodingDNA (introns).• After transcription, during RNA processing, intronsare removed and the exons are ligated together toform the mature mRNA that appears in the cytoplasm.• DNA in each chromosome is exactly replicated accordingto the rules of base pairing during the Sphase of the cell cycle.• Each strand of the double helix is replicated simultaneouslybut by somewhat different mechanisms. Acomplex of proteins, including DNA polymerase,replicates the leading strand continuously in the 5′ to3′ direction. The lagging strand is replicated discontinuously,in short pieces of 150–250 nucleotides, inthe 3′ to 5′ direction.• DNA replication occurs at several sites—called replicationbubbles—in each chromosome. The entireprocess takes about 9 hours in a typical cell.• A variety of mechanisms employing different enzymesrepair damaged DNA, as after exposure tochemical mutagens or ultraviolet radiation.REFERENCESDePamphilis ML: Origins of DNA replication in metazoan chromosomes.J Biol Chem 1993;268:1.Hartwell LH, Kastan MB: Cell cycle control and cancer. Science1994;266:1821.Jenuwein T, Allis CD: Translating the histone code. Science 2001;293:1074.Lander ES et al: Initial sequencing and analysis of the humangenome. Nature 2001;409:860.Luger L et al: Crystal structure of the nucleosome core particle at2.8 Å resolution. Nature 1997;398:251.

340 / CHAPTER 36Marians KJ: Prokaryotic DNA replication. Annu Rev Biochem1992;61:673.Michelson RJ, Weinart T: Closing the gaps among a web of DNArepair disorders. Bioessays J 2002;22:966.Moll UM, Erster S, Zaika A: p53, p63 and p73—solos, alliancesand feuds among family members. Biochim Biophys Acta2001;1552:47.Mouse Genome Sequencing Consortium: Initial sequencing andcomparative analysis of the mouse genome. Nature 2002;420:520.Narlikar GJ et al: Cooperation between complexes that regulatechromatin structure and transcription. Cell 2002;108:475.Sullivan et al: Determining centromere identity: cyclical stories andforking paths. Nat Rev Genet 2001;2:584.van Holde K, Zlatanova J: Chromatin higher order: chasing a mirage?J Biol Chem 1995;270:8373.Venter JC et al: The sequence of the human genome. Science2002;291:1304.Wallace DC: Mitochondrial DNA in aging and disease. Sci Am1997 Aug;277:40.Wood RD: Nucleotide excision repair in mammalian cells. J BiolChem 1997;272:23465.

RNA Synthesis, Processing,& Modification 37Daryl K. Granner, MD, & P. Anthony Weil, PhDBIOMEDICAL IMPORTANCEThe synthesis of an RNA molecule from DNA is acomplex process involving one of the group of RNApolymerase enzymes and a number of associated proteins.The general steps required to synthesize the primarytranscript are initiation, elongation, and termination.Most is known about initiation. A number ofDNA regions (generally located upstream from the initiationsite) and protein factors that bind to these sequencesto regulate the initiation of transcription havebeen identified. Certain RNAs—mRNAs in particular—havevery different life spans in a cell. It is importantto understand the basic principles of messengerRNA synthesis and metabolism, for modulation of thisprocess results in altered rates of protein synthesis andthus a variety of metabolic changes. This is how all organismsadapt to changes of environment. It is also howdifferentiated cell structures and functions are establishedand maintained. The RNA molecules synthesizedin mammalian cells are made as precursor moleculesthat have to be processed into mature, activeRNA. Errors or changes in synthesis, processing, andsplicing of mRNA transcripts are a cause of disease.RNA EXISTS IN FOUR MAJOR CLASSESAll eukaryotic cells have four major classes of RNA: ribosomalRNA (rRNA), messenger RNA (mRNA), transferRNA (tRNA), and small nuclear RNA (snRNA).The first three are involved in protein synthesis, andsnRNA is involved in mRNA splicing. As shown inTable 37–1, these various classes of RNA are differentin their diversity, stability, and abundance in cells.RNA IS SYNTHESIZED FROM A DNATEMPLATE BY AN RNA POLYMERASEThe processes of DNA and RNA synthesis are similarin that they involve (1) the general steps of initiation,elongation, and termination with 5′ to 3′ polarity; (2)large, multicomponent initiation complexes; and (3)adherence to Watson-Crick base-pairing rules. Theseprocesses differ in several important ways, including thefollowing: (1) ribonucleotides are used in RNA synthesisrather than deoxyribonucleotides; (2) U replaces Tas the complementary base pair for A in RNA; (3) aprimer is not involved in RNA synthesis; (4) only a verysmall portion of the genome is transcribed or copiedinto RNA, whereas the entire genome must be copiedduring DNA replication; and (5) there is no proofreadingfunction during RNA transcription.The process of synthesizing RNA from a DNA templatehas been characterized best in prokaryotes. Althoughin mammalian cells the regulation of RNA synthesisand the processing of the RNA transcripts aredifferent from those in prokaryotes, the process of RNAsynthesis per se is quite similar in these two classes oforganisms. Therefore, the description of RNA synthesisin prokaryotes, where it is better understood, is applicableto eukaryotes even though the enzymes involvedand the regulatory signals are different.The Template Strand of DNAIs TranscribedThe sequence of ribonucleotides in an RNA molecule iscomplementary to the sequence of deoxyribonucleotidesin one strand of the double-stranded DNAmolecule (Figure 35–8). The strand that is transcribedor copied into an RNA molecule is referred to as thetemplate strand of the DNA. The other DNA strand isfrequently referred to as the coding strand of that gene.It is called this because, with the exception of T for Uchanges, it corresponds exactly to the sequence of theprimary transcript, which encodes the protein productof the gene. In the case of a double-stranded DNA moleculecontaining many genes, the template strand foreach gene will not necessarily be the same strand of theDNA double helix (Figure 37–1). Thus, a given strandof a double-stranded DNA molecule will serve as thetemplate strand for some genes and the coding strandof other genes. Note that the nucleotide sequence of anRNA transcript will be the same (except for U replacingT) as that of the coding strand. The information in thetemplate strand is read out in the 3′ to 5′ direction.341

342 / CHAPTER 37Table 37–1. Classes of eukaryotic RNA.RNA Types Abundance StabilityRibosomal 28S, 18S, 5.8S, 5S 80% of total Very stable(rRNA)Messenger ~10 5 different 2–5% of total Unstable to(mRNA) species verystableTransfer ~60 different ~15% of total Very stable(tRNA) speciesSmall nuclear ~30 different ≤ 1% of total Very stable(snRNA) speciesDNA-Dependent RNA PolymeraseInitiates Transcription at a DistinctSite, the PromoterDNA-dependent RNA polymerase is the enzyme responsiblefor the polymerization of ribonucleotides intoa sequence complementary to the template strand ofthe gene (see Figures 37–2 and 37–3). The enzyme attachesat a specific site—the promoter—on the templatestrand. This is followed by initiation of RNA synthesisat the starting point, and the process continuesuntil a termination sequence is reached (Figure 37–3).A transcription unit is defined as that region of DNAthat includes the signals for transcription initiation,elongation, and termination. The RNA product, whichis synthesized in the 5′ to 3′ direction, is the primarytranscript. In prokaryotes, this can represent the productof several contiguous genes; in mammalian cells, itusually represents the product of a single gene. The 5′terminals of the primary RNA transcript and the maturecytoplasmic RNA are identical. Thus, the startingpoint of transcription corresponds to the 5 nucleotideof the mRNA. This is designated position +1,as is the corresponding nucleotide in the DNA. TheGene A Gene B Gene C5′ 3′3′ 5′Template strandsGene DFigure 37–1. This figure illustrates that genes can betranscribed off both strands of DNA. The arrowheads indicatethe direction of transcription (polarity). Note thatthe template strand is always read in the 3′ to 5′ direction.The opposite strand is called the coding strand becauseit is identical (except for T for U changes) to themRNA transcript (the primary transcript in eukaryoticcells) that encodes the protein product of the gene.5′ P-P-P3′5′RNA transcriptβ3′ OHαβ′σRNAP complexαTranscriptionFigure 37–2. RNA polymerase (RNAP) catalyzes thepolymerization of ribonucleotides into an RNA sequencethat is complementary to the template strandof the gene. The RNA transcript has the same polarity(5′ to 3′) as the coding strand but contains U ratherthan T. E coli RNAP consists of a core complex of twoα subunits and two β subunits (β and β′). The holoenzymecontains the σ subunit bound to the α 2 ββ′ coreassembly. The ω subunit is not shown. The transcription“bubble” is an approximately 20-bp area of meltedDNA, and the entire complex covers 30–75 bp, dependingon the conformation of RNAP.pppApNRNAPpp(5) Chain terminationand RNAP releasepppApN(1) Template bindingpppApNATP + NTP(2) Chain initiationpppApN(4) Chain elongationNTPsNTPs(3) PromoterclearanceFigure 37–3. The transcription cycle in bacteria. BacterialRNA transcription is described in four steps:(1) Template binding: RNA polymerase (RNAP) bindsto DNA and locates a promoter (P) melts the two DNAstrands to form a preinitiation complex (PIC). (2) Chaininitiation: RNAP holoenzyme (core + one of multiplesigma factors) catalyzes the coupling of the first base(usually ATP or GTP) to a second ribonucleosidetriphosphate to form a dinucleotide. (3) Chain elongation:Successive residues are added to the 3′-OH terminusof the nascent RNA molecule. (4) Chain terminationand release: The completed RNA chain and RNAPare released from the template. The RNAP holoenzymere-forms, finds a promoter, and the cycle is repeated.5′3′

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 343numbers increase as the sequence proceeds downstream.This convention makes it easy to locate particular regions,such as intron and exon boundaries. The nucleotidein the promoter adjacent to the transcriptioninitiation site is designated −1, and these negative numbersincrease as the sequence proceeds upstream, awayfrom the initiation site. This provides a conventionalway of defining the location of regulatory elements inthe promoter.The primary transcripts generated by RNA polymeraseII—one of three distinct nuclear DNA-dependentRNA polymerases in eukaryotes—are promptlycapped by 7-methylguanosine triphosphate caps (Figure35–10) that persist and eventually appear on the 5′end of mature cytoplasmic mRNA. These caps are necessaryfor the subsequent processing of the primarytranscript to mRNA, for the translation of the mRNA,and for protection of the mRNA against exonucleolyticattack.Bacterial DNA-Dependent RNAPolymerase Is a Multisubunit EnzymeThe DNA-dependent RNA polymerase (RNAP) of thebacterium Escherichia coli exists as an approximately400 kDa core complex consisting of two identical αsubunits, similar but not identical β and β′ subunits,and an ω subunit. Beta is thought to be the catalyticsubunit (Figure 37–2). RNAP, a metalloenzyme, alsocontains two zinc molecules. The core RNA polymeraseassociates with a specific protein factor (the sigma [σ]factor) that helps the core enzyme recognize and bindto the specific deoxynucleotide sequence of the promoterregion (Figure 37–5) to form the preinitiationcomplex (PIC). Sigma factors have a dual role in theprocess of promoter recognition; σ association withcore RNA polymerase decreases its affinity for nonpromoterDNA while simultaneously increasing holoenzymeaffinity for promoter DNA. Bacteria contain multipleσ factors, each of which acts as a regulatoryprotein that modifies the promoter recognition specificityof the RNA polymerase. The appearance of differentσ factors can be correlated temporally with variousprograms of gene expression in prokaryotic systemssuch as bacteriophage development, sporulation, andthe response to heat shock.Table 37–2. Nomenclature and properties ofmammalian nuclear DNA-dependent RNApolymerases.Form of RNA Sensitivity toPolymerase -Amanitin Major ProductsI (A) Insensitive rRNAII (B) High sensitivity mRNAIII (C) Intermediate sensitivity tRNA/5S rRNAMammalian Cells Possess ThreeDistinct Nuclear DNA-DependentRNA PolymerasesThe properties of mammalian polymerases are describedin Table 37–2. Each of these DNA-dependentRNA polymerases is responsible for transcription of differentsets of genes. The sizes of the RNA polymerasesrange from MW 500,000 to MW 600,000. These enzymesare much more complex than prokaryotic RNApolymerases. They all have two large subunits and anumber of smaller subunits—as many as 14 in the caseof RNA pol III. The eukaryotic RNA polymerases haveextensive amino acid homologies with prokaryoticRNA polymerases. This homology has been shown recentlyto extend to the level of three-dimensional structures.The functions of each of the subunits are not yetfully understood. Many could have regulatory functions,such as serving to assist the polymerase in therecognition of specific sequences like promoters andtermination signals.One peptide toxin from the mushroom Amanitaphalloides, α-amanitin, is a specific differential inhibitorof the eukaryotic nuclear DNA-dependent RNA polymerasesand as such has proved to be a powerful researchtool (Table 37–2). α-Amanitin blocks the translocationof RNA polymerase during transcription.RNA SYNTHESIS IS A CYCLICAL PROCESS& INVOLVES INITIATION, ELONGATION,& TERMINATIONThe process of RNA synthesis in bacteria—depicted inFigure 37–3—involves first the binding of the RNAholopolymerase molecule to the template at the promotersite to form a PIC. Binding is followed by a conformationalchange of the RNAP, and the first nucleotide(almost always a purine) then associates withthe initiation site on the β subunit of the enzyme. Inthe presence of the appropriate nucleotide, the RNAPcatalyzes the formation of a phosphodiester bond, andthe nascent chain is now attached to the polymerizationsite on the β subunit of RNAP. (The analogy to the Aand P sites on the ribosome should be noted; see Figure38–9.)Initiation of formation of the RNA molecule at its5′ end then follows, while elongation of the RNA mole-

344 / CHAPTER 37cule from the 5′ to its 3′ end continues cyclically, antiparallelto its template. The enzyme polymerizes theribonucleotides in a specific sequence dictated by thetemplate strand and interpreted by Watson-Crick basepairingrules. Pyrophosphate is released in the polymerizationreaction. This pyrophosphate (PP i ) is rapidlydegraded to 2 mol of inorganic phosphate (P i ) by ubiquitouspyrophosphatases, thereby providing irreversibilityon the overall synthetic reaction. In both prokaryotesand eukaryotes, a purine ribonucleotide is usuallythe first to be polymerized into the RNA molecule. Aswith eukaryotes, 5′ triphosphate of this first nucleotideis maintained in prokaryotic mRNA.As the elongation complex containing the coreRNA polymerase progresses along the DNA molecule,DNA unwinding must occur in order to provide accessfor the appropriate base pairing to the nucleotides ofthe coding strand. The extent of this transcription bubble(ie, DNA unwinding) is constant throughout transcriptionand has been estimated to be about 20 basepairs per polymerase molecule. Thus, it appears that thesize of the unwound DNA region is dictated by thepolymerase and is independent of the DNA sequence inthe complex. This suggests that RNA polymerase hasassociated with it an “unwindase” activity that opensthe DNA helix. The fact that the DNA double helixmust unwind and the strands part at least transientlyfor transcription implies some disruption of the nucleosomestructure of eukaryotic cells. Topoisomerase bothprecedes and follows the progressing RNAP to preventthe formation of superhelical complexes.Termination of the synthesis of the RNA moleculein bacteria is signaled by a sequence in the templatestrand of the DNA molecule—a signal that is recognizedby a termination protein, the rho (ρ) factor. Rhois an ATP-dependent RNA-stimulated helicase thatdisrupts the nascent RNA-DNA complex. After terminationof synthesis of the RNA molecule, the enzymeseparates from the DNA template and probably dissociatesto free core enzyme and free σ factor. With theassistance of another σ factor, the core enzyme thenrecognizes a promoter at which the synthesis of a newRNA molecule commences. In eukaryotic cells, terminationis less well defined. It appears to be somehowlinked both to initiation and to addition of the 3′polyA tail of mRNA and could involve destabilizationof the RNA-DNA complex at a region of A–U basepairs. More than one RNA polymerase molecule maytranscribe the same template strand of a gene simultaneously,but the process is phased and spaced in such away that at any one moment each is transcribing a differentportion of the DNA sequence. An electron micrographof extremely active RNA synthesis is shownin Figure 37–4.Figure 37–4. Electron photomicrograph of multiplecopies of amphibian ribosomal RNA genes in theprocess of being transcribed. The magnification isabout 6000 ×. Note that the length of the transcripts increasesas the RNA polymerase molecules progressalong the individual ribosomal RNA genes; transcriptionstart sites (filled circles) to transcription terminationsites (open circles). RNA polymerase I (not visualizedhere) is at the base of the nascent rRNA transcripts.Thus, the proximal end of the transcribed gene hasshort transcripts attached to it, while much longer transcriptsare attached to the distal end of the gene. Thearrows indicate the direction (5′ to 3′) of transcription.(Reproduced with permission, from Miller OL Jr, Beatty BR:Portrait of a gene. J Cell Physiol 1969;74[Suppl 1]:225.)THE FIDELITY & FREQUENCY OFTRANSCRIPTION IS CONTROLLEDBY PROTEINS BOUND TO CERTAINDNA SEQUENCESThe DNA sequence analysis of specific genes has allowedthe recognition of a number of sequences importantin gene transcription. From the large number ofbacterial genes studied it is possible to construct consensusmodels of transcription initiation and terminationsignals.

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 345The question, “How does RNAP find the correctsite to initiate transcription?” is not trivial when thecomplexity of the genome is considered. E coli has4 × 10 3 transcription initiation sites in 4 × 10 6 basepairs (bp) of DNA. The situation is even more complexin humans, where perhaps 10 5 transcription initiationsites are distributed throughout in 3 × 10 9 bp of DNA.RNAP can bind to many regions of DNA, but it scansthe DNA sequence—at a rate of ≥ 10 3 bp/s—until itrecognizes certain specific regions of DNA to which itbinds with higher affinity. This region is called the promoter,and it is the association of RNAP with the promoterthat ensures accurate initiation of transcription.The promoter recognition-utilization process is the targetfor regulation in both bacteria and humans.Bacterial Promoters Are Relatively SimpleBacterial promoters are approximately 40 nucleotides(40 bp or four turns of the DNA double helix) inlength, a region small enough to be covered by anE coli RNA holopolymerase molecule. In this consensuspromoter region are two short, conserved sequence elements.Approximately 35 bp upstream of the transcriptionstart site there is a consensus sequence of eight nucleotidepairs (5′-TGTTGACA-3′) to which the RNAPbinds to form the so-called closed complex. Moreproximal to the transcription start site—about ten nucleotidesupstream—is a six-nucleotide-pair A+T-richsequence (5′-TATAAT-3′). These conserved sequenceelements comprising the promoter are shown schematicallyin Figure 37–5. The latter sequence has a lowmelting temperature because of its deficiency of GCnucleotide pairs. Thus, the TATA box is thought toease the dissociation between the two DNA strands sothat RNA polymerase bound to the promoter regioncan have access to the nucleotide sequence of its immediatelydownstream template strand. Once this processoccurs, the combination of RNA polymerase plus promoteris called the open complex. Other bacteria haveslightly different consensus sequences in their promoters,but all generally have two components to the promoter;these tend to be in the same position relative tothe transcription start site, and in all cases the sequencesbetween the boxes have no similarity but still providecritical spacing functions facilitating recognition of −35and −10 sequence by RNA polymerase holoenzyme.Within a bacterial cell, different sets of genes are oftenTRANSCRIPTION UNITCoding strand 5′Template strand 3′TGTTGACA−35regionPromoter5′ FlankingsequencesTATAAT−10regionTranscriptionstart site+1PPP5′Transcribed regionTerminationsignalsOHRNA3′3′ Flankingsequences3′5′ DNAFigure 37–5. Bacterial promoters, such as that from E coli shown here,share two regions of highly conserved nucleotide sequence. These regionsare located 35 and 10 bp upstream (in the 5′ direction of the coding strand)from the start site of transcription, which is indicated as +1. By convention,all nucleotides upstream of the transcription initiation site (at +1) are numberedin a negative sense and are referred to as 5′-flanking sequences. Alsoby convention, the DNA regulatory sequence elements (TATA box, etc) aredescribed in the 5′ to 3′ direction and as being on the coding strand. Theseelements function only in double-stranded DNA, however. Note that thetranscript produced from this transcription unit has the same polarity or“sense” (ie, 5′ to 3′ orientation) as the coding strand. Termination ciselementsreside at the end of the transcription unit (see Figure 37–6 formore detail). By convention the sequences downstream of the site at whichtranscription termination occurs are termed 3′-flanking sequences.

346 / CHAPTER 37coordinately regulated. One important way that this isaccomplished is through the fact that these co-regulatedgenes share unique −35 and −10 promoter sequences.These unique promoters are recognized by different σfactors bound to core RNA polymerase.Rho-dependent transcription termination signalsin E coli also appear to have a distinct consensus sequence,as shown in Figure 37–6. The conserved consensussequence, which is about 40 nucleotide pairs inlength, can be seen to contain a hyphenated or interruptedinverted repeat followed by a series of AT basepairs. As transcription proceeds through the hyphenated,inverted repeat, the generated transcript can formthe intramolecular hairpin structure, also depicted inFigure 37–6.Transcription continues into the AT region, andwith the aid of the ρ termination protein the RNApolymerase stops, dissociates from the DNA template,and releases the nascent transcript.Eukaryotic Promoters Are More ComplexIt is clear that the signals in DNA which control transcriptionin eukaryotic cells are of several types. Twotypes of sequence elements are promoter-proximal. Oneof these defines where transcription is to commencealong the DNA, and the other contributes to the mechanismsthat control how frequently this event is to occur.For example, in the thymidine kinase gene of the herpessimplex virus, which utilizes transcription factors of itsmammalian host for gene expression, there is a singleunique transcription start site, and accurate transcriptionfrom this start site depends upon a nucleotide sequencelocated 32 nucleotides upstream from the start site (ie, at−32) (Figure 37–7). This region has the sequence ofTATAAAAG and bears remarkable similarity to thefunctionally related TATA box that is located about 10bp upstream from the prokaryotic mRNA start site (Figure37–5). Mutation or inactivation of the TATA boxmarkedly reduces transcription of this and many othergenes that contain this consensus cis element (see Figures37–7, 37–8). Most mammalian genes have a TATA boxthat is usually located 25–30 bp upstream from the transcriptionstart site. The consensus sequence for a TATAbox is TATAAA, though numerous variations have beencharacterized. The TATA box is bound by 34 kDaTATA binding protein (TBP), which in turn binds severalother proteins called TBP-associated factors(TAFs). This complex of TBP and TAFs is referred to asTFIID. Binding of TFIID to the TATA box sequence isthought to represent the first step in the formation of thetranscription complex on the promoter.A small number of genes lack a TATA box. In suchinstances, two additional cis elements, an initiator sequence(Inr) and the so-called downstream promoterelement (DPE), direct RNA polymerase II to the promoterand in so doing provide basal transcription startingfrom the correct site. The Inr element spans the startDirection of transcriptionCoding strand 5′Template strand 3′AGCCCGCTCGGGCGGCGGGCTCGCCCGATTTTTTTTAAAAAAAA3′DNA5′Coding strand 5′Template strand 3′5′AGCCCGCGGGCGTTTTTTTTAAAAAAAAU U UUUUUU-3′UCRNA transcript3′5′DNAFigure 37–6. The predominant bacterial transcription termination signal contains an inverted, hyphenated repeat(the two boxed areas) followed by a stretch of AT base pairs (top figure). The inverted repeat, when transcribedinto RNA, can generate the secondary structure in the RNA transcript shown at the bottom of the figure.Formation of this RNA hairpin causes RNA polymerase to pause and subsequently the ρ termination factor interactswith the paused polymerase and somehow induces chain termination.

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 347Promoter proximalupstream elementsSp1PromoterTFIID+1Sp1GC CAAT GC TATA box tk coding regionCTFFigure 37–7. Transcription elements and binding factors in the herpes simplex virus thymidine kinase(tk) gene. DNA-dependent RNA polymerase II binds to the region of the TATA box (which is boundby transcription factor TFIID) to form a multicomponent preinitiation complex capable of initiatingtranscription at a single nucleotide (+1). The frequency of this event is increased by the presence of upstreamcis-acting elements (the GC and CAAT boxes). These elements bind trans-acting transcriptionfactors, in this example Sp1 and CTF (also called C/EBP, NF1, NFY). These cis elements can function independentlyof orientation (arrows).−25Regulated expressionDistalregulatoryelementsPromoterproximalelements“Basal” expressionPromoterOtherregulatoryelementsEnhancer (+)andrepressor (−)elementsPromoterproximalelements(GC/CAAT, etc)TATA+1Inr DPECoding regionFigure 37–8. Schematic diagram showing the transcription control regions in a hypothetical class II(mRNA-producing) eukaryotic gene. Such a gene can be divided into its coding and regulatory regions,as defined by the transcription start site (arrow; +1). The coding region contains the DNA sequence thatis transcribed into mRNA, which is ultimately translated into protein. The regulatory region consists oftwo classes of elements. One class is responsible for ensuring basal expression. These elements generallyhave two components. The proximal component, generally the TATA box, or Inr or DPE elements directRNA polymerase II to the correct site (fidelity). In TATA-less promoters, an initiator (Inr) element thatspans the initiation site (+1) may direct the polymerase to this site. Another component, the upstreamelements, specifies the frequency of initiation. Among the best studied of these is the CAAT box, butseveral other elements (Sp1, NF1, AP1, etc) may be used in various genes. A second class of regulatorycis-acting elements is responsible for regulated expression. This class consists of elements that enhanceor repress expression and of others that mediate the response to various signals, including hormones,heat shock, heavy metals, and chemicals. Tissue-specific expression also involves specific sequences ofthis sort. The orientation dependence of all the elements is indicated by the arrows within the boxes. Forexample, the proximal element (the TATA box) must be in the 5′ to 3′ orientation. The upstream elementswork best in the 5′ to 3′ orientation, but some of them can be reversed. The locations of some elementsare not fixed with respect to the transcription start site. Indeed, some elements responsible forregulated expression can be located either interspersed with the upstream elements, or they can be locateddownstream from the start site.

348 / CHAPTER 37site (from −3 to +5) and consists of the general consensussequence TCA +1 G/T T T/C which is similar to theinitiation site sequence per se. (A+1 indicates the firstnucleotide transcribed.) The proteins that bind to Inr inorder to direct pol II binding include TFIID. Promotersthat have both a TATA box and an Inr may be strongerthan those that have just one of these elements. TheDPE has the consensus sequence A/GGA/T CGTG andis localized about 25 bp downstream of the +1 start site.Like the Inr, DPE sequences are also bound by the TAFsubunits of TFIID. In a survey of over 200 eukaryoticgenes, roughly 30% contained a TATA box and Inr,25% contained Inr and DPE, 15% contained all threeelements, while ~30% contained just the Inr.Sequences farther upstream from the start site determinehow frequently the transcription event occurs.Mutations in these regions reduce the frequency oftranscriptional starts tenfold to twentyfold. Typical ofthese DNA elements are the GC and CAAT boxes, sonamed because of the DNA sequences involved. As illustratedin Figure 37–7, each of these boxes binds aprotein, Sp1 in the case of the GC box and CTF (orC/EPB,NF1,NFY) by the CAAT box; both bindthrough their distinct DNA binding domains (DBDs).The frequency of transcription initiation is a consequenceof these protein-DNA interactions and complexinteractions between particular domains of the transcriptionfactors (distinct from the DBD domains—socalledactivation domains; ADs) of these proteins andthe rest of the transcription machinery (RNA polymeraseII and the basal factors TFIIA, B, D, E, F). (Seebelow and Figures 37–9 and 37–10). The protein-DNA interaction at the TATA box involving RNApolymerase II and other components of the basal transcriptionmachinery ensures the fidelity of initiation.Together, then, the promoter and promoter-proximalcis-active upstream elements confer fidelity and frequencyof initiation upon a gene. The TATA box has aparticularly rigid requirement for both position and orientation.Single-base changes in any of these cis elementshave dramatic effects on function by reducingthe binding affinity of the cognate trans factors (eitherTFIID/TBP or Sp1, CTF, and similar factors). Thespacing of these elements with respect to the transcriptionstart site can also be critical. This is particularlytrue for the TATA box Inr and DPE.A third class of sequence elements can either increaseor decrease the rate of transcription initiation of eukaryoticgenes. These elements are called either enhancers orrepressors (or silencers), depending on which effectthey have. They have been found in a variety of locationsboth upstream and downstream of the transcription startsite and even within the transcribed portions of somegenes. In contrast to proximal and upstream promoter elements,enhancers and silencers can exert their effectswhen located hundreds or even thousands of bases awayfrom transcription units located on the same chromosome.Surprisingly, enhancers and silencers can functionin an orientation-independent fashion. Literally hundredsof these elements have been described. In somecases, the sequence requirements for binding are rigidlyconstrained; in others, considerable sequence variation isHFATATADBEpol II–50 –30–10 +10 +30+50Figure 37–9. The eukaryotic basal transcription complex. Formation of the basal transcription complex beginswhen TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA andprotein-protein interactions. The entire complex spans DNA from position −30 to +30 relative to the initiation site(+1, marked by bent arrow). The atomic level, x-ray-derived structures of RNA polymerase II alone and of TBPbound to TATA promoter DNA in the presence of either TFIIB or TFIIA have all been solved at 3 Å resolution. Thestructure of TFIID complexes have been determined by electron microscopy at 30 Å resolution. Thus, the molecularstructures of the transcription machinery are beginning to be elucidated. Much of this structural information isconsistent with the models presented here.

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 349TAFRate oftranscriptionTBPBasalcomplexRate oftranscriptionCCAATTAFTBPCTFTAFBasal complexCAAT TATA nil+ CTFCCAATCTFTAFCTFTBPBasalcomplexTAF TAFCAATTATAnilTAFTBPBasal complexTAFTBPBasalcomplexCAATCTFAFigure 37–10. Two models for assembly of the active transcription complex and for how activators and coactivatorsmight enhance transcription. Shown here as a small oval is TBP, which contains TFIID, a large oval that containsall the components of the basal transcription complex illustrated in Figure 37–9 (ie, RNAP II and TFIIA, TFIIB,TFIIE, TFIIF, and TFIIH). Panel A: The basal transcription complex is assembled on the promoter after the TBP subunitof TFIID is bound to the TATA box. Several TAFs (coactivators) are associated with TBP. In this example, a transcriptionactivator, CTF, is shown bound to the CAAT box, forming a loop complex by interacting with a TAFbound to TBP. Panel B: The recruitment model. The transcription activator CTF binds to the CAAT box and interactswith a coactivator (TAF in this case). This allows for an interaction with the preformed TBP-basal transcriptioncomplex. TBP can now bind to the TATA box, and the assembled complex is fully active.BTATAallowed. Some sequences bind only a single protein, butthe majority bind several different proteins. Similarly, asingle protein can bind to more than one element.Hormone response elements (for steroids, T 3 , retinoicacid, peptides, etc) act as—or in conjunction with—enhancers or silencers (Chapter 43). Other processesthat enhance or silence gene expression—such as the responseto heat shock, heavy metals (Cd 2+ and Zn 2+ ),and some toxic chemicals (eg, dioxin)—are mediatedthrough specific regulatory elements. Tissue-specific expressionof genes (eg, the albumin gene in liver, the hemoglobingene in reticulocytes) is also mediated by specificDNA sequences.Specific Signals RegulateTranscription TerminationThe signals for the termination of transcription byeukaryotic RNA polymerase II are very poorly understood.However, it appears that the termination signalsexist far downstream of the coding sequence of eukaryoticgenes. For example, the transcription terminationsignal for mouse β-globin occurs at several positions1000–2000 bases beyond the site at which the poly(A)tail will eventually be added. Little is known about thetermination process or whether specific terminationfactors similar to the bacterial ρ factor are involved.However, it is known that the mRNA 3′ terminal isgenerated posttranscriptionally, is somehow coupled toevents or structures formed at the time and site of initiation,depends on a special structure in one of the subunitsof RNA polymerase II (the CTD; see below), andappears to involve at least two steps. After RNA polymeraseII has traversed the region of the transcriptionunit encoding the 3′ end of the transcript, an RNA endonucleasecleaves the primary transcript at a positionabout 15 bases 3′ of the consensus sequence AAUAAAthat serves in eukaryotic transcripts as a cleavage signal.

350 / CHAPTER 37Finally, this newly formed 3′ terminal is polyadenylatedin the nucleoplasm, as described below.THE EUKARYOTICTRANSCRIPTION COMPLEXA complex apparatus consisting of as many as 50unique proteins provides accurate and regulatable transcriptionof eukaryotic genes. The RNA polymerase enzymes(pol I, pol II, and pol III for class I, II, and IIIgenes, respectively) transcribe information contained inthe template strand of DNA into RNA. These polymerasesmust recognize a specific site in the promoter inorder to initiate transcription at the proper nucleotide.In contrast to the situation in prokaryotes, eukaryoticRNA polymerases alone are not able to discriminate betweenpromoter sequences and other regions of DNA;thus, other proteins known as general transcription factorsor GTFs facilitate promoter-specific binding ofthese enzymes and formation of the preinitiation complex(PIC). This combination of components can catalyzebasal or (non)-unregulated transcription in vitro.Another set of proteins—coactivators—help regulatethe rate of transcription initiation by interacting withtranscription activators that bind to upstream DNA elements(see below).Formation of the BasalTranscription ComplexIn bacteria, a σ factor–polymerase complex selectivelybinds to DNA in the promoter forming the PIC. Thesituation is more complex in eukaryotic genes. Class IIgenes—those transcribed by pol II to make mRNA—are described as an example. In class II genes, the functionof σ factors is assumed by a number of proteins.Basal transcription requires, in addition to pol II, anumber of GTFs called TFIIA, TFIIB, TFIID,TFIIE, TFIIF, and TFIIH. These GTFs serve to promoteRNA polymerase II transcription on essentially allgenes. Some of these GTFs are composed of multiplesubunits. TFIID, which binds to the TATA box promoterelement, is the only one of these factors capableof binding to specific sequences of DNA. As describedabove, TFIID consists of TATA bindingprotein (TBP) and 14 TBP-associated factors (TAFs).TBP binds to the TATA box in the minor groove ofDNA (most transcription factors bind in the majorgroove) and causes an approximately 100-degree bendor kink of the DNA helix. This bending is thought tofacilitate the interaction of TBP-associated factors withother components of the transcription initiation complexand possibly with factors bound to upstream elements.Although defined as a component of class IIgene promoters, TBP, by virtue of its association withdistinct, polymerase-specific sets of TAFs, is also an importantcomponent of class I and class III initiationcomplexes even if they do not contain TATA boxes.The binding of TBP marks a specific promoter fortranscription and is the only step in the assembly processthat is entirely dependent on specific, high-affinity protein-DNAinteraction. Of several subsequent in vitrosteps, the first is the binding of TFIIB to the TFIIDpromotercomplex. This results in a stable ternary complexwhich is then more precisely located and moretightly bound at the transcription initiation site. Thiscomplex then attracts and tethers the pol II-TFIIF complexto the promoter. TFIIF is structurally and functionallysimilar to the bacterial σ factor and is requiredfor the delivery of pol II to the promoter. TFIIA bindsto this assembly and may allow the complex to respondto activators, perhaps by the displacement of repressors.Addition of TFIIE and TFIIH is the final step in the assemblyof the PIC. TFIIE appears to join the complexwith pol II-TFIIF, and TFIIH is then recruited. Each ofthese binding events extends the size of the complex sothat finally about 60 bp (from −30 to +30 relative to +1,the nucleotide from which transcription commences)are covered (Figure 37–9). The PIC is now completeand capable of basal transcription initiated from the correctnucleotide. In genes that lack a TATA box, thesame factors, including TBP, are required. In such cases,an Inr or the DPEs (see Figure 37–8) position the complexfor accurate initiation of transcription.Phosphorylation Activates Pol IIEukaryotic pol II consists of 12 subunits. The twolargest subunits, both about 200 kDa, are homologousto the bacterial β and β′ subunits. In addition to the increasednumber of subunits, eukaryotic pol II differsfrom its prokaryotic counterpart in that it has a series ofheptad repeats with consensus sequence Tyr-Ser-Pro-Thr-Ser-Pro-Ser at the carboxyl terminal of the largestpol II subunit. This carboxyl terminal repeat domain(CTD) has 26 repeated units in brewers’ yeast and 52units in mammalian cells. The CTD is both a substratefor several kinases, including the kinase component ofTFIIH, and a binding site for a wide array of proteins.The CTD has been shown to interact with RNA processingenzymes; such binding may be involved withRNA polyadenylation. The association of the factorswith the CTD of RNA polymerase II (and other componentsof the basal machinery) somehow serves tocouple initiation with mRNA 3′ end formation. Pol IIis activated when phosphorylated on the Ser and Thrresidues and displays reduced activity when the CTD isdephosphorylated. Pol II lacking the CTD tail is incapableof activating transcription, which underscores theimportance of this domain.

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 351Pol II associates with other proteins to form aholoenzyme complex. In yeast, at least nine gene products—calledSrb (for suppressor of RNA polymeraseB)—bind to the CTD. The Srb proteins—or mediators,as they are also called—are essential for pol IItranscription, though their exact role in this process hasnot been defined. Related proteins comprising evenmore complex forms of RNA polymerase II have beendescribed in human cells.The Role of Transcription Activators& CoactivatorsTFIID was originally considered to be a single protein.However, several pieces of evidence led to the importantdiscovery that TFIID is actually a complex consistingof TBP and the 14 TAFs. The first evidence thatTFIID was more complex than just the TBP moleculescame from the observation that TBP binds to a 10-bpsegment of DNA, immediately over the TATA box ofthe gene, whereas native holo-TFIID covers a 35 bp orlarger region (Figure 37–9). Second, TBP has a molecularmass of 20–40 kDa (depending on the species),whereas the TFIID complex has a mass of about 1000kDa. Finally, and perhaps most importantly, TBP supportsbasal transcription but not the augmented transcriptionprovided by certain activators, eg, Sp1 boundto the GC box. TFIID, on the other hand, supportsboth basal and enhanced transcription by Sp1, Oct1,AP1, CTF, ATF, etc. (Table 37–3). The TAFs are essentialfor this activator-enhanced transcription. It isnot yet clear whether there are one or several forms ofTFIID that might differ slightly in their complement ofTable 37–3. Some of the transcription controlelements, their consensus sequences, and thefactors that bind to them which are found inmammalian genes transcribed by RNApolymerase II. A complete list would includedozens of examples. The asterisks mean thatthere are several members of this family.Element Consensus Sequence FactorTATA box TATAAA TBPCAAT box CCAATC C/EBP*, NF-Y*GC box GGGCGG Sp1*CAACTGACMyo DT/CGGA/CN 5 GCCAA NF1*lg octamer ATGCAAAT Oct1, 2, 4, 6*AP1 TGAG/CTC/AA Jun, Fos, ATF*Serum response GATGCCCATA SRFHeat shock (NGAAN) 3 HSFTAFs. It is conceivable that different combinations ofTAFs with TBP—or one of several recently discoveredTBP-like factors (TLFs)—may bind to different promoters,and recent reports suggest that this may accountfor selective activation noted in various promotersand for the different strengths of certain promoters.TAFs, since they are required for the action of activators,are often called coactivators. There are thusthree classes of transcription factors involved in the regulationof class II genes: basal factors, coactivators, andactivator-repressors (Table 37–4). How these classes ofproteins interact to govern both the site and frequencyof transcription is a question of central importance.Two Models Explain the Assemblyof the Preinitiation ComplexThe formation of the PIC described above is based onthe sequential addition of purified components in invitro experiments. An essential feature of this model isthat the assembly takes place on the DNA template.Accordingly, transcription activators, which have autonomousDNA binding and activation domains (seeChapter 39), are thought to function by stimulating eitherPIC formation or PIC function. The TAF coactivatorsare viewed as bridging factors that communicatebetween the upstream activators, the proteins associatedwith pol II, or the many other components of TFIID.This view, which assumes that there is stepwise assemblyof the PIC—promoted by various interactions betweenactivators, coactivators, and PIC components—is illustrated in panel A of Figure 37–10. This modelwas supported by observations that many of these proteinscould indeed bind to one another in vitro.Recent evidence suggests that there is another possiblemechanism of PIC formation and transcription regulation.First, large preassembled complexes of GTFsand pol II are found in cell extracts, and this complexcan associate with a promoter in a single step. Second,the rate of transcription achieved when activators areadded to limiting concentrations of pol II holoenzymecan be matched by increasing the concentration of thepol II holoenzyme in the absence of activators. Thus,Table 37–4. Three classes of transcription factorsin class II genes.General MechanismsBasal componentsCoactivatorsActivatorsSpecific ComponentsTBP, TFIIA, B, E, F, and HTAFs (TBP + TAFs) = TFIID; SrbsSP1, ATF, CTF, AP1, etc

352 / CHAPTER 37activators are not in themselves absolutely essential forPIC formation. These observations led to the “recruitment”hypothesis, which has now been tested experimentally.Simply stated, the role of activators andcoactivators may be solely to recruit a preformedholoenzyme-GTF complex to the promoter. The requirementfor an activation domain is circumventedwhen either a component of TFIID or the pol IIholoenzyme is artificially tethered, using recombinantDNA techniques, to the DNA binding domain (DBD)of an activator. This anchoring, through the DBDcomponent of the activator molecule, leads to a transcriptionallycompetent structure, and there is no furtherrequirement for the activation domain of the activator.In this view, the role of activation domains andTAFs is to form an assembly that directs the preformedholoenzyme-GTF complex to the promoter; they donot assist in PIC assembly (see panel B, Figure 37–10).The efficiency of this recruitment process determinesthe rate of transcription at a given promoter.Hormones—and other effectors that serve to transmitinformation related to the extracellular environment—modulategene expression by influencing the assemblyand activity of the activator and coactivatorcomplexes and the subsequent formation of the PIC atthe promoter of target genes (see Chapter 43). The numerouscomponents involved provide for an abundanceof possible combinations and therefore a range of transcriptionalactivity of a given gene. It is important tonote that the two models are not mutually exclusive—stepwise versus holoenzyme-mediated PIC formation.Indeed, one can envision various more complex modelsinvoking elements of both models operating on a gene.RNA MOLECULES ARE USUALLYPROCESSED BEFORE THEYBECOME FUNCTIONALIn prokaryotic organisms, the primary transcripts ofmRNA-encoding genes begin to serve as translationtemplates even before their transcription has been completed.This is because the site of transcription is notcompartmentalized into a nucleus as it is in eukaryoticorganisms. Thus, transcription and translation are coupledin prokaryotic cells. Consequently, prokaryoticmRNAs are subjected to little processing prior to carryingout their intended function in protein synthesis. Indeed,appropriate regulation of some genes (eg, the Trpoperon) relies upon this coupling of transcription andtranslation. Prokaryotic rRNA and tRNA molecules aretranscribed in units considerably longer than the ultimatemolecule. In fact, many of the tRNA transcriptionunits contain more than one molecule. Thus, inprokaryotes the processing of these rRNA and tRNAprecursor molecules is required for the generation ofthe mature functional molecules.Nearly all eukaryotic RNA primary transcripts undergoextensive processing between the time they aresynthesized and the time at which they serve their ultimatefunction, whether it be as mRNA or as a componentof the translation machinery such as rRNA,5S RNA, or tRNA or RNA processing machinery,snRNAs. Processing occurs primarily within the nucleusand includes nucleolytic cleavage to smaller moleculesand coupled nucleolytic and ligation reactions(splicing of exons). In mammalian cells, 50–75% ofthe nuclear RNA does not contribute to the cytoplasmicmRNA. This nuclear RNA loss is significantlygreater than can be reasonably accounted for by the lossof intervening sequences alone (see below). Thus, theexact function of the seemingly excessive transcripts inthe nucleus of a mammalian cell is not known.The Coding Portions (Exons)of Most Eukaryotic GenesAre Interrupted by IntronsInterspersed within the amino acid-coding portions(exons) of many genes are long sequences of DNA thatdo not contribute to the genetic information ultimatelytranslated into the amino acid sequence of a proteinmolecule (see Chapter 36). In fact, these sequences actuallyinterrupt the coding region of structural genes.These intervening sequences (introns) exist withinmost but not all mRNA encoding genes of higher eukaryotes.The primary transcripts of the structural genescontain RNA complementary to the interspersed sequences.However, the intron RNA sequences arecleaved out of the transcript, and the exons of the transcriptare appropriately spliced together in the nucleusbefore the resulting mRNA molecule appears in the cytoplasmfor translation (Figures 37–11 and 37–12).One speculation is that exons, which often encode anactivity domain of a protein, represent a convenientmeans of shuffling genetic information, permitting organismsto quickly test the results of combining novelprotein functional domains.Introns Are Removed & ExonsAre Spliced TogetherThe mechanisms whereby introns are removed fromthe primary transcript in the nucleus, exons are ligatedto form the mRNA molecule, and the mRNA moleculeis transported to the cytoplasm are being elucidated.Four different splicing reaction mechanisms have beendescribed. The one most frequently used in eukaryoticcells is described below. Although the sequences of nu-

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 353Exon 1Exon 2Intron5′ Cap G GA GA n 3′ Primary transcriptCapG GA GA nNucleophilic attackat 5′ end of intronGCapG OHA GA nLariat formationCapGOHGGA nCut at 3′ end of intronACapG—GA n andGALigation of 3′ end of exon1 to 5′ end of exon 2Intron is digestedFigure 37–11. The processing of the primary transcript to mRNA. In this hypotheticaltranscript, the 5′ (left) end of the intron is cut (↓) and a lariat formsbetween the G at the 5′ end of the intron and an A near the 3′ end, in the consensussequence UACUAAC. This sequence is called the branch site, and it is the3′ most A that forms the 5′–2’ bond with the G. The 3′ (right) end of the intron isthen cut (⇓). This releases the lariat, which is digested, and exon 1 is joined toexon 2 at G residues.cleotides in the introns of the various eukaryotic transcripts—andeven those within a single transcript—arequite heterogeneous, there are reasonably conserved sequencesat each of the two exon-intron (splice) junctionsand at the branch site, which is located 20–40 nucleotidesupstream from the 3′ splice site (see consensussequences in Figure 37–12). A special structure, thespliceosome, is involved in converting the primarytranscript into mRNA. Spliceosomes consist of the primarytranscript, five small nuclear RNAs (U1, U2, U5,U4, and U6) and more than 60 proteins. Collectively,these form a small nucleoprotein (snRNP) complex,sometimes called a “snurp.” It is likely that this pentasnRNPspliceosome forms prior to interaction withmRNA precursors. Snurps are thought to position theRNA segments for the necessary splicing reactions. Thesplicing reaction starts with a cut at the junction of the5′ exon (donor or left) and intron (Figure 37–11). ThisAConsensus sequences5′ AG G UAAGUUACUAAC 28-37 nucleotides C AG G3′C5′ Exon IntronExon 3′Figure 37–12. Consensus sequences at splice junctions. The 5′ (donor or left) and 3′ (acceptoror right) sequences are shown. Also shown is the yeast consensus sequence(UACUAAC) for the branch site. In mammalian cells, this consensus sequence is PyNPyPy-PuAPy, where Py is a pyrimidine, Pu is a purine, and N is any nucleotide. The branch site is located20–40 nucleotides upstream from the 3′ site.

354 / CHAPTER 37is accomplished by a nucleophilic attack by an adenylylresidue in the branch point sequence located just upstreamfrom the 3′ end of this intron. The free 5′ terminalthen forms a loop or lariat structure that is linkedby an unusual 5′–2′ phosphodiester bond to the reactiveA in the PyNPyPyPuAPy branch site sequence(Figure 37–12). This adenylyl residue is typically located28–37 nucleotides upstream from the 3′ end ofthe intron being removed. The branch site identifiesthe 3′ splice site. A second cut is made at the junctionof the intron with the 3′ exon (donor on right). In thissecond transesterification reaction, the 3′ hydroxyl ofthe upstream exon attacks the 5′ phosphate at thedownstream exon-intron boundary, and the lariatstructure containing the intron is released and hydrolyzed.The 5′ and 3′ exons are ligated to form a continuoussequence.The snRNAs and associated proteins are requiredfor formation of the various structures and intermediates.U1 within the snRNP complex binds first by basepairing to the 5′ exon-intron boundary. U2 within thesnRNP complex then binds by base pairing to thebranch site, and this exposes the nucleophilic A residue.U5/U4/U6 within the snRNP complex mediates anATP-dependent protein-mediated unwinding that resultsin disruption of the base-paired U4-U6 complexwith the release of U4. U6 is then able to interact firstwith U2, then with U1. These interactions serve to approximatethe 5′ splice site, the branch point with itsreactive A, and the 3′ splice site. This alignment is enhancedby U5. This process also results in the formationof the loop or lariat structure. The two ends arecleaved, probably by the U2-U6 within the snRNPcomplex. U6 is certainly essential, since yeasts deficientin this snRNA are not viable. It is important to notethat RNA serves as the catalytic agent. This sequence isthen repeated in genes containing multiple introns. Insuch cases, a definite pattern is followed for each gene,and the introns are not necessarily removed in sequence—1,then 2, then 3, etc.The relationship between hnRNA and the correspondingmature mRNA in eukaryotic cells is now apparent.The hnRNA molecules are the primary transcriptsplus their early processed products, which, afterthe addition of caps and poly(A) tails and removal ofthe portion corresponding to the introns, are transportedto the cytoplasm as mature mRNA molecules.Alternative Splicing Providesfor Different mRNAsThe processing of hnRNA molecules is a site for regulationof gene expression. Alternative patterns ofRNA splicing result from tissue-specific adaptive anddevelopmental control mechanisms. As mentionedabove, the sequence of exon-intron splicing events generallyfollows a hierarchical order for a given gene. Thefact that very complex RNA structures are formed duringsplicing—and that a number of snRNAs and proteinsare involved—affords numerous possibilities for achange of this order and for the generation of differentmRNAs. Similarly, the use of alternative terminationcleavage-polyadenylationsites also results in mRNAheterogeneity. Some schematic examples of theseprocesses, all of which occur in nature, are shown inFigure 37–13.Faulty splicing can cause disease. At least oneform of β-thalassemia, a disease in which the β-globingene of hemoglobin is severely underexpressed, appearsto result from a nucleotide change at an exon-intronjunction, precluding removal of the intron and thereforeleading to diminished or absent synthesis of theβ-chain protein. This is a consequence of the fact thatthe normal translation reading frame of the mRNA isdisrupted—a defect in this fundamental process (splicing)that underscores the accuracy which the process ofRNA-RNA splicing must achieve.Alternative Promoter UtilizationProvides a Form of RegulationTissue-specific regulation of gene expression can beprovided by control elements in the promoter or by themRNA precursor1Selective splicing1Alternative 5′ donor site1′Alternative 3′ acceptor site112 3 AAUAA AAUAA (A) n2 3 AAUAA AAUAA (A) n222′Alternative polyadenylation site3 AAUAA AAUAA (A) n3 AAUAA AAUAA (A) n3 AAUAA (A) nFigure 37–13. Mechanisms of alternative processingof mRNA precursors. This form of RNA processinginvolves the selective inclusion or exclusion of exons,the use of alternative 5′ donor or 3′ acceptor sites, andthe use of different polyadenylation sites.

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 355use of alternative promoters. The glucokinase (GK)gene consists of ten exons interrupted by nine introns.The sequence of exons 2–10 is identical in liver andpancreatic B cells, the primary tissues in which GK proteinis expressed. Expression of the GK gene is regulatedvery differently—by two different promoters—in thesetwo tissues. The liver promoter and exon 1L are locatednear exons 2–10; exon 1L is ligated directly to exon 2.In contrast, the pancreatic B cell promoter is locatedabout 30 kbp upstream. In this case, the 3′ boundary ofexon 1B is ligated to the 5′ boundary of exon 2. Theliver promoter and exon 1L are excluded and removedduring the splicing reaction (see Figure 37–14). The existenceof multiple distinct promoters allows for cellandtissue-specific expression patterns of a particulargene (mRNA).Both Ribosomal RNAs & MostTransfer RNAs Are ProcessedFrom Larger PrecursorsIn mammalian cells, the three rRNA molecules aretranscribed as part of a single large precursor molecule.The precursor is subsequently processed in the nucleolusto provide the RNA component for the ribosomesubunits found in the cytoplasm. The rRNAgenes are located in the nucleoli of mammalian cells.Hundreds of copies of these genes are present in everycell. This large number of genes is required to synthesizesufficient copies of each type of rRNA to form the10 7 ribosomes required for each cell replication.Whereas a single mRNA molecule may be copied into10 5 protein molecules, providing a large amplification,the rRNAs are end products. This lack of amplificationrequires a large number of genes. Similarly, transferRNAs are often synthesized as precursors, with extra sequencesboth 5′ and 3′ of the sequences comprising themature tRNA. A small fraction of tRNAs even containintrons.RNAS CAN BE EXTENSIVELY MODIFIEDEssentially all RNAs are covalently modified after transcription.It is clear that at least some of these modificationsare regulatory.Messenger RNA (mRNA) Is Modifiedat the 5 & 3 EndsAs mentioned above, mammalian mRNA moleculescontain a 7-methylguanosine cap structure at their 5′terminal, and most have a poly(A) tail at the 3′ terminal.The cap structure is added to the 5′ end of thenewly transcribed mRNA precursor in the nucleusprior to transport of the mRNA molecule to the cytoplasm.The 5 cap of the RNA transcript is requiredboth for efficient translation initiation and protectionof the 5′ end of mRNA from attack by 5′ →3′ exonucleases.The secondary methylations of mRNA molecules,those on the 2′-hydroxy and the N 6 of adenylylresidues, occur after the mRNA molecule has appearedin the cytoplasm.Poly(A) tails are added to the 3′ end of mRNA moleculesin a posttranscriptional processing step. ThemRNA is first cleaved about 20 nucleotides downstreamfrom an AAUAA recognition sequence. Anotherenzyme, poly(A) polymerase, adds a poly(A) tail whichis subsequently extended to as many as 200 A residues.The poly(A) tail appears to protect the 3′ end ofmRNA from 3′ →5′ exonuclease attack. The presenceor absence of the poly(A) tail does not determinewhether a precursor molecule in the nucleus appears inthe cytoplasm, because all poly(A)-tailed hnRNA moleculesdo not contribute to cytoplasmic mRNA, nor doall cytoplasmic mRNA molecules contain poly(A) tailsLiver(˜30 kb)1B 1L 2A 2 3 4 5 6 7 8 9 10B cell/pituitary(˜30 kb)1B 1L 2A 2 3 4 5 6 7 8 9 10Figure 37–14. Alternative promoter use in the liver and pancreatic B cell glucokinasegenes. Differential regulation of the glucokinase (GK) gene is accomplished by the use oftissue-specific promoters. The B cell GK gene promoter and exon 1B are located about30 kbp upstream from the liver promoter and exon 1L. Each promoter has a uniquestructure and is regulated differently. Exons 2–10 are identical in the two genes, and theGK proteins encoded by the liver and B cell mRNAs have identical kinetic properties.

356 / CHAPTER 37(the histones are most notable in this regard). Cytoplasmicenzymes in mammalian cells can both add and removeadenylyl residues from the poly(A) tails; thisprocess has been associated with an alteration of mRNAstability and translatability.The size of some cytoplasmic mRNA molecules,even after the poly(A) tail is removed, is still considerablygreater than the size required to code for the specificprotein for which it is a template, often by a factorof 2 or 3. The extra nucleotides occur in untranslated(non-protein coding) regions both 5′ and 3′ ofthe coding region; the longest untranslated sequencesare usually at the 3′ end. The exact function of these sequencesis unknown, but they have been implicated inRNA processing, transport, degradation, and translation;each of these reactions potentially contributes additionallevels of control of gene expression.RNA Editing Changes mRNAAfter TranscriptionThe central dogma states that for a given gene and geneproduct there is a linear relationship between the codingsequence in DNA, the mRNA sequence, and theprotein sequence (Figure 36–7). Changes in the DNAsequence should be reflected in a change in the mRNAsequence and, depending on codon usage, in protein sequence.However, exceptions to this dogma have beenrecently documented. Coding information can bechanged at the mRNA level by RNA editing. In suchcases, the coding sequence of the mRNA differs fromthat in the cognate DNA. An example is the apolipoproteinB (apoB) gene and mRNA. In liver, the singleapoB gene is transcribed into an mRNA that directs thesynthesis of a 100-kDa protein, apoB100. In the intestine,the same gene directs the synthesis of the primarytranscript; however, a cytidine deaminase converts aCAA codon in the mRNA to UAA at a single specificsite. Rather than encoding glutamine, this codon becomesa termination signal, and a 48-kDa protein(apoB48) is the result. ApoB100 and apoB48 have differentfunctions in the two organs. A growing numberof other examples include a glutamine to argininechange in the glutamate receptor and several changesin trypanosome mitochondrial mRNAs, generally involvingthe addition or deletion of uridine. The exactextent of RNA editing is unknown, but current estimatessuggest that < 0.01% of mRNAs are edited inthis fashion.Transfer RNA (tRNA) Is ExtensivelyProcessed & ModifiedAs described in Chapters 35 and 38, the tRNA moleculesserve as adapter molecules for the translation ofmRNA into protein sequences. The tRNAs containmany modifications of the standard bases A, U, G, andC, including methylation, reduction, deamination, andrearranged glycosidic bonds. Further modification ofthe tRNA molecules includes nucleotide alkylationsand the attachment of the characteristic CpCpAOH terminalat the 3′ end of the molecule by the enzyme nucleotidyltransferase. The 3′ OH of the A ribose is thepoint of attachment for the specific amino acid that isto enter into the polymerization reaction of proteinsynthesis. The methylation of mammalian tRNA precursorsprobably occurs in the nucleus, whereas thecleavage and attachment of CpCpAOH are cytoplasmicfunctions, since the terminals turn over more rapidlythan do the tRNA molecules themselves. Enzymeswithin the cytoplasm of mammalian cells are requiredfor the attachment of amino acids to the CpCpAOHresidues. (See Chapter 38.)RNA CAN ACT AS A CATALYSTIn addition to the catalytic action served by thesnRNAs in the formation of mRNA, several otherenzymatic functions have been attributed to RNA.Ribozymes are RNA molecules with catalytic activity.These generally involve transesterification reactions,and most are concerned with RNA metabolism (splicingand endoribonuclease). Recently, a ribosomal RNAcomponent was noted to hydrolyze an aminoacyl esterand thus to play a central role in peptide bond function(peptidyl transferases; see Chapter 38). These observations,made in organelles from plants, yeast, viruses,and higher eukaryotic cells, show that RNA can act asan enzyme. This has revolutionized thinking about enzymeaction and the origin of life itself.SUMMARY• RNA is synthesized from a DNA template by the enzymeRNA polymerase.• There are three distinct nuclear DNA-dependentRNA polymerases in mammals: RNA polymerases I,II, and III. These enzymes control the transcriptionalfunction—the transcription of rRNA, mRNA, andsmall RNA (tRNA/5S rRNA, snRNA) genes, respectively.• RNA polymerases interact with unique cis-active regionsof genes, termed promoters, in order to formpreinitiation complexes (PICs) capable of initiation.In eukaryotes the process of PIC formation is facilitatedby multiple general transcription factors(GTFs), TFIIA, B, D, E, F, and H.• Eukaryotic PIC formation can occur either stepwise—bythe sequential, ordered interactions of

RNA SYNTHESIS, PROCESSING, & MODIFICATION / 357GTFs and RNA polymerase with promoters—or inone step by the recognition of the promoter by a preformedGTF-RNA polymerase holoenzyme complex.• Transcription exhibits three phases: initiation, elongation,and termination. All are dependent upon distinctDNA cis-elements and can be modulated bydistinct trans-acting protein factors.• Most eukaryotic RNAs are synthesized as precursorsthat contain excess sequences which are removedprior to the generation of mature, functional RNA.• Eukaryotic mRNA synthesis results in a pre-mRNAprecursor that contains extensive amounts of excessRNA (introns) that must be precisely removed byRNA splicing to generate functional, translatablemRNA composed of exonic coding and noncodingsequences.• All steps—from changes in DNA template, sequence,and accessibility in chromatin to RNA stability—aresubject to modulation and hence are potential controlsites for eukaryotic gene regulation.REFERENCESBusby S, Ebright RH: Promoter structure, promoter recognition,and transcription activation in prokaryotes. Cell 1994;79:743.Cramer P, Bushnell DA, Kornberg R: Structural basis of transcription:RNA polymerase II at 2.8 angstrom resolution. Science2001;292:1863.Fedor MJ: Ribozymes. Curr Biol 1998;8:R441.Gott JM, Emeson RB: Functions and mechanisms of RNA editing.Ann Rev Genet 2000;34:499.Hirose Y, Manley JL: RNA polymerase II and the integration ofnuclear events. Genes Dev 2000;14:1415.Keaveney M, Struhl K: Activator-mediated recruitment of theRNA polymerase machinery is the predominant mechanismfor transcriptional activation in yeast. Mol Cell 1998;1:917.Lemon B, Tjian R: Orchestrated response: a symphony of transcriptionfactors for gene control. Genes Dev 2000;14:2551.Maniatis T, Reed R: An extensive network of coupling among geneexpression machines. Nature 2002;416:499.Orphanides G, Reinberg D: A unified theory of gene expression.Cell 2002;108:439.Shatkin AJ, Manley JL: The ends of the affair: capping and polyadenylation.Nat Struct Biol 2000;7:838.Stevens SW et al: Composition and functional characterization ofthe yeast spliceosomal penta-snRNP. Mol Cell 2002;9:31.Tucker M, Parker R: Mechanisms and control of mRNA decappingin Saccharomyces cerevisiae. Ann Rev Biochem 2000;69:571.Woychik NA, Hampsey M: The RNA polymerase II machinery:structure illuminates function. Cell 2002;108:453.

Protein Synthesis & theGenetic Code 38Daryl K. Granner, MDBIOMEDICAL IMPORTANCEThe letters A, G, T, and C correspond to the nucleotidesfound in DNA. They are organized into threelettercode words called codons, and the collection ofthese codons makes up the genetic code. It was impossibleto understand protein synthesis—or to explainmutations—before the genetic code was elucidated.The code provides a foundation for explaining the wayin which protein defects may cause genetic disease andfor the diagnosis and perhaps the treatment of thesedisorders. In addition, the pathophysiology of manyviral infections is related to the ability of these agents todisrupt host cell protein synthesis. Many antibacterialagents are effective because they selectively disrupt proteinsynthesis in the invading bacterial cell but do notaffect protein synthesis in eukaryotic cells.GENETIC INFORMATION FLOWSFROM DNA TO RNA TO PROTEINThe genetic information within the nucleotide sequenceof DNA is transcribed in the nucleus into thespecific nucleotide sequence of an RNA molecule. Thesequence of nucleotides in the RNA transcript is complementaryto the nucleotide sequence of the templatestrand of its gene in accordance with the base-pairingrules. Several different classes of RNA combine to directthe synthesis of proteins.In prokaryotes there is a linear correspondence betweenthe gene, the messenger RNA (mRNA) transcribedfrom the gene, and the polypeptide product.The situation is more complicated in higher eukaryoticcells, in which the primary transcript is much largerthan the mature mRNA. The large mRNA precursorscontain coding regions (exons) that will form the maturemRNA and long intervening sequences (introns)that separate the exons. The hnRNA is processedwithin the nucleus, and the introns, which often makeup much more of this RNA than the exons, are removed.Exons are spliced together to form maturemRNA, which is transported to the cytoplasm, where itis translated into protein.The cell must possess the machinery necessary totranslate information accurately and efficiently fromthe nucleotide sequence of an mRNA into the sequenceof amino acids of the corresponding specific protein.Clarification of our understanding of this process,which is termed translation, awaited deciphering of thegenetic code. It was realized early that mRNA moleculesthemselves have no affinity for amino acids and,therefore, that the translation of the information in themRNA nucleotide sequence into the amino acid sequenceof a protein requires an intermediate adaptermolecule. This adapter molecule must recognize a specificnucleotide sequence on the one hand as well as aspecific amino acid on the other. With such an adaptermolecule, the cell can direct a specific amino acid intothe proper sequential position of a protein during itssynthesis as dictated by the nucleotide sequence of thespecific mRNA. In fact, the functional groups of theamino acids do not themselves actually come into contactwith the mRNA template.THE NUCLEOTIDE SEQUENCEOF AN mRNA MOLECULE CONSISTSOF A SERIES OF CODONS THAT SPECIFYTHE AMINO ACID SEQUENCE OF THEENCODED PROTEINTwenty different amino acids are required for the synthesisof the cellular complement of proteins; thus,there must be at least 20 distinct codons that make upthe genetic code. Since there are only four different nucleotidesin mRNA, each codon must consist of morethan a single purine or pyrimidine nucleotide. Codonsconsisting of two nucleotides each could provide foronly 16 (4 2 ) specific codons, whereas codons of threenucleotides could provide 64 (4 3 ) specific codons.It is now known that each codon consists of a sequenceof three nucleotides; ie, it is a triplet code(see Table 38–1). The deciphering of the genetic codedepended heavily on the chemical synthesis of nucleotidepolymers, particularly triplets in repeated sequence.358

PROTEIN SYNTHESIS & THE GENETIC CODE / 359Table 38–1. The genetic code (codonassignments in mammalian messenger RNA). 1First Second ThirdNucleotide Nucleotide NucleotideU C A GPhe Ser Tyr Cys UPhe Ser Tyr Cys CU Leu Ser Term Term 2 ALeu Ser Term Trp GLeu Pro His Arg ULeu Pro His Arg CC Leu Pro Gln Arg ALeu Pro Gln Arg GIle Thr Asn Ser UIle Thr Asn Ser CA Ile 2 Thr Lys Arg 2 AMet Thr Lys Arg 2 GVal Ala Asp Gly UVal Ala Asp Gly CG Val Ala Glu Gly AVal Ala Glu Gly G1 The terms first, second, and third nucleotide refer to the individualnucleotides of a triplet codon. U, uridine nucleotide;C, cytosine nucleotide; A, adenine nucleotide; G, guanine nucleotide;Term, chain terminator codon. AUG, which codes forMet, serves as the initiator codon in mammalian cells and encodesfor internal methionines in a protein. (Abbreviations ofamino acids are explained in Chapter 3.)2 In mammalian mitochondria, AUA codes for Met and UGA forTrp, and AGA and AGG serve as chain terminators.THE GENETIC CODE IS DEGENERATE,UNAMBIGUOUS, NONOVERLAPPING,WITHOUT PUNCTUATION, & UNIVERSALThree of the 64 possible codons do not code for specificamino acids; these have been termed nonsense codons.These nonsense codons are utilized in the cell as terminationsignals; they specify where the polymerizationof amino acids into a protein molecule is to stop. Theremaining 61 codons code for 20 amino acids (Table38–1). Thus, there must be “degeneracy” in the geneticcode—ie, multiple codons must decode the sameamino acid. Some amino acids are encoded by severalcodons; for example, six different codons specify serine.Other amino acids, such as methionine and tryptophan,have a single codon. In general, the third nucleotidein a codon is less important than the first twoin determining the specific amino acid to be incorporated,and this accounts for most of the degeneracy ofthe code. However, for any specific codon, only a singleamino acid is indicated; with rare exceptions, the geneticcode is unambiguous—ie, given a specific codon,only a single amino acid is indicated. The distinctionbetween ambiguity and degeneracy is an importantconcept.The unambiguous but degenerate code can be explainedin molecular terms. The recognition of specificcodons in the mRNA by the tRNA adapter molecules isdependent upon their anticodon region and specificbase-pairing rules. Each tRNA molecule contains a specificsequence, complementary to a codon, which istermed its anticodon. For a given codon in the mRNA,only a single species of tRNA molecule possesses theproper anticodon. Since each tRNA molecule can becharged with only one specific amino acid, each codontherefore specifies only one amino acid. However, sometRNA molecules can utilize the anticodon to recognizemore than one codon. With few exceptions, given aspecific codon, only a specific amino acid will be incorporated—although,given a specific amino acid,more than one codon may be used.As discussed below, the reading of the genetic codeduring the process of protein synthesis does not involveany overlap of codons. Thus, the genetic code isnonoverlapping. Furthermore, once the reading iscommenced at a specific codon, there is no punctuationbetween codons, and the message is read in a continuingsequence of nucleotide triplets until a translationstop codon is reached.Until recently, the genetic code was thought to beuniversal. It has now been shown that the set of tRNAmolecules in mitochondria (which contain their ownseparate and distinct set of translation machinery) fromlower and higher eukaryotes, including humans, readsfour codons differently from the tRNA molecules inthe cytoplasm of even the same cells. As noted in Table38–1, the codon AUA is read as Met, and UGA codesfor Trp in mammalian mitochondria. In addition, inmitochondria, the codons AGA and AGG are read asstop or chain terminator codons rather than as Arg. Asa result, mitochondria require only 22 tRNA moleculesto read their genetic code, whereas the cytoplasmictranslation system possesses a full complement of 31tRNA species. These exceptions noted, the geneticcode is universal. The frequency of use of each aminoacid codon varies considerably between species andamong different tissues within a species. The specifictRNA levels generally mirror these codon usage biases.Thus, a particular abundantly used codon is decodedby a similarly abundant specific tRNA which recognizesthat particular codon. Tables of codon usage are becomingmore accurate as more genes are sequenced.This is of considerable importance because investigators

360 / CHAPTER 38Table 38–2. Features of the genetic code.• Degenerate• Unambiguous• Nonoverlapping• Not punctuated• Universaloften need to deduce mRNA structure from the primarysequence of a portion of protein in order to synthesizean oligonucleotide probe and initiate a recombinantDNA cloning project. The main features of thegenetic code are listed in Table 38–2.AT LEAST ONE SPECIES OF TRANSFERRNA (tRNA) EXISTS FOR EACH OF THE20 AMINO ACIDStRNA molecules have extraordinarily similar functionsand three-dimensional structures. The adapter functionof the tRNA molecules requires the charging of eachspecific tRNA with its specific amino acid. Since thereis no affinity of nucleic acids for specific functionalgroups of amino acids, this recognition must be carriedout by a protein molecule capable of recognizing both aspecific tRNA molecule and a specific amino acid. Atleast 20 specific enzymes are required for these specificrecognition functions and for the proper attachment ofthe 20 amino acids to specific tRNA molecules. Theprocess of recognition and attachment (charging)proceeds in two steps by one enzyme for each of the 20amino acids. These enzymes are termed aminoacyltRNAsynthetases. They form an activated intermediateof aminoacyl-AMP-enzyme complex (Figure 38–1).The specific aminoacyl-AMP-enzyme complex thenrecognizes a specific tRNA to which it attaches theaminoacyl moiety at the 3′-hydroxyl adenosyl terminal.The charging reactions have an error rate of less than10 −4 and so are extremely accurate. The amino acid remainsattached to its specific tRNA in an ester linkageuntil it is polymerized at a specific position in the fabricationof a polypeptide precursor of a protein molecule.The regions of the tRNA molecule referred to inChapter 35 (and illustrated in Figure 35–11) now becomeimportant. The thymidine-pseudouridine-cytidine(TΨC) arm is involved in binding of the aminoacyl-tRNAto the ribosomal surface at the site ofprotein synthesis. The D arm is one of the sites importantfor the proper recognition of a given tRNA speciesby its proper aminoacyl-tRNA synthetase. The acceptorarm, located at the 3′-hydroxyl adenosyl terminal, is thesite of attachment of the specific amino acid.The anticodon region consists of seven nucleotides,and it recognizes the three-letter codon in mRNA (Figure38–2). The sequence read from the 3′ to 5′ directionin that anticodon loop consists of a variablebase–modified purine–XYZ–pyrimidine–pyrimidine-5′. Note that this direction of reading the anticodon is3′ to 5′, whereas the genetic code in Table 38–1 is read5′ to 3′, since the codon and the anticodon loop of themRNA and tRNA molecules, respectively, are antiparallelin their complementarity just like all other intermolecularinteractions between nucleic acid strands.The degeneracy of the genetic code resides mostly inthe last nucleotide of the codon triplet, suggesting thatthe base pairing between this last nucleotide and thecorresponding nucleotide of the anticodon is not strictlyATPPP iAMP + EnzOOHOOCHCREnz • Adenosine O P O C CH RH 2 NOHNH 2Amino acid (aa)Enzyme (Enz)AMINOACYLtRNASYNTHETASEEnz•AMP-aa(Activated amino acid)Aminoacyl-AMP-enzymecomplextRNAtRNA-aaAminoacyl-tRNAFigure 38–1. Formation of aminoacyl-tRNA. A two-step reaction, involving the enzymeaminoacyl-tRNA synthetase, results in the formation of aminoacyl-tRNA. The first reaction involvesthe formation of an AMP-amino acid-enzyme complex. This activated amino acid is nexttransferred to the corresponding tRNA molecule. The AMP and enzyme are released, and the lattercan be reutilized. The charging reactions have an error rate of less than 10 –4 and so are extremelyaccurate.

PROTEIN SYNTHESIS & THE GENETIC CODE / 361mRNA5′TψCarmPhenylalanyltRNAN• Pu * •CodonU • U • UAnticodonA • A • A• Py•Anticodon Pyarm3′C•C•A5′Acceptor armPheDarmFigure 38–2. Recognition of the codon by the anticodon.One of the codons for phenylalanine is UUU.tRNA charged with phenylalanine (Phe) has the complementarysequence AAA; hence, it forms a base-paircomplex with the codon. The anticodon region typicallyconsists of a sequence of seven nucleotides: variable(N), modified purine ((Pu*), X, Y, Z, and two pyrimidines(Py) in the 3′ to 5′ direction.by the Watson-Crick rule. This is called wobble; thepairing of the codon and anticodon can “wobble” at thisspecific nucleotide-to-nucleotide pairing site. For example,the two codons for arginine, AGA and AGG, canbind to the same anticodon having a uracil at its 5′ end(UCU). Similarly, three codons for glycine—GGU,GGC, and GGA—can form a base pair from one anticodon,CCI. I is an inosine nucleotide, another of thepeculiar bases appearing in tRNA molecules.MUTATIONS RESULT WHEN CHANGESOCCUR IN THE NUCLEOTIDE SEQUENCEAlthough the initial change may not occur in the templatestrand of the double-stranded DNA molecule forthat gene, after replication, daughter DNA moleculeswith mutations in the template strand will segregateand appear in the population of organisms.Some Mutations Occurby Base SubstitutionSingle-base changes (point mutations) may be transitionsor transversions. In the former, a given pyrimidineis changed to the other pyrimidine or a given3′purine is changed to the other purine. Transversions arechanges from a purine to either of the two pyrimidinesor the change of a pyrimidine into either of the twopurines, as shown in Figure 38–3.If the nucleotide sequence of the gene containingthe mutation is transcribed into an RNA molecule,then the RNA molecule will possess a complementarybase change at this corresponding locus.Single-base changes in the mRNA molecules mayhave one of several effects when translated into protein:(1) There may be no detectable effect because of thedegeneracy of the code. This would be more likely ifthe changed base in the mRNA molecule were to be atthe third nucleotide of a codon; such mutations areoften referred to as silent mutations. Because of wobble,the translation of a codon is least sensitive to achange at the third position.(2) A missense effect will occur when a differentamino acid is incorporated at the corresponding site inthe protein molecule. This mistaken amino acid—ormissense, depending upon its location in the specificprotein—might be acceptable, partially acceptable, orunacceptable to the function of that protein molecule.From a careful examination of the genetic code, onecan conclude that most single-base changes would resultin the replacement of one amino acid by anotherwith rather similar functional groups. This is an effectivemechanism to avoid drastic change in the physicalproperties of a protein molecule. If an acceptable missenseeffect occurs, the resulting protein molecule maynot be distinguishable from the normal one. A partiallyacceptable missense will result in a protein moleculewith partial but abnormal function. If an unacceptablemissense effect occurs, then the protein molecule willnot be capable of functioning in its assigned role.(3) A nonsense codon may appear that would thenresult in the premature termination of amino acid incorporationinto a peptide chain and the production ofonly a fragment of the intended protein molecule. Theprobability is high that a prematurely terminated proteinmolecule or peptide fragment will not function inits assigned role.TATransitionsCGTCAGAGTransversionsFigure 38–3. Diagrammatic representation of transitionmutations and transversion mutations.TC

362 / CHAPTER 38Hemoglobin Illustrates the Effects ofSingle-Base Changes in Structural GenesSome mutations have no apparent effect. The genesystem that encodes hemoglobin is one of the beststudiedin humans. The lack of effect of a single-basechange is demonstrable only by sequencing the nucleotidesin the mRNA molecules or structural genes.The sequencing of a large number of hemoglobinmRNAs and genes from many individuals has shownthat the codon for valine at position 67 of the β chainof hemoglobin is not identical in all persons who possessa normally functional β chain of hemoglobin. HemoglobinMilwaukee has at position 67 a glutamicacid; hemoglobin Bristol contains aspartic acid at position67. In order to account for the amino acid changeby the change of a single nucleotide residue in thecodon for amino acid 67, one must infer that themRNA encoding hemoglobin Bristol possessed a GUUor GUC codon prior to a later change to GAU orGAC, both codons for aspartic acid. However, themRNA encoding hemoglobin Milwaukee would haveto possess at position 67 a codon GUA or GUG inorder that a single nucleotide change could provide forthe appearance of the glutamic acid codons GAA orGAG. Hemoglobin Sydney, which contains an alanineat position 67, could have arisen by the change of a singlenucleotide in any of the four codons for valine(GUU, GUC, GUA, or GUG) to the alanine codons(GCU, GCC, GCA, or GCG, respectively).Substitution of Amino Acids CausesMissense MutationsA. ACCEPTABLE MISSENSE MUTATIONSAn example of an acceptable missense mutation (Figure38–4, top) in the structural gene for the β chain of hemoglobincould be detected by the presence of an electrophoreticallyaltered hemoglobin in the red cells of anapparently healthy individual. Hemoglobin Hikari hasbeen found in at least two families of Japanese people.This hemoglobin has asparagine substituted for lysineat the 61 position in the β chain. The correspondingProtein molecule Amino acid CodonsHb A, β chain61 LysineAAAorAAGAcceptablemissenseHb Hikari, β chainAsparagineAAUorAACPartiallyacceptablemissenseHb A, β chain6 GlutamateGAAorGAGHb S, β chainValineGUAorGUGHb A, α chain58 HistidineCAUorCACUnacceptablemissenseHb M (Boston), α chainTyrosineUAUorUACFigure 38–4. Examples of three types of missense mutations resulting in abnormal hemoglobinchains. The amino acid alterations and possible alterations in the respective codons are indicated.The hemoglobin Hikari β-chain mutation has apparently normal physiologic propertiesbut is electrophoretically altered. Hemoglobin S has a β-chain mutation and partial function; hemoglobinS binds oxygen but precipitates when deoxygenated. Hemoglobin M Boston, anα-chain mutation, permits the oxidation of the heme ferrous iron to the ferric state and so willnot bind oxygen at all.

PROTEIN SYNTHESIS & THE GENETIC CODE / 363transversion might be either AAA or AAG changed toeither AAU or AAC. The replacement of the specific lysinewith asparagine apparently does not alter the normalfunction of the β chain in these individuals.B. PARTIALLY ACCEPTABLE MISSENSE MUTATIONSA partially acceptable missense mutation (Figure 38–4,center) is best exemplified by hemoglobin S, which isfound in sickle cell anemia. Here glutamic acid, thenormal amino acid in position 6 of the β chain, hasbeen replaced by valine. The corresponding single nucleotidechange within the codon would be GAA orGAG of glutamic acid to GUA or GUG of valine.Clearly, this missense mutation hinders normal functionand results in sickle cell anemia when the mutantgene is present in the homozygous state. The glutamate-to-valinechange may be considered to be partiallyacceptable because hemoglobin S does bind and releaseoxygen, although abnormally.C. UNACCEPTABLE MISSENSE MUTATIONSAn unacceptable missense mutation (Figure 38–4, bottom)in a hemoglobin gene generates a nonfunctioninghemoglobin molecule. For example, the hemoglobin Mmutations generate molecules that allow the Fe 2+ of theheme moiety to be oxidized to Fe 3+ , producing methemoglobin.Methemoglobin cannot transport oxygen(see Chapter 6).Frameshift Mutations Result FromDeletion or Insertion of Nucleotides inDNA That Generates Altered mRNAsThe deletion of a single nucleotide from the codingstrand of a gene results in an altered reading frame inthe mRNA. The machinery translating the mRNA doesnot recognize that a base was missing, since there is nopunctuation in the reading of codons. Thus, a major alterationin the sequence of polymerized amino acids, asdepicted in example 1, Figure 38–5, results. Alteringthe reading frame results in a garbled translation of themRNA distal to the single nucleotide deletion. Notonly is the sequence of amino acids distal to this deletiongarbled, but reading of the message can also resultin the appearance of a nonsense codon and thus theproduction of a polypeptide both garbled and prematurelyterminated (example 3, Figure 38–5).If three nucleotides or a multiple of three are deletedfrom a coding region, the corresponding mRNA whentranslated will provide a protein from which is missingthe corresponding number of amino acids (example 2,Figure 38–5). Because the reading frame is a triplet, thereading phase will not be disturbed for those codonsdistal to the deletion. If, however, deletion of one ortwo nucleotides occurs just prior to or within the normaltermination codon (nonsense codon), the readingof the normal termination signal is disturbed. Such adeletion might result in reading through a terminationsignal until another nonsense codon is encountered (example1, Figure 38–5). Examples of this phenomenonare described in discussions of hemoglobinopathies.Insertions of one or two or nonmultiples of three nucleotidesinto a gene result in an mRNA in which thereading frame is distorted upon translation, and the sameeffects that occur with deletions are reflected in themRNA translation. This may result in garbled aminoacid sequences distal to the insertion and the generationof a nonsense codon at or distal to the insertion, or perhapsreading through the normal termination codon.Following a deletion in a gene, an insertion (or viceversa) can reestablish the proper reading frame (example4, Figure 38–5). The corresponding mRNA, whentranslated, would contain a garbled amino acid sequencebetween the insertion and deletion. Beyond the reestablishmentof the reading frame, the amino acid sequencewould be correct. One can imagine that different combinationsof deletions, of insertions, or of both wouldresult in formation of a protein wherein a portion is abnormal,but this portion is surrounded by the normalamino acid sequences. Such phenomena have beendemonstrated convincingly in a number of diseases.Suppressor Mutations Can CounteractSome of the Effects of Missense,Nonsense, & Frameshift MutationsThe above discussion of the altered protein products ofgene mutations is based on the presence of normallyfunctioning tRNA molecules. However, in prokaryoticand lower eukaryotic organisms, abnormally functioningtRNA molecules have been discovered that arethemselves the results of mutations. Some of these abnormaltRNA molecules are capable of binding to anddecoding altered codons, thereby suppressing the effectsof mutations in distant structural genes. These suppressortRNA molecules, usually formed as the resultof alterations in their anticodon regions, are capable ofsuppressing missense mutations, nonsense mutations,and frameshift mutations. However, since the suppressortRNA molecules are not capable of distinguishingbetween a normal codon and one resulting from a genemutation, their presence in a cell usually results in decreasedviability. For instance, the nonsense suppressortRNA molecules can suppress the normal terminationsignals to allow a read-through when it is not desirable.Frameshift suppressor tRNA molecules may read a normalcodon plus a component of a juxtaposed codon toprovide a frameshift, also when it is not desirable. SuppressortRNA molecules may exist in mammalian cells,since read-through transcription occurs.

364 / CHAPTER 38NormalWild typemRNA 5'... UAG UUUG AUG GCC UCU UGC AAA GGC UAU AGU AGU UAG... 3'PolypeptideMet Ala Ser Cys Lys Gly Tyr Ser Ser STOPExample 1Deletion (–1)–1 UmRNA 5'... UAG UUUG AUG GCC CUU GCA AAG GCU AUA GUA GUU AG... 3'PolypeptideMet Ala Leu Ala Lys Ala Thr Val Val SerExample 2Deletion (– 3)–3 UGCGarbledmRNA 5'... UAG UUUG AUG GCC UCU AAA GGC UAU AGU AGU UAG... 3'PolypeptideMet Ala Ser Lys Gly Try Ser Ser STOPExample 3Insertion (+1)+1 CmRNA 5'... UAG UUUG AUG GCC CUC UUG CAA AGG CUA UAG UAG UUAG... 3'PolypeptideMet Ala Leu Leu Gln Arg Leu STOPExample 4Insertion (+1)Deletion (–1)Garbled+1 U –1 CmRNA 5'... UAG UUUG AUG GCC UCU UUG CAA AGG UAU AGU AGU UAG... 3'PolypeptideMet Ala Ser Leu Gln Arg Tyr Ser Ser STOPGarbledFigure 38–5. Examples of the effects of deletions and insertions in a gene on the sequence of the mRNAtranscript and of the polypeptide chain translated therefrom. The arrows indicate the sites of deletions or insertions,and the numbers in the ovals indicate the number of nucleotide residues deleted or inserted. Blue typeindicates amino acids in correct order.LIKE TRANSCRIPTION, PROTEINSYNTHESIS CAN BE DESCRIBEDIN THREE PHASES: INITIATION,ELONGATION, & TERMINATIONThe general structural characteristics of ribosomes andtheir self-assembly process are discussed in Chapter 37.These particulate entities serve as the machinery onwhich the mRNA nucleotide sequence is translated intothe sequence of amino acids of the specified protein.The translation of the mRNA commences near its 5′terminal with the formation of the correspondingamino terminal of the protein molecule. The message isread from 5′ to 3′, concluding with the formation ofthe carboxyl terminal of the protein. Again, the conceptof polarity is apparent. As described in Chapter 37, thetranscription of a gene into the corresponding mRNAor its precursor first forms the 5′ terminal of the RNAmolecule. In prokaryotes, this allows for the beginningof mRNA translation before the transcription of thegene is completed. In eukaryotic organisms, the process

PROTEIN SYNTHESIS & THE GENETIC CODE / 365of transcription is a nuclear one; mRNA translation occursin the cytoplasm. This precludes simultaneoustranscription and translation in eukaryotic organismsand makes possible the processing necessary to generatemature mRNA from the primary transcript—hnRNA.Initiation Involves Several Protein-RNAComplexes (Figure 38–6)Initiation of protein synthesis requires that an mRNAmolecule be selected for translation by a ribosome.Once the mRNA binds to the ribosome, the latter findsthe correct reading frame on the mRNA, and translationbegins. This process involves tRNA, rRNA,mRNA, and at least ten eukaryotic initiation factors(eIFs), some of which have multiple (three to eight)subunits. Also involved are GTP, ATP, and aminoacids. Initiation can be divided into four steps: (1) dissociationof the ribosome into its 40S and 60S subunits;(2) binding of a ternary complex consisting ofmet-tRNA i , GTP, and eIF-2 to the 40S ribosome toform a preinitiation complex; (3) binding of mRNA tothe 40S preinitiation complex to form a 43S initiationcomplex; and (4) combination of the 43S initiationcomplex with the 60S ribosomal subunit to form the80S initiation complex.A. RIBOSOMAL DISSOCIATIONTwo initiation factors, eIF-3 and eIF-1A, bind to thenewly dissociated 40S ribosomal subunit. This delaysits reassociation with the 60S subunit and allows othertranslation initiation factors to associate with the 40Ssubunit.B. FORMATION OF THE 43S PREINITIATION COMPLEXThe first step in this process involves the binding ofGTP by eIF-2. This binary complex then binds to mettRNAi , a tRNA specifically involved in binding to theinitiation codon AUG. (There are two tRNAs for methionine.One specifies methionine for the initiatorcodon, the other for internal methionines. Each has aunique nucleotide sequence.) This ternary complexbinds to the 40S ribosomal subunit to form the 43Spreinitiation complex, which is stabilized by associationwith eIF-3 and eIF-1A.eIF-2 is one of two control points for protein synthesisinitiation in eukaryotic cells. eIF-2 consists ofα, β, and γ subunits. eIF-2α is phosphorylated (onserine 51) by at least four different protein kinases(HCR, PKR, PERK, and GCN2) that are activatedwhen a cell is under stress and when the energy expenditurerequired for protein synthesis would be deleterious.Such conditions include amino acid and glucosestarvation, virus infection, misfolded proteins, serumdeprivation, hyperosmolality, and heat shock. PKR isparticularly interesting in this regard. This kinase is activatedby viruses and provides a host defense mechanismthat decreases protein synthesis, thereby inhibitingviral replication. Phosphorylated eIF-2α bindstightly to and inactivates the GTP-GDP recycling proteineIF-2B. This prevents formation of the 43S preinitiationcomplex and blocks protein synthesis.C. FORMATION OF THE 43S INITIATION COMPLEXThe 5′ terminals of most mRNA molecules in eukaryoticcells are “capped,” as described in Chapter 37. Thismethyl-guanosyl triphosphate cap facilitates the bindingof mRNA to the 43S preinitiation complex. A capbinding protein complex, eIF-4F (4F), which consistsof eIF-4E and the eIF-4G (4G)-eIF4A (4A) complex,binds to the cap through the 4E protein. Then eIF-4A(4A) and eIF-4B (4B) bind and reduce the complex secondarystructure of the 5′ end of the mRNA throughATPase and ATP-dependent helicase activities. The associationof mRNA with the 43S preinitiation complexto form the 48S initiation complex requires ATP hydrolysis.eIF-3 is a key protein because it binds withhigh affinity to the 4G component of 4F, and it linksthis complex to the 40S ribosomal subunit. Followingassociation of the 43S preinitiation complex with themRNA cap and reduction (“melting”) of the secondarystructure near the 5′ end of the mRNA, the complexscans the mRNA for a suitable initiation codon. Generallythis is the 5′-most AUG, but the precise initiationcodon is determined by so-called Kozak consensus sequencesthat surround the AUG:− 3 −1Most preferred is the presence of a purine at positions−3 and +4 relative to the AUG.D. ROLE OF THE POLY(A) TAIL IN INITIATIONBiochemical and genetic experiments in yeast have revealedthat the 3′ poly(A) tail and its binding protein,Pab1p, are required for efficient initiation of proteinsynthesis. Further studies showed that the poly(A) tailstimulates recruitment of the 40S ribosomal subunit tothe mRNA through a complex set of interactions.Pab1p, bound to the poly(A) tail, interacts with eIF-4G,which in turn binds to eIF-4E that is bound to the capstructure. It is possible that a circular structure isformed and that this helps direct the 40S ribosomalsubunit to the 5′ end of the mRNA. This helps explainhow the cap and poly(A) tail structures have a synergisticeffect on protein synthesis. It appears that a similarmechanism is at work in mammalian cells.+4GCCA / GCCAUGG

Ternary complexformationFormation of the 80Sinitiation complexActivation of mRNA80S31ARibosomaldissociation60SeIF-2CMet240S3 1ACapAUG(A) n2MetTernarycomplexATP4F = 4E + 4G 4A2B31A243S Preinitiationcomplex4FCapATPAUG4A 4B(A) nMetADP + P i4FCap4A4BAUG(A) n2B24FCapAUG(A) nGDP31A2GTPMetATP2B2ADP + P iCap3AUG1A 2(A) n48S Initiationcomplex2BMet2+ P i +1A + 3eIF-5Cap(A) nP siteA siteMet80S InitiationcomplexElongationFigure 38–6. Diagrammatic representation of the initiation of protein synthesis on the mRNA template containinga 5′ cap (G m TP-5′) and 3′ poly(A) terminal [3′(A) n ]. This process proceeds in three steps: (1) activation of mRNA;(2) formation of the ternary complex consisting of tRNAmet i , initiation factor eIF-2, and GTP; and (3) formation of theactive 80S initiation complex. (See text for details.) GTP, •; GDP, . The various initiation factors appear in abbreviatedform as circles or squares, eg, eIF-3 (3 ), eIF-4F ( 4F ). 4•F is a complex consisting of 4E and 4A bound to 4G (seeFigure 38–7). The constellation of protein factors and the 40S ribosomal subunit comprise the 43S preinitiation complex.When bound to mRNA, this forms the 48S preinitiation complex.

PROTEIN SYNTHESIS AND THE GENETIC CODE / 367E. FORMATION OF THE 80S INITIATION COMPLEXThe binding of the 60S ribosomal subunit to the 48Sinitiation complex involves hydrolysis of the GTPbound to eIF-2 by eIF-5. This reaction results in releaseof the initiation factors bound to the 48S initiationcomplex (these factors then are recycled) and the rapidassociation of the 40S and 60S subunits to form the80S ribosome. At this point, the met-tRNA i is on the Psite of the ribosome, ready for the elongation cycle tocommence.The Regulation of eIF-4E Controlsthe Rate of InitiationThe 4F complex is particularly important in controllingthe rate of protein translation. As described above, 4F isa complex consisting of 4E, which binds to the m 7 Gcap structure at the 5′ end of the mRNA, and 4G,which serves as a scaffolding protein. In addition tobinding 4E, 4G binds to eIF-3, which links the complexto the 40S ribosomal subunit. It also binds 4A and4B, the ATPase-helicase complex that helps unwind theRNA (Figure 38–7).4E is responsible for recognition of the mRNA capstructure, which is a rate-limiting step in translation.This process is regulated at two levels. Insulin and mitogenicgrowth factors result in the phosphorylation of4E on ser 209 (or thr 210). Phosphorylated 4E binds tothe cap much more avidly than does the nonphosphorylatedform, thus enhancing the rate of initiation. Acomponent of the MAP kinase pathway (see Figure43–8) appears to be involved in this phosphorylationreaction.The activity of 4E is regulated in a second way, andthis also involves phosphorylation. A recently discoveredset of proteins bind to and inactivate 4E. Theseproteins include 4E-BP1 (BP1, also known as PHAS-1)and the closely related proteins 4E-BP2 and 4E-BP3.BP1 binds with high affinity to 4E. The [4E]•[BP1] associationprevents 4E from binding to 4G (to form 4F).Since this interaction is essential for the binding of 4Fto the ribosomal 40S subunit and for correctly positioningthis on the capped mRNA, BP-1 effectively inhibitstranslation initiation.Insulin and other growth factors result in the phosphorylationof BP-1 at five unique sites. Phosphorylationof BP-1 results in its dissociation from 4E, and itcannot rebind until critical sites are dephosphorylated.The protein kinase responsible has not been identified,but it appears to be different from the one that phosphorylates4E. A kinase in the mammalian target ofrapamycin (mTOR) pathway, perhaps mTOR itself, isinvolved. These effects on the activation of 4E explainin part how insulin causes a marked posttranscriptional4E-BP 4E-BPeIF-4E4FCapInsulin(kinaseactivation)PO 4AUGeIF-4EPO 4eIF-4GeIF-4AeIF-4G eIF-4FcomplexeIF-4EeIF-4APO 4(A) nFigure 38–7. Activation of eIF-4E by insulin and formationof the cap binding eIF-4F complex. The 4F-capmRNA complex is depicted as in Figure 38–6. The 4Fcomplex consists of eIF-4E (4E), eIF-4A, and eIF-4G. 4E isinactive when bound by one of a family of binding proteins(4E-BPs). Insulin and mitogenic factors (eg, IGF-1,PDGF, interleukin-2, and angiotensin II) activate a serineprotein kinase in the mTOR pathway, and this results inthe phosphorylation of 4E-BP. Phosphorylated 4E-BPdissociates from 4E, and the latter is then able to formthe 4F complex and bind to the mRNA cap. Thesegrowth peptides also phosphorylate 4E itself by activatinga component of the MAP kinase pathway. Phosphorylated4E binds much more avidly to the cap thandoes nonphosphorylated 4E.increase of protein synthesis in liver, adipose tissue, andmuscle.Elongation Also Is a Multistep Process(Figure 38–8)Elongation is a cyclic process on the ribosome in whichone amino acid at a time is added to the nascent peptidechain. The peptide sequence is determined by the orderof the codons in the mRNA. Elongation involves severalsteps catalyzed by proteins called elongation factors (EFs).These steps are (1) binding of aminoacyl-tRNA to the Asite, (2) peptide bond formation, and (3) translocation.

368 / CHAPTER 38GTPG m TP—5′+P sitePeptidyltRNAm etnn-1n-2n+n+1GTP3′ (A) nA siteA. BINDING OF AMINOACYL-TRNA TO THE A SITEIn the complete 80S ribosome formed during theprocess of initiation, the A site (aminoacyl or acceptorsite) is free. The binding of the proper aminoacyltRNAin the A site requires proper codon recognition.Elongation factor EF1A forms a ternary complex withGTP and the entering aminoacyl-tRNA (Figure 38–8).This complex then allows the aminoacyl-tRNA to enterthe A site with the release of EF1A•GDP and phosphate.GTP hydrolysis is catalyzed by an active site onthe ribosome. As shown in Figure 38–8, EF1A-GDPthen recycles to EF1A-GTP with the aid of other solubleprotein factors and GTP.GDPEFIAGTPn+15′P i + GDP +EFIAE sitemetnn-1n-2nn+1n+1n+13′B. PEPTIDE BOND FORMATIONThe α-amino group of the new aminoacyl-tRNA in theA site carries out a nucleophilic attack on the esterifiedcarboxyl group of the peptidyl-tRNA occupying the Psite (peptidyl or polypeptide site). At initiation, this siteis occupied by aminoacyl-tRNA met i . This reaction iscatalyzed by a peptidyltransferase, a component of the28S RNA of the 60S ribosomal subunit. This is anotherexample of ribozyme activity and indicates an important—andpreviously unsuspected—direct role forRNA in protein synthesis (Table 38–3). Because theamino acid on the aminoacyl-tRNA is already “activated,”no further energy source is required for this reaction.The reaction results in attachment of the growingpeptide chain to the tRNA in the A site.5′GTP + +EF2G m TP—5′nm etnn-1n-2n+1n+1n+1nn+23′Codon3′ (A) nC. TRANSLOCATIONThe now deacylated tRNA is attached by its anticodonto the P site at one end and by the open CCA tail to anexit (E) site on the large ribosomal subunit (Figure38–8). At this point, elongation factor 2 (EF2) bindsto and displaces the peptidyl tRNA from the A site tothe P site. In turn, the deacylated tRNA is on the E site,from which it leaves the ribosome. The EF2-GTP complexis hydrolyzed to EF2-GDP, effectively moving themRNA forward by one codon and leaving the A siteopen for occupancy by another ternary complex ofamino acid tRNA-EF1A-GTP and another cycle ofelongation.P i + GDP + +EF2metn+1nn-1n-2Figure 38–8. Diagrammatic representation of the peptideelongation process of protein synthesis. The small circles labeledn − 1, n, n + 1, etc, represent the amino acid residues ofthe newly formed protein molecule. EFIA and EF2 representelongation factors 1 and 2, respectively. The peptidyl-tRNA andaminoacyl-tRNA sites on the ribosome are represented by P siteand A site, respectively.

PROTEIN SYNTHESIS & THE GENETIC CODE / 369Termination(stop)codonG m TP–5′3′ (A) nP siteA site+Releasing factor (RF1)C+GTPReleasing factor (RF3)N5′3′GTPCH 2 ONG m TP–5′3′ (A) n+ + + 60S + + GDP +N C40S P iPeptidetRNAFigure 38–9. Diagrammatic representation of the termination process of protein synthesis. The peptidyl-tRNA andaminoacyl-tRNA sites are indicated as P site and A site, respectively. The termination (stop) codon is indicated by thethree vertical bars. Releasing factor RF1 binds to the stop codon. Releasing factor RF3, with bound GTP, binds to RF1.Hydrolysis of the peptidyl-tRNA complex is shown by the entry of H 2 O. N and C indicate the amino and carboxyl terminalamino acids, respectively, and illustrate the polarity of protein synthesis.RF1RF3

370 / CHAPTER 38Table 38–3. Evidence that rRNAis peptidyltransferase.• Ribosomes can make peptide bonds even when proteinsare removed or inactivated.• Certain parts of the rRNA sequence are highly conserved inall species.• These conserved regions are on the surface of the RNAmolecule.• RNA can be catalytic.• Mutations that result in antibiotic resistance at the level ofprotein synthesis are more often found in rRNA than in theprotein components of the ribosome.The charging of the tRNA molecule with theaminoacyl moiety requires the hydrolysis of an ATP toan AMP, equivalent to the hydrolysis of two ATPs totwo ADPs and phosphates. The entry of the aminoacyltRNAinto the A site results in the hydrolysis of oneGTP to GDP. Translocation of the newly formed peptidyl-tRNAin the A site into the P site by EF2 similarlyresults in hydrolysis of GTP to GDP and phosphate.Thus, the energy requirements for the formation of onepeptide bond include the equivalent of the hydrolysis oftwo ATP molecules to ADP and of two GTP moleculesto GDP, or the hydrolysis of four high-energy phosphatebonds. A eukaryotic ribosome can incorporate asmany as six amino acids per second; prokaryotic ribosomesincorporate as many as 18 per second. Thus, theprocess of peptide synthesis occurs with great speed andaccuracy until a termination codon is reached.Termination Occurs When a StopCodon Is Recognized (Figure 38–9)In comparison to initiation and elongation, terminationis a relatively simple process. After multiple cyclesof elongation culminating in polymerization of the specificamino acids into a protein molecule, the stop orterminating codon of mRNA (UAA, UAG, UGA) appearsin the A site. Normally, there is no tRNA with ananticodon capable of recognizing such a terminationsignal. Releasing factor RF1 recognizes that a stopcodon resides in the A site (Figure 38–9). RF1 is boundby a complex consisting of releasing factor RF3 withbound GTP. This complex, with the peptidyl transferase,promotes hydrolysis of the bond between thepeptide and the tRNA occupying the P site. Thus, awater molecule rather than an amino acid is added.This hydrolysis releases the protein and the tRNA fromthe P site. Upon hydrolysis and release, the 80S ribosomedissociates into its 40S and 60S subunits, whichare then recycled. Therefore, the releasing factors areproteins that hydrolyze the peptidyl-tRNA bond whena stop codon occupies the A site. The mRNA is then releasedfrom the ribosome, which dissociates into itscomponent 40S and 60S subunits, and another cyclecan be repeated.Polysomes Are Assemblies of RibosomesMany ribosomes can translate the same mRNA moleculesimultaneously. Because of their relatively largesize, the ribosome particles cannot attach to an mRNAany closer than 35 nucleotides apart. Multiple ribosomeson the same mRNA molecule form a polyribosome,or “polysome.” In an unrestricted system, thenumber of ribosomes attached to an mRNA (and thusthe size of polyribosomes) correlates positively with thelength of the mRNA molecule. The mass of the mRNAmolecule is, of course, quite small compared with themass of even a single ribosome.A single mammalian ribosome is capable of synthesizingabout 400 peptide bonds each minute. Polyribosomesactively synthesizing proteins can exist as freeparticles in the cellular cytoplasm or may be attached tosheets of membranous cytoplasmic material referred toas endoplasmic reticulum. Attachment of the particulatepolyribosomes to the endoplasmic reticulum is responsiblefor its “rough” appearance as seen by electronmicroscopy. The proteins synthesized by the attachedpolyribosomes are extruded into the cisternal space betweenthe sheets of rough endoplasmic reticulum andare exported from there. Some of the protein productsof the rough endoplasmic reticulum are packaged bythe Golgi apparatus into zymogen particles for eventualexport (see Chapter 46). The polyribosomal particlesfree in the cytosol are responsible for the synthesis ofproteins required for intracellular functions.The Machinery of Protein Synthesis CanRespond to Environmental ThreatsFerritin, an iron-binding protein, prevents ionized iron(Fe 2+ ) from reaching toxic levels within cells. Elementaliron stimulates ferritin synthesis by causing the release ofa cytoplasmic protein that binds to a specific region inthe 5′ nontranslated region of ferritin mRNA. Disruptionof this protein-mRNA interaction activates ferritinmRNA and results in its translation. This mechanismprovides for rapid control of the synthesis of a proteinthat sequesters Fe 2+ , a potentially toxic molecule.Many Viruses Co-opt the Host CellProtein Synthesis MachineryThe protein synthesis machinery can also be modifiedin deleterious ways. Viruses replicate by using host

PROTEIN SYNTHESIS & THE GENETIC CODE / 371cell processes, including those involved in protein synthesis.Some viral mRNAs are translated much more efficientlythan those of the host cell (eg, encephalomyocarditisvirus). Others, such as reovirus and vesicularstomatitis virus, replicate abundantly, and their mRNAshave a competitive advantage over host cell mRNAs forlimited translation factors. Other viruses inhibit hostcell protein synthesis by preventing the association ofmRNA with the 40S ribosome.Poliovirus and other picornaviruses gain a selectiveadvantage by disrupting the function of the 4F complexto their advantage. The mRNAs of these viruses do nothave a cap structure to direct the binding of the 40S ribosomalsubunit (see above). Instead, the 40S ribosomalsubunit contacts an internal ribosomal entry site(IRES) in a reaction that requires 4G but not 4E. Thevirus gains a selective advantage by having a protease thatattacks 4G and removes the amino terminal 4E bindingsite. Now the 4E-4G complex (4F) cannot form, so the40S ribosomal subunit cannot be directed to cappedmRNAs. Host cell translation is thus abolished. The 4Gfragment can direct binding of the 40S ribosomal subunitto IRES-containing mRNAs, so viral mRNA translationis very efficient (Figure 38–10). These viruses alsopromote the dephosphorylation of BP1 (PHAS-1),thereby decreasing cap (4E)-dependent translation.4E4E4GPoliovirusprotease4G4ECapCap4E4G4GIRES4GIRESAUGAUGNilAUGAUGFigure 38–10. Picornaviruses disrupt the 4F complex.The 4E-4G complex (4F) directs the 40S ribosomalsubunit to the typical capped mRNA (see text). 4Galone is sufficient for targeting the 40S subunit to theinternal ribosomal entry site (IRES) of viral mRNAs. Togain selective advantage, certain viruses (eg, poliovirus)have a protease that cleaves the 4E binding site fromthe amino terminal end of 4G. This truncated 4G can directthe 40S ribosomal subunit to mRNAs that have anIRES but not to those that have a cap. The widths of thearrows indicate the rate of translation initiation fromthe AUG codon in each example.POSTTRANSLATIONAL PROCESSINGAFFECTS THE ACTIVITY OFMANY PROTEINSSome animal viruses, notably poliovirus and hepatitis Avirus, synthesize long polycistronic proteins from onelong mRNA molecule. These protein molecules aresubsequently cleaved at specific sites to provide the severalspecific proteins required for viral function. In animalcells, many proteins are synthesized from themRNA template as a precursor molecule, which thenmust be modified to achieve the active protein. Theprototype is insulin, which is a low-molecular-weightprotein having two polypeptide chains with interchainand intrachain disulfide bridges. The molecule is synthesizedas a single chain precursor, or prohormone,which folds to allow the disulfide bridges to form. Aspecific protease then clips out the segment that connectsthe two chains which form the functional insulinmolecule (see Figure 42–12).Many other peptides are synthesized as proproteinsthat require modifications before attaining biologic activity.Many of the posttranslational modifications involvethe removal of amino terminal amino acidresidues by specific aminopeptidases. Collagen, anabundant protein in the extracellular spaces of highereukaryotes, is synthesized as procollagen. Three procollagenpolypeptide molecules, frequently not identical insequence, align themselves in a particular way that isdependent upon the existence of specific amino terminalpeptides. Specific enzymes then carry out hydroxylationsand oxidations of specific amino acid residueswithin the procollagen molecules to provide cross-linksfor greater stability. Amino terminal peptides arecleaved off the molecule to form the final product—astrong, insoluble collagen molecule. Many other posttranslationalmodifications of proteins occur. Covalentmodification by acetylation, phosphorylation, methylation,ubiquitinylation, and glycosylation is common,for example.MANY ANTIBIOTICS WORK BECAUSETHEY SELECTIVELY INHIBIT PROTEINSYNTHESIS IN BACTERIARibosomes in bacteria and in the mitochondria ofhigher eukaryotic cells differ from the mammalian ribosomedescribed in Chapter 35. The bacterial ribosomeis smaller (70S rather than 80S) and has a different,somewhat simpler complement of RNA and protein

372 / CHAPTER 38molecules. This difference is exploited for clinical purposesbecause many effective antibiotics interact specificallywith the proteins and RNAs of prokaryotic ribosomesand thus inhibit protein synthesis. This results ingrowth arrest or death of the bacterium. The most usefulmembers of this class of antibiotics (eg, tetracyclines,lincomycin, erythromycin, and chloramphenicol)do not interact with components of eukaryoticribosomal particles and thus are not toxic to eukaryotes.Tetracycline prevents the binding of aminoacyl-tRNAsto the A site. Chloramphenicol and the macrolide classof antibiotics work by binding to 23S rRNA, which isinteresting in view of the newly appreciated role ofrRNA in peptide bond formation through its peptidyltransferaseactivity. It should be mentioned that theclose similarity between prokaryotic and mitochondrialribosomes can lead to complications in the use of someantibiotics.tRNANNHOCH 2 OH HH HNH OHO C CH CH 2OO –PON(CH 3 ) 2NNH 2OCH 3NNNO CH 2 OH HH HO OHO C CH CH 2NH 2NH 2Figure 38–11. The comparative structures of the antibioticpuromycin (top) and the 3′ terminal portion oftyrosinyl-tRNA (bottom).NNOHOther antibiotics inhibit protein synthesis on all ribosomes(puromycin) or only on those of eukaryoticcells (cycloheximide). Puromycin (Figure 38–11) is astructural analog of tyrosinyl-tRNA. Puromycin is incorporatedvia the A site on the ribosome into the carboxylterminal position of a peptide but causes the prematurerelease of the polypeptide. Puromycin, as atyrosinyl-tRNA analog, effectively inhibits protein synthesisin both prokaryotes and eukaryotes. Cycloheximideinhibits peptidyltransferase in the 60S ribosomalsubunit in eukaryotes, presumably by binding to anrRNA component.Diphtheria toxin, an exotoxin of Corynebacteriumdiphtheriae infected with a specific lysogenic phage, catalyzesthe ADP-ribosylation of EF-2 on the uniqueamino acid diphthamide in mammalian cells. Thismodification inactivates EF-2 and thereby specificallyinhibits mammalian protein synthesis. Many animals(eg, mice) are resistant to diphtheria toxin. This resistanceis due to inability of diphtheria toxin to cross thecell membrane rather than to insensitivity of mouseEF-2 to diphtheria toxin-catalyzed ADP-ribosylationby NAD.Ricin, an extremely toxic molecule isolated from thecastor bean, inactivates eukaryotic 28S ribosomal RNAby providing the N-glycolytic cleavage or removal of asingle adenine.Many of these compounds—puromycin and cycloheximidein particular—are not clinically useful buthave been important in elucidating the role of proteinsynthesis in the regulation of metabolic processes, particularlyenzyme induction by hormones.SUMMARY• The flow of genetic information follows the sequenceDNA → RNA → protein.• The genetic information in the structural region of agene is transcribed into an RNA molecule such thatthe sequence of the latter is complementary to that inthe DNA.• Several different types of RNA, including ribosomalRNA (rRNA), transfer RNA (tRNA), and messengerRNA (mRNA), are involved in protein synthesis.• The information in mRNA is in a tandem array ofcodons, each of which is three nucleotides long.• The mRNA is read continuously from a start codon(AUG) to a termination codon (UAA, UAG, UGA).• The open reading frame of the mRNA is the series ofcodons, each specifying a certain amino acid, that determinesthe precise amino acid sequence of the protein.• Protein synthesis, like DNA and RNA synthesis, followsa 5′ to 3′ polarity and can be divided into three

PROTEIN SYNTHESIS & THE GENETIC CODE / 373processes: initiation, elongation, and termination.Mutant proteins arise when single-base substitutionsresult in codons that specify a different amino acid ata given position, when a stop codon results in a truncatedprotein, or when base additions or deletionsalter the reading frame, so different codons are read.• A variety of compounds, including several antibiotics,inhibit protein synthesis by affecting one ormore of the steps involved in protein synthesis.REFERENCESCrick F et al: The genetic code. Nature 1961;192:1227.Green R, Noller HF: Ribosomes and translation. Annu RevBiochem 1997;66:679.Kozak M: Structural features in eukaryotic mRNAs that modulatethe initiation of translation. J Biol Chem 1991;266:1986.Lawrence JC, Abraham RT: PHAS/4E-BPs as regulators of mRNAtranslation and cell proliferation. Trends Biochem Sci1997;22:345.Sachs AB, Buratowski S: Common themes in translational andtranscriptional regulation. Trends Biochem Sci 1997;22:189.Sachs AB, Sarnow P, Hentze MW: Starting at the beginning, middleand end: translation initiation in eukaryotes. Cell 1997;98:831.Weatherall DJ et al: The hemoglobinopathies. In: The Metabolicand Molecular Bases of Inherited Disease, 8th ed. Scriver CR etal (editors). McGraw-Hill, 2001.

Regulation of Gene Expression 39Daryl K. Granner, MD, & P. Anthony Weil, PhDBIOMEDICAL IMPORTANCEOrganisms adapt to environmental changes by alteringgene expression. The process of alteration of gene expressionhas been studied in detail and often involvesmodulation of gene transcription. Control of transcriptionultimately results from changes in the interactionof specific binding regulatory proteins with various regionsof DNA in the controlled gene. This can have apositive or negative effect on transcription. Transcriptioncontrol can result in tissue-specific gene expression,and gene regulation is influenced by hormones,heavy metals, and chemicals. In addition to transcriptionlevel controls, gene expression can also be modulatedby gene amplification, gene rearrangement, posttranscriptionalmodifications, and RNA stabilization.Many of the mechanisms that control gene expressionare used to respond to hormones and therapeuticagents. Thus, a molecular understanding of theseprocesses will lead to development of agents that alterpathophysiologic mechanisms or inhibit the function orarrest the growth of pathogenic organisms.374REGULATED EXPRESSION OF GENESIS REQUIRED FOR DEVELOPMENT,DIFFERENTIATION, & ADAPTATIONThe genetic information present in each somatic cell ofa metazoan organism is practically identical. The exceptionsare found in those few cells that have amplified orrearranged genes in order to perform specialized cellularfunctions. Expression of the genetic information mustbe regulated during ontogeny and differentiation of theorganism and its cellular components. Furthermore, inorder for the organism to adapt to its environment andto conserve energy and nutrients, the expression ofgenetic information must be cued to extrinsic signalsand respond only when necessary. As organisms haveevolved, more sophisticated regulatory mechanismshave appeared which provide the organism and its cellswith the responsiveness necessary for survival in a complexenvironment. Mammalian cells possess about 1000times more genetic information than does the bacteriumEscherichia coli. Much of this additional geneticinformation is probably involved in regulation of geneexpression during the differentiation of tissues and biologicprocesses in the multicellular organism and in ensuringthat the organism can respond to complex environmentalchallenges.In simple terms, there are only two types of generegulation: positive regulation and negative regulation(Table 39–1). When the expression of genetic informationis quantitatively increased by the presence ofa specific regulatory element, regulation is said to bepositive; when the expression of genetic information isdiminished by the presence of a specific regulatory element,regulation is said to be negative. The element ormolecule mediating negative regulation is said to be anegative regulator or repressor; that mediating positiveregulation is a positive regulator or activator. However,a double negative has the effect of acting as a positive.Thus, an effector that inhibits the function of a negativeregulator will appear to bring about a positive regulation.Many regulated systems that appear to be inducedare in fact derepressed at the molecular level.(See Chapter 9 for explanation of these terms.)BIOLOGIC SYSTEMS EXHIBIT THREETYPES OF TEMPORAL RESPONSESTO A REGULATORY SIGNALFigure 39–1 depicts the extent or amount of gene expressionin three types of temporal response to an inducingsignal. A type A response is characterized by anincreased extent of gene expression that is dependentupon the continued presence of the inducing signal.When the inducing signal is removed, the amount ofgene expression diminishes to its basal level, but theamount repeatedly increases in response to the reappearanceof the specific signal. This type of response iscommonly observed in prokaryotes in response to suddenchanges of the intracellular concentration of a nutrient.It is also observed in many higher organismsafter exposure to inducers such as hormones, nutrients,or growth factors (Chapter 43).A type B response exhibits an increased amount ofgene expression that is transient even in the continuedpresence of the regulatory signal. After the regulatorysignal has terminated and the cell has been allowed torecover, a second transient response to a subsequentregulatory signal may be observed. This phenomenonof response-desensitization-recovery characterizes theaction of many pharmacologic agents, but it is also a

REGULATION OF GENE EXPRESSION / 375Table 39–1. Effects of positive and negativeregulation on gene expression.Rate of Gene ExpressionNegative PositiveRegulation RegulationRegulator present Decreased IncreasedRegulator absent Increased DecreasedGene expressionGene expressionGene expressionType ASignalType BSignalType CSignalRecoveryTimeTimeTimeFigure 39–1. Diagrammatic representations of theresponses of the extent of expression of a gene to specificregulatory signals such as a hormone.feature of many naturally occurring processes. This typeof response commonly occurs during development ofan organism, when only the transient appearance of aspecific gene product is required although the signalpersists.The type C response pattern exhibits, in responseto the regulatory signal, an increased extent of gene expressionthat persists indefinitely even after terminationof the signal. The signal acts as a trigger in this pattern.Once expression of the gene is initiated in the cell, itcannot be terminated even in the daughter cells; it istherefore an irreversible and inherited alteration. Thistype of response typically occurs during the developmentof differentiated function in a tissue or organ.Prokaryotes Provide Models for the Studyof Gene Expression in Mammalian CellsAnalysis of the regulation of gene expression inprokaryotic cells helped establish the principle that informationflows from the gene to a messenger RNA to aspecific protein molecule. These studies were aided bythe advanced genetic analyses that could be performedin prokaryotic and lower eukaryotic organisms. In recentyears, the principles established in these early studies,coupled with a variety of molecular biology techniques,have led to remarkable progress in the analysisof gene regulation in higher eukaryotic organisms, includingmammals. In this chapter, the initial discussionwill center on prokaryotic systems. The impressive geneticstudies will not be described, but the physiologyof gene expression will be discussed. However, nearlyall of the conclusions about this physiology have beenderived from genetic studies and confirmed by moleculargenetic and biochemical studies.Some Features of Prokaryotic GeneExpression Are UniqueBefore the physiology of gene expression can be explained,a few specialized genetic and regulatory termsmust be defined for prokaryotic systems. In prokaryotes,the genes involved in a metabolic pathway areoften present in a linear array called an operon, eg, thelac operon. An operon can be regulated by a single promoteror regulatory region. The cistron is the smallestunit of genetic expression. As described in Chapter 9,some enzymes and other protein molecules are composedof two or more nonidentical subunits. Thus, the“one gene, one enzyme” concept is not necessarilyvalid. The cistron is the genetic unit coding for thestructure of the subunit of a protein molecule, acting asit does as the smallest unit of genetic expression. Thus,the one gene, one enzyme idea might more accurately

376 / CHAPTER 39be regarded as a one cistron, one subunit concept. Asingle mRNA that encodes more than one separatelytranslated protein is referred to as a polycistronicmRNA. For example, the polycistronic lac operonmRNA is translated into three separate proteins (seebelow). Operons and polycistronic mRNAs are commonin bacteria but not in eukaryotes.An inducible gene is one whose expression increasesin response to an inducer or activator, a specific positiveregulatory signal. In general, inducible genes haverelatively low basal rates of transcription. By contrast,genes with high basal rates of transcription are oftensubject to down-regulation by repressors.The expression of some genes is constitutive, meaningthat they are expressed at a reasonably constant rateand not known to be subject to regulation. These areoften referred to as housekeeping genes. As a result ofmutation, some inducible gene products become constitutivelyexpressed. A mutation resulting in constitutiveexpression of what was formerly a regulated gene iscalled a constitutive mutation.Analysis of Lactose Metabolism in E coliLed to the Operon HypothesisJacob and Monod in 1961 described their operonmodel in a classic paper. Their hypothesis was to alarge extent based on observations on the regulation oflactose metabolism by the intestinal bacterium E coli.The molecular mechanisms responsible for the regulationof the genes involved in the metabolism of lactoseare now among the best-understood in any organism.β-Galactosidase hydrolyzes the β-galactoside lactose togalactose and glucose. The structural gene for β-galactosidase(lacZ) is clustered with the genes responsiblefor the permeation of galactose into the cell (lacY) andfor thiogalactoside transacetylase (lacA). The structuralgenes for these three enzymes, along with the lac promoterand lac operator (a regulatory region), are physicallyassociated to constitute the lac operon as depictedin Figure 39–2. This genetic arrangement of the structuralgenes and their regulatory genes allows for coordinateexpression of the three enzymes concerned withlactose metabolism. Each of these linked genes is transcribedinto one large mRNA molecule that containsmultiple independent translation start (AUG) and stop(UAA) codons for each cistron. Thus, each protein istranslated separately, and they are not processed from asingle large precursor protein. This type of mRNA moleculeis called a polycistronic mRNA. PolycistronicmRNAs are predominantly found in prokaryotic organisms.It is now conventional to consider that a gene includesregulatory sequences as well as the region thatPromotersitelacIOperatorlacZlac operonlacYlacAFigure 39–2. The positional relationships of thestructural and regulatory genes of the lac operon. lacZencodes β-galactosidase, lacY encodes a permease, andlacA encodes a thiogalactoside transacetylase. lacI encodesthe lac operon repressor protein.encodes the primary transcript. Although there aremany historical exceptions, a gene is generally italicizedin lower case and the encoded protein, when abbreviated,is expressed in roman type with the first letter capitalized.For example, the gene lacI encodes the repressorprotein LacI. When E coli is presented with lactoseor some specific lactose analogs under appropriate nonrepressingconditions (eg, high concentrations of lactose,no or very low glucose in media; see below), theexpression of the activities of β-galactosidase, galactosidepermease, and thiogalactoside transacetylase is increased100-fold to 1000-fold. This is a type A response,as depicted in Figure 39–1. The kinetics ofinduction can be quite rapid; lac-specific mRNAs arefully induced within 5–6 minutes after addition of lactoseto a culture; β-galactosidase protein is maximalwithin 10 minutes. Under fully induced conditions,there can be up to 5000 β-galactosidase molecules percell, an amount about 1000 times greater than thebasal, uninduced level. Upon removal of the signal, ie,the inducer, the synthesis of these three enzymes declines.When E coli is exposed to both lactose and glucoseas sources of carbon, the organisms first metabolizethe glucose and then temporarily stop growing until thegenes of the lac operon become induced to provide theability to metabolize lactose as a usable energy source.Although lactose is present from the beginning of thebacterial growth phase, the cell does not induce thoseenzymes necessary for catabolism of lactose until theglucose has been exhausted. This phenomenon was firstthought to be attributable to repression of the lacoperon by some catabolite of glucose; hence, it wastermed catabolite repression. It is now known thatcatabolite repression is in fact mediated by a catabolitegene activator protein (CAP) in conjunction withcAMP (Figure 18–5). This protein is also referred to asthe cAMP regulatory protein (CRP). The expression ofmany inducible enzyme systems or operons in E coliand other prokaryotes is sensitive to catabolite repression,as discussed below.

REGULATION OF GENE EXPRESSION / 377AOperatorPromoterlacI gene lacZ gene lacY gene lacA geneNo induceror↑ RNApolymeraseRNA polymerasecannot transcribeoperator or distalgenes (Z,Y, A)InducerandglucoseRepressorsubunitsRepressor(tetramer)BCAP-cAMPlacI lacZ lacY lacAWith inducerandno glucoseInactiverepressorRNA polymerases transcribing genesmRNAInducersβ-GalactosidaseproteinPermeaseproteinTransacetylaseproteinFigure 39–3. The mechanism of repression and derepression of the lac operon. When either no inducer ispresent or inducer is present with glucose (A), the lacI gene products that are synthesized constitutively forma repressor tetramer molecule which binds at the operator locus to prevent the efficient initiation of transcriptionby RNA polymerase at the promoter locus and thus to prevent the subsequent transcription of the lacZ,lacY, and lacA structural genes. When inducer is present (B), the constitutively expressed lacI gene forms repressormolecules that are conformationally altered by the inducer and cannot efficiently bind to the operatorlocus (affinity of binding reduced > 1000-fold). In the presence of cAMP and its binding protein (CAP), the RNApolymerase can transcribe the structural genes lacZ, lacY, and lacA, and the polycistronic mRNA moleculeformed can be translated into the corresponding protein molecules β-galactosidase, permease, andtransacetylase, allowing for the catabolism of lactose.The physiology of induction of the lac operon iswell understood at the molecular level (Figure 39–3).Expression of the normal lacI gene of the lac operon isconstitutive; it is expressed at a constant rate, resultingin formation of the subunits of the lac repressor. Fouridentical subunits with molecular weights of 38,000 assembleinto a lac repressor molecule. The LacI repressorprotein molecule, the product of lacI, has a high affinity(K d about 10 −13 mol/L) for the operator locus. The operatorlocus is a region of double-stranded DNA 27base pairs long with a twofold rotational symmetry andan inverted palindrome (indicated by solid lines aboutthe dotted axis) in a region that is 21 base pairs long, asshown below::5′ − AAT TGTGAGC G GATAACAATT3′− TTA ACACTCG C CTATTGTTAA:The minimum effective size of an operator for LacIrepressor binding is 17 base pairs (boldface letters in the

378 / CHAPTER 39above sequence). At any one time, only two subunits ofthe repressors appear to bind to the operator, and withinthe 17-base-pair region at least one base of each basepair is involved in LacI recognition and binding. Thebinding occurs mostly in the major groove without interruptingthe base-paired, double-helical nature of theoperator DNA. The operator locus is between the promotersite, at which the DNA-dependent RNA polymeraseattaches to commence transcription, and the transcriptioninitiation site of the lacZ gene, the structuralgene for β-galactosidase (Figure 39–2). When attachedto the operator locus, the LacI repressor molecule preventstranscription of the operator locus as well as of thedistal structural genes, lacZ, lacY, and lacA. Thus, theLacI repressor molecule is a negative regulator; in itspresence (and in the absence of inducer; see below), expressionfrom the lacZ, lacY, and lacA genes is prevented.There are normally 20–40 repressor tetramermolecules in the cell, a concentration of tetramer sufficientto effect, at any given time, > 95% occupancy ofthe one lac operator element in a bacterium, thus ensuringlow (but not zero) basal lac operon gene transcriptionin the absence of inducing signals.A lactose analog that is capable of inducing the lacoperon while not itself serving as a substrate for β-galactosidaseis an example of a gratuitous inducer. An exampleis isopropylthiogalactoside (IPTG). The additionof lactose or of a gratuitous inducer such as IPTG tobacteria growing on a poorly utilized carbon source(such as succinate) results in prompt induction of thelac operon enzymes. Small amounts of the gratuitous induceror of lactose are able to enter the cell even in theabsence of permease. The LacI repressor molecules—both those attached to the operator loci and those free inthe cytosol—have a high affinity for the inducer. Bindingof the inducer to a repressor molecule attached tothe operator locus induces a conformational change inthe structure of the repressor and causes it to dissociatefrom the DNA because its affinity for the operator isnow 10 3 times lower (K d about 10 −9 mol/L) than that ofLacI in the absence of IPTG. If DNA-dependent RNApolymerase has already attached to the coding strand atthe promoter site, transcription will begin. The polymerasegenerates a polycistronic mRNA whose 5′ terminalis complementary to the template strand of the operator.In such a manner, an inducer derepresses the lacoperon and allows transcription of the structural genesfor β-galactosidase, galactoside permease, and thiogalactosidetransacetylase. Translation of the polycistronicmRNA can occur even before transcription is completed.Derepression of the lac operon allows the cell tosynthesize the enzymes necessary to catabolize lactose asan energy source. Based on the physiology just described,IPTG-induced expression of transfected plasmidsbearing the lac operator-promoter ligated to appropriatebioengineered constructs is commonly used to expressmammalian recombinant proteins in E coli.In order for the RNA polymerase to efficiently forma PIC at the promoter site, there must also be presentthe catabolite gene activator protein (CAP) to whichcAMP is bound. By an independent mechanism, thebacterium accumulates cAMP only when it is starvedfor a source of carbon. In the presence of glucose—orof glycerol in concentrations sufficient for growth—thebacteria will lack sufficient cAMP to bind to CAP becausethe glucose inhibits adenylyl cyclase, the enzymethat converts ATP to cAMP (see Chapter 42). Thus, inthe presence of glucose or glycerol, cAMP-saturatedCAP is lacking, so that the DNA-dependent RNApolymerase cannot initiate transcription of the lacoperon. In the presence of the CAP-cAMP complex,which binds to DNA just upstream of the promotersite, transcription then occurs (Figure 39–3). Studiesindicate that a region of CAP contacts the RNA polymeraseα subunit and facilitates binding of this enzyme tothe promoter. Thus, the CAP-cAMP regulator is actingas a positive regulator because its presence is requiredfor gene expression. The lac operon is therefore controlledby two distinct, ligand-modulated DNA bindingtrans factors; one that acts positively (cAMP-CRPcomplex) and one that acts negatively (LacI repressor).Maximal activity of the lac operon occurs when glucoselevels are low (high cAMP with CAP activation) andlactose is present (LacI is prevented from binding to theoperator).When the lacI gene has been mutated so that itsproduct, LacI, is not capable of binding to operatorDNA, the organism will exhibit constitutive expressionof the lac operon. In a contrary manner, an organismwith a lacI gene mutation that produces a LacI proteinwhich prevents the binding of an inducer to therepressor will remain repressed even in the presence ofthe inducer molecule, because the inducer cannot bindto the repressor on the operator locus in order to derepressthe operon. Similarly, bacteria harboring mutationsin their lac operator locus such that the operatorsequence will not bind a normal repressor moleculeconstitutively express the lac operon genes. Mechanismsof positive and negative regulation comparable to thosedescribed here for the lac system have been observed ineukaryotic cells (see below).The Genetic Switch of BacteriophageLambda () Provides a Paradigmfor Protein-DNA Interactionsin Eukaryotic CellsLike some eukaryotic viruses (eg, herpes simplex, HIV),some bacterial viruses can either reside in a dormantstate within the host chromosomes or can replicate

REGULATION OF GENE EXPRESSION / 379within the bacterium and eventually lead to lysis andkilling of the bacterial host. Some E coli harbor such a“temperate” virus, bacteriophage lambda (λ). Whenlambda infects an organism of that species it injects its45,000-bp, double-stranded, linear DNA genome intothe cell (Figure 39–4). Depending upon the nutritionalstate of the cell, the lambda DNA will either integrateinto the host genome (lysogenic pathway) and remaindormant until activated (see below), or it will commencereplicating until it has made about 100 copiesof complete, protein-packaged virus, at which point itcauses lysis of its host (lytic pathway). The newly generatedvirus particles can then infect other susceptiblehosts.When integrated into the host genome in its dormantstate, lambda will remain in that state until activatedby exposure of its lysogenic bacterial host toDNA-damaging agents. In response to such a noxiousstimulus, the dormant bacteriophage becomes “induced”and begins to transcribe and subsequently translatethose genes of its own genome which are necessaryfor its excision from the host chromosome, its DNAreplication, and the synthesis of its protein coat andlysis enzymes. This event acts like a trigger or type C(Figure 39–1) response; ie, once lambda has committeditself to induction, there is no turning back until thecell is lysed and the replicated bacteriophage released.This switch from a dormant or prophage state to alytic infection is well understood at the genetic andmolecular levels and will be described in detail here.The switching event in lambda is centered aroundan 80-bp region in its double-stranded DNA genomereferred to as the “right operator” (O R ) (Figure 39–5A).The right operator is flanked on its left side by thestructural gene for the lambda repressor protein, the cIprotein, and on its right side by the structural gene encodinganother regulatory protein called Cro. Whenlambda is in its prophage state—ie, integrated into thehost genome—the cI repressor gene is the only lambdagene cI protein that is expressed. When the bacteriophageis undergoing lytic growth, the cI repressor geneis not expressed, but the cro gene—as well as manyother genes in lambda—is expressed. That is, when therepressor gene is on, the cro gene is off, and whenthe cro gene is on, the repressor gene is off. As weshall see, these two genes regulate each other’s expressionand thus, ultimately, the decision between lyticand lysogenic growth of lambda. This decision betweenrepressor gene transcription and cro genetranscription is a paradigmatic example of a molecularswitch.The 80-bp λ right operator, O R , can be subdividedinto three discrete, evenly spaced, 17-bp cis-activeDNA elements that represent the binding sites for eitherof two bacteriophage λ regulatory proteins. Impor-45 10Ultravioletradiation9123LysogenicpathwayInductionLyticpathwayFigure 39–4. Infection of the bacterium E coli byphage lambda begins when a virus particle attaches itselfto the bacterial cell (1) and injects its DNA (shadedline) into the cell (2, 3). Infection can take either of twocourses depending on which of two sets of viral genesis turned on. In the lysogenic pathway, the viral DNAbecomes integrated into the bacterial chromosome (4,5), where it replicates passively as the bacterial cell divides.The dormant virus is called a prophage, and thecell that harbors it is called a lysogen. In the alternativelytic mode of infection, the viral DNA replicates itself (6)and directs the synthesis of viral proteins (7). About 100new virus particles are formed. The proliferating viruseslyse, or burst, the cell (8). A prophage can be “induced”by a DNA damaging agent such as ultraviolet radiation(9). The inducing agent throws a switch, so that a differentset of genes is turned on. Viral DNA loops out of thechromosome (10) and replicates; the virus proceedsalong the lytic pathway. (Reproduced, with permission,from Ptashne M, Johnson AD, Pabo CO: A genetic switchin a bacterial virus. Sci Am [Nov] 1982;247:128.)687

380 / CHAPTER 39AGene for repressor (cl)Gene for CroO RRepressor RNAO R 3 O R 2 O R 1BRepressor promotercro Promotercro RNACT A C C T C T G G C G G T G A TA T G G A G A C C G C C A C T AFigure 39–5. Right operator (O R ) is shown in increasing detail in this series of drawings.The operator is a region of the viral DNA some 80 base pairs long (A). To its left lies thegene encoding lambda repressor (cI), to its right the gene (cro) encoding the regulator proteinCro. When the operator region is enlarged (B), it is seen to include three subregions,O R 1, O R 2, and O R 3, each 17 base pairs long. They are recognition sites to which both repressorand Cro can bind. The recognition sites overlap two promoters—sequences of bases towhich RNA polymerase binds in order to transcribe these genes into mRNA (wavy lines),that are translated into protein. Site O R 1 is enlarged (C) to show its base sequence. Notethat in this region of the λ chromosome, both strands of DNA act as a template for transcription(Chapter 39). (Reproduced, with permission, from Ptashne M, Johnson AD, Pabo CO:A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)ATtantly, the nucleotide sequences of these three tandemlyarranged sites are similar but not identical (Figure39–5B). The three related cis elements, termed operatorsO R 1, O R 2, and O R 3, can be bound by either cI orCro proteins. However, the relative affinities of cI andCro for each of the sites varies, and this differentialbinding affinity is central to the appropriate operationof the λ phage lytic or lysogenic “molecular switch.”The DNA region between the cro and repressor genesalso contains two promoter sequences that direct thebinding of RNA polymerase in a specified orientation,where it commences transcribing adjacent genes. Onepromoter directs RNA polymerase to transcribe in therightward direction and, thus, to transcribe cro andother distal genes, while the other promoter directs thetranscription of the repressor gene in the leftward direction(Figure 39–5B).The product of the repressor gene, the 236-aminoacid,27 kDa repressor protein, exists as a twodomainmolecule in which the amino terminal domainbinds to operator DNA and the carboxyl terminaldomain promotes the association of one repressorprotein with another to form a dimer. A dimer of repressormolecules binds to operator DNA much moretightly than does the monomeric form (Figure 39–6Ato 39–6C).The product of the cro gene, the 66-amino-acid,9kDa Cro protein, has a single domain but also bindsthe operator DNA more tightly as a dimer (Figure39–6D). The Cro protein’s single domain mediatesboth operator binding and dimerization.In a lysogenic bacterium—ie, a bacterium containinga lambda prophage—the lambda repressor dimer bindspreferentially to O R 1 but in so doing, by a cooperativeinteraction, enhances the binding (by a factor of 10) ofanother repressor dimer to O R 2 (Figure 39–7). Theaffinity of repressor for O R 3 is the least of the three operatorsubregions. The binding of repressor to O R 1 has twomajor effects. The occupation of O R 1 by repressorblocks the binding of RNA polymerase to the rightwardpromoter and in that way prevents expression ofcro. Second, as mentioned above, repressor dimer boundto O R 1 enhances the binding of repressor dimer to O R 2.The binding of repressor to O R 2 has the importantadded effect of enhancing the binding of RNA polymeraseto the leftward promoter that overlaps O R 2 andthereby enhances transcription and subsequent expressionof the repressor gene. This enhancement of transcriptionis apparently mediated through direct proteinproteininteractions between promoter-bound RNApolymerase and O R 2-bound repressor. Thus, the lambdarepressor is both a negative regulator, by preventingtranscription of cro, and a positive regulator, by enhancingtranscription of its own gene, the repressor gene.This dual effect of repressor is responsible for the stablestate of the dormant lambda bacteriophage; not onlydoes the repressor prevent expression of the genes necessaryfor lysis, but it also promotes expression of itself to

REGULATION OF GENE EXPRESSION / 381A B C DCOOHNH 2Amino acids132 – 236Amino acids1 – 92COOH COOHCOOH COOHNH 2 NH 2 NH 2 NH 2CroO R 1 O R 3Figure 39–6. Schematic molecular structures of cI (lambda repressor, shown in A, B, and C)and Cro (D). Lambda repressor protein is a polypeptide chain 236 amino acids long. The chainfolds itself into a dumbbell shape with two substructures: an amino terminal (NH 2 ) domain anda carboxyl terminal (COOH) domain. The two domains are linked by a region of the chain that issusceptible to cleavage by proteases (indicated by the two arrows in A). Single repressor molecules(monomers) tend to associate to form dimers (B); a dimer can dissociate to formmonomers again. A dimer is held together mainly by contact between the carboxyl terminaldomains (hatching). Repressor dimers bind to (and can dissociate from) the recognition sites inthe operator region; they display the greatest affinity for site O R 1 (C). It is the amino terminaldomain of the repressor molecule that makes contact with the DNA (hatching). Cro (D) has asingle domain with sites that promote dimerization and other sites that promote binding ofdimers to operator, preferentially to O R 3. (Reproduced, with permission, from Ptashne M, JohnsonAD, Pabo CO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)stabilize this state of differentiation. In the event that intracellularrepressor protein concentration becomes veryhigh, this excess repressor will bind to O R 3 and by sodoing diminish transcription of the repressor gene fromthe leftward promoter until the repressor concentrationdrops and repressor dissociates itself from O R 3.With such a stable, repressive, cI-mediated, lysogenicstate, one might wonder how the lytic cycle couldever be entered. However, this process does occur quiteefficiently. When a DNA-damaging signal, such as ultravioletlight, strikes the lysogenic host bacterium,fragments of single-stranded DNA are generated thatactivate a specific protease coded by a bacterial geneand referred to as recA (Figure 39–7). The activatedrecA protease hydrolyzes the portion of the repressorprotein that connects the amino terminal and carboxylterminal domains of that molecule (see Figure 39–6A).Such cleavage of the repressor domains causes the repressordimers to dissociate, which in turn causes dissociationof the repressor molecules from O R 2 andeventually from O R 1. The effects of removal of repressorfrom O R 1 and O R 2 are predictable. RNA polymeraseimmediately has access to the rightward promoterand commences transcribing the cro gene, and the enhancementeffect of the repressor at O R 2 on leftwardtranscription is lost (Figure 39–7).The resulting newly synthesized Cro protein alsobinds to the operator region as a dimer, but its order ofpreference is opposite to that of repressor (Figure39–7). That is, Cro binds most tightly to O R 3, butthere is no cooperative effect of Cro at O R 3 on thebinding of Cro to O R 2. At increasingly higher concentrationsof Cro, the protein will bind to O R 2 and eventuallyto O R 1.Occupancy of O R 3 by Cro immediately turns offtranscription from the leftward promoter and in thatway prevents any further expression of the repressorgene. The molecular switch is thus completely“thrown” in the lytic direction. The cro gene is now expressed,and the repressor gene is fully turned off. Thisevent is irreversible, and the expression of other lambdagenes begins as part of the lytic cycle. When Cro repressorconcentration becomes quite high, it will eventuallyoccupy O R 1 and in so doing reduce the expression of itsown gene, a process that is necessary in order to effectthe final stages of the lytic cycle.The three-dimensional structures of Cro and of thelambda repressor protein have been determined byx-ray crystallography, and models for their binding and effectingthe above-described molecular and genetic eventshave been proposed and tested. Both bind to DNA usinghelix-turn-helix DNA binding domain motifs (see below).

ProphageO R 3O R 2 O R 1RNA polymeraseRepressor promotercro promoterInduction (1)O R 3 O R 2 O R 1RNA polymeraserecARepressor promotercro promoterInduction (2)Ultraviolet radiationO R 3 O R 2 O R 1RNA polymeraseEarly lytic growthRepressor promoterO R 3 O R 2 O R 1cro promoterRNA polymeraseRepressor promotercro promoterFigure 39–7. Configuration of the switch is shown at four stages of lambda’s life cycle. The lysogenic pathway (inwhich the virus remains dormant as a prophage) is selected when a repressor dimer binds to O R 1, thereby making itlikely that O R 2 will be filled immediately by another dimer. In the prophage (top), the repressor dimers bound at O R 1and O R 2 prevent RNA polymerase from binding to the rightward promoter and so block the synthesis of Cro (negativecontrol). The repressors also enhance the binding of polymerase to the leftward promoter (positive control),with the result that the repressor gene is transcribed into RNA (wavy line) and more repressor is synthesized, maintainingthe lysogenic state. The prophage is induced when ultraviolet radiation activates the protease recA, whichcleaves repressor monomers. The equilibrium of free monomers, free dimers, and bound dimers is thereby shifted,and dimers leave the operator sites. RNA polymerase is no longer encouraged to bind to the leftward promoter, sothat repressor is no longer synthesized. As induction proceeds, all the operator sites become vacant, and so polymerasecan bind to the rightward promoter and Cro is synthesized. During early lytic growth, a single Cro dimer bindsto O R 3 shaded circles, the site for which it has the highest affinity. Consequently, RNA polymerase cannot bind to theleftward promoter, but the rightward promoter remains accessible. Polymerase continues to bind there, transcribingcro and other early lytic genes. Lytic growth ensues. (Reproduced, with permission, from Ptashne M, Johnson AD, PaboCO: A genetic switch in a bacterial virus. Sci Am [Nov] 1982;247:128.)382

REGULATION OF GENE EXPRESSION / 383To date, this system provides the best understanding ofthe molecular events involved in gene regulation.Detailed analysis of the lambda repressor led to theimportant concept that transcription regulatory proteinshave several functional domains. For example, lambdarepressor binds to DNA with high affinity. Repressormonomers form dimers, dimers interact with eachother, and repressor interacts with RNA polymerase.The protein-DNA interface and the three proteinproteininterfaces all involve separate and distinct domainsof the repressor molecule. As will be noted below(see Figure 39–17), this is a characteristic shared bymost (perhaps all) molecules that regulate transcription.SPECIAL FEATURES ARE INVOLVEDIN REGULATION OF EUKARYOTICGENE TRANSCRIPTIONMost of the DNA in prokaryotic cells is organized intogenes, and the templates can always be transcribed. Avery different situation exists in mammalian cells, inwhich relatively little of the total DNA is organizedinto genes and their associated regulatory regions. Thefunction of the extra DNA is unknown. In addition, asdescribed in Chapter 36, the DNA in eukaryotic cells isextensively folded and packed into the protein-DNAcomplex called chromatin. Histones are an importantpart of this complex since they both form the structuresknown as nucleosomes (see Chapter 36) and also factorsignificantly into gene regulatory mechanisms as outlinedbelow.Chromatin Remodeling Is an ImportantAspect of Eukaryotic Gene ExpressionChromatin structure provides an additional level ofcontrol of gene transcription. As discussed in Chapter36, large regions of chromatin are transcriptionally inactivewhile others are either active or potentially active.With few exceptions, each cell contains the same complementof genes (antibody-producing cells are a notableexception). The development of specialized organs, tissues,and cells and their function in the intact organismdepend upon the differential expression of genes.Some of this differential expression is achieved byhaving different regions of chromatin available for transcriptionin cells from various tissues. For example, theDNA containing the β-globin gene cluster is in “active”chromatin in the reticulocyte but in “inactive” chromatinin muscle cells. All the factors involved in the determinationof active chromatin have not been elucidated.The presence of nucleosomes and of complexes ofhistones and DNA (see Chapter 36) certainly provides abarrier against the ready association of transcription factorswith specific DNA regions. The dynamics of the formationand disruption of nucleosome structure are thereforean important part of eukaryotic gene regulation.Histone acetylation and deacetylation is an importantdeterminant of gene activity. The surprisingdiscovery that histone acetylase activity is associatedwith TAFs and the coactivators involved in hormonalregulation of gene transcription (see Chapter 43) hasprovided a new concept of gene regulation. Acetylationis known to occur on lysine residues in the amino terminaltails of histone molecules. This modification reducesthe positive charge of these tails and decreases thebinding affinity of histone for the negatively chargedDNA. Accordingly, the acetylation of histone could resultin disruption of nucleosomal structure and allowreadier access of transcription factors to cognate regulatoryDNA elements. As discussed previously, thiswould enhance binding of the basal transcription machineryto the promoter. Histone deacetylation wouldhave the opposite effect. Different proteins with specificacetylase and deacetylase activities are associated withvarious components of the transcription apparatus. Thespecificity of these processes is under investigation, asare a variety of mechanisms of action. Some specific examplesare illustrated in Chapter 43.There is evidence that the methylation of deoxycytidineresidues (in the sequence 5′- m CpG-3′) in DNAmay effect gross changes in chromatin so as to precludeits active transcription, as described in Chapter 36. Forexample, in mouse liver, only the unmethylated ribosomalgenes can be expressed, and there is evidence thatmany animal viruses are not transcribed when theirDNA is methylated. Acute demethylation of deoxycytidineresidues in a specific region of the tyrosine aminotransferasegene—in response to glucocorticoid hormones—hasbeen associated with an increased rate oftranscription of the gene. However, it is not possible togeneralize that methylated DNA is transcriptionally inactive,that all inactive chromatin is methylated, or thatactive DNA is not methylated.Finally, the binding of specific transcription factorsto cognate DNA elements may result in disruption ofnucleosomal structure. Many eukaryotic genes havemultiple protein-binding DNA elements. The serialbinding of transcription factors to these elements—in acombinatorial fashion—may either directly disrupt thestructure of the nucleosome or prevent its re-formationor recruit, via protein-protein interactions, multiproteincoactivator complexes that have the ability to covalentlymodify or remodel nucleosomes. These reactionsresult in chromatin-level structural changes that in theend increase DNA accessibility to other factors and thetranscription machinery.Eukaryotic DNA that is in an “active” region ofchromatin can be transcribed. As in prokaryotic cells, a

384 / CHAPTER 39promoter dictates where the RNA polymerase will initiatetranscription, but this promoter cannot be neatlydefined as containing a −35 and −10 box, particularlyin mammalian cells (Chapter 37). In addition, thetrans-acting factors generally come from other chromosomes(and so act in trans), whereas this considerationis moot in the case of the single chromosome-containingprokaryotic cells. Additional complexity is added byelements or factors that enhance or repress transcription,define tissue-specific expression, and modulate theactions of many effector molecules.Certain DNA Elements Enhance or RepressTranscription of Eukaryotic GenesIn addition to gross changes in chromatin affectingtranscriptional activity, certain DNA elements facilitateor enhance initiation at the promoter. For example, insimian virus 40 (SV40) there exists about 200 bp upstreamfrom the promoter of the early genes a region oftwo identical, tandem 72-bp lengths that can greatly increasethe expression of genes in vivo. Each of these72-bp elements can be subdivided into a series ofsmaller elements; therefore, some enhancers have a verycomplex structure. Enhancer elements differ from thepromoter in two remarkable ways. They can exert theirpositive influence on transcription even when separatedby thousands of base pairs from a promoter; they workwhen oriented in either direction; and they can workupstream (5′) or downstream (3′) from the promoter.Enhancers are promiscuous; they can stimulate anypromoter in the vicinity and may act on more than onepromoter. The SV40 enhancer element can exert an influenceon, for example, the transcription of β-globinby increasing its transcription 200-fold in cells containingboth the enhancer and the β-globin gene on thesame plasmid (see below and Figure 39–8). The enhancerelement does not produce a product that in turnacts on the promoter, since it is active only when it existswithin the same DNA molecule as (ie, cis to) thepromoter. Enhancer binding proteins are responsiblefor this effect. The exact mechanisms by which thesetranscription activators work are subject to much debate.Certainly, enhancer binding trans factors havebeen shown to interact with a plethora of other transcriptionproteins. These interactions include chromatin-modifyingcoactivators as well as the individualcomponents of the basal RNA polymerase II transcriptionmachinery. Ultimately, trans-factor-enhancer DNAbinding events result in an increase in the binding ofthe basal transcription machinery to the promoter. Enhancerelements and associated binding proteins oftenconvey nuclease hypersensitivity to those regions wherethey reside (Chapter 36). A summary of the propertiesof enhancers is presented in Table 39–2. One of the(Enhancerresponse element)ABCDSV40SV40mtGREPromoterβ globinβ globintkPEPCKStructural geneβ globinβ globinhGHCATFigure 39–8. A schematic explanation of the actionof enhancers and other cis-acting regulatory elements.These model chimeric genes consist of a reporter(structural) gene that encodes a protein which can bereadily assayed, a promoter that ensures accurate initiationof transcription, and the putative regulatory elements.In all cases, high-level transcription from the indicatedchimeras depends upon the presence ofenhancers, which stimulate transcription ≥ 100-foldover basal transcriptional levels (ie, transcription of thesame chimeric genes containing just promoters fusedto the structural genes). Examples A and B illustrate thefact that enhancers (eg, SV40) work in either orientationand upon a heterologous promoter. Example C illustratesthat the metallothionein (mt) regulatory element(which under the influence of cadmium or zinc inducestranscription of the endogenous mt gene and hencethe metal-binding mt protein) will work through thethymidine kinase (tk) promoter to enhance transcriptionof the human growth hormone (hGH) gene. Theengineered genetic constructions were introduced intothe male pronuclei of single-cell mouse embryos andthe embryos placed into the uterus of a surrogatemother to develop as transgenic animals. Offspringhave been generated under these conditions, and insome the addition of zinc ions to their drinking watereffects an increase in liver growth hormone. In thiscase, these transgenic animals have responded to thehigh levels of growth hormone by becoming twice aslarge as their normal litter mates. Example D illustratesthat a glucocorticoid response element (GRE) will workthrough homologous (PEPCK gene) or heterologouspromoters (not shown; ie, tk promoter, SV40 promoter,β-globin promoter, etc).

REGULATION OF GENE EXPRESSION / 385Table 39–2. Summary of the propertiesof enhancers.• Work when located long distances from the promoter• Work when upstream or downstream from the promoter• Work when oriented in either direction• Work through heterologous promoters• Work by binding one or more proteins• Work by facilitating binding of the basal transcription complexto the promoterbest-understood mammalian enhancer systems is thatof the β-interferon gene. This gene is induced uponviral infection of mammalian cells. One goal of the cell,once virally infected, is to attempt to mount an antiviralresponse—if not to save the infected cell, then tohelp to save the entire organism from viral infection.Interferon production is one mechanism by which thisis accomplished. This family of proteins is secreted byvirally infected cells. They interact with neighboringcells to cause an inhibition of viral replication by a varietyof mechanisms, thereby limiting the extent of viralinfection. The enhancer element controlling inductionof this gene, located between nucleotides −110 and −45of the β-interferon gene, is well characterized. This enhanceris composed of four distinct clustered cis elements,each of which is bound by distinct trans factors.One cis element is bound by the trans-acting factorNF-κB, one by a member of the IRF (interferon regulatoryfactor) family of trans factors, and a third by theheterodimeric leucine zipper factor ATF-2/c-Jun. Thefourth factor is the ubiquitous, architectural transcriptionfactor known as HMG I(Y). Upon binding to itsdegenerate, A+T-rich binding sites, HMG I(Y) inducesa significant bend in the DNA. There are four suchHMG I(Y) binding sites interspersed throughout theenhancer. These sites play a critical role in forming theenhanceosome, along with the aforementioned threetrans factors, by inducing a series of critically spacedDNA bends. Consequently, HMG I(Y) induces the cooperativeformation of a unique, stereospecific, threedimensional structure within which all four factors areactive when viral infection signals are sensed by the cell.The structure formed by the cooperative assembly ofthese four factors is termed the β-interferon enhanceosome(see Figure 39–9), so named because of its obviousstructural similarity to the nucleosome, also aunique three-dimensional protein DNA structure thatwraps DNA about an assembly of proteins (see Figures36–1 and 36–2). The enhanceosome, once formed, inducesa large increase in β-interferon gene transcriptionupon virus infection. It is not simply the protein occupancyof the linearly apposed cis element sites that inducesβ-interferon gene transcription—rather, it is theformation of the enhanceosome proper that providesappropriate surfaces for the recruitment of coactivatorsthat results in the enhanced formation of the PIC onthe cis-linked promoter and thus transcription activation.The cis-acting elements that decrease or repress theexpression of specific genes have also been identified.Because fewer of these elements have been studied, it isnot possible to formulate generalizations about theirmechanism of action—though again, as for gene activation,chromatin level covalent modifications of histonesand other proteins by (repressor)-recruited multisubunitcorepressors have been implicated.Tissue-Specific Expression MayResult From the Actionof Enhancers or RepressorsMany genes are now recognized to harbor enhancer oractivator elements in various locations relative to theircoding regions. In addition to being able to enhancegene transcription, some of these enhancer elementsclearly possess the ability to do so in a tissue-specificmanner. Thus, the enhancer element associated withthe immunoglobulin genes between the J and C regionsenhances the expression of those genes preferentially inlymphoid cells. Similarly to the SV40 enhancer, whichis capable of promiscuously activating a variety of cislinkedgenes, enhancer elements associated with thegenes for pancreatic enzymes are capable of enhancingeven unrelated but physically linked genes preferentiallyin the pancreatic cells of mice into which the specificallyengineered gene constructions were introducedmicrosurgically at the single-cell embryo stage. Thistransgenic animal approach has proved useful instudying tissue-specific gene expression. For example,DNA containing a pancreatic B cell tissue-specific enhancer(from the insulin gene), when ligated in a vectorto polyoma large-T antigen, an oncogene, producedB cell tumors in transgenic mice. Tumors did not developin any other tissue. Tissue-specific gene expressionmay therefore be mediated by enhancers or enhancer-likeelements.Reporter Genes Are Used to DefineEnhancers & Other Regulatory ElementsBy ligating regions of DNA suspected of harboring regulatorysequences to various reporter genes (the reporteror chimeric gene approach) (Figures 39–10and 39–11), one can determine which regions in thevicinity of structural genes have an influence on theirexpression. Pieces of DNA thought to harbor regulatoryelements are ligated to a suitable reporter gene and

386 / CHAPTER 39HMG PRDIV HMG PRDI-III PRDII HMG NRDI HMGHMGI-YATF-2cJunNF-κBIRF(IRF3/7)HMGIcJunATF-2HMGIIRF3NF-κBHMGIIRF7HMGIFigure 39–9. Formation and putative structure of the enhanceosome formed on the human β-interferon geneenhancer. Diagramatically represented at the top is the distribution of the multiple cis-elements (HMG, PRDIV,PRDI-III, PRDII, NRDI) composing the β-interferon gene enhancer. The intact enhancer mediates transcriptional inductionof the β-interferon gene (over 100-fold) upon virus infection of human cells. The cis-elements of this modularenhancer represent the binding sites for the trans-factors HMG I(Y), cJun-ATF-2, IRF3, IRF7, and NF-κB, respectively.The factors interact with these DNA elements in an obligatory, ordered, and highly cooperative fashion asindicated by the arrow. Initial binding of four HMG I(Y) proteins induces sharp DNA bends in the enhancer, causingthe entire 70–80 bp region to assume a high level of curvature. This curvature is integral to the subsequent highlycooperative binding of the other trans-factors since this enables the DNA-bound factors to make important, directprotein-protein interactions that both contribute to the formation and stability of the enhanceosome and generatea unique three-dimensional surface that serves to recruit chromatin-modifying activities (eg, Swi/Snf and P/CAF) aswell as the general transcription machinery (RNA polymerase II and GTFs). Although four of the five cis-elements(PRDIV, PRDI-III, PRDII, NRDI) independently can modestly stimulate (~tenfold) transcription of a reporter gene intransfected cells (see Figures 39–10 and 39–12), all five cis-elements, in appropriate order, are required to form anenhancer that can appropriately stimulate mRNA gene transcription (ie, ≥ 100-fold) in response to viral infection of ahuman cell. This distinction indicates the strict requirement for appropriate enhanceosome architecture for efficienttrans-activation. Similar enhanceosomes, involving distinct cis- and trans-factors, are proposed to form on manyother mammalian genes.introduced into a host cell (Figure 39–10). Basal expressionof the reporter gene will be increased if theDNA contains an enhancer. Addition of a hormone orheavy metal to the culture medium will increase expressionof the reporter gene if the DNA contains a hormoneor metal response element (Figure 39–11). Thelocation of the element can be pinpointed by using progressivelyshorter pieces of DNA, deletions, or pointmutations (Figure 39–11).This strategy, using transfected cells in cultureand transgenic animals, has led to the identificationof dozens of enhancers, repressors, tissue-specific elements,and hormone, heavy metal, and drug-responseelements. The activity of a gene at any moment reflectsthe interaction of these numerous cis-actingDNA elements with their respective trans-acting factors.The challenge now is to figure out how this occurs.

REGULATION OF GENE EXPRESSION / 387Test promoterGENEENHANCER-PROMOTERCellsControlCATIdentification ofcontrol elementsHormonesReporter gene5′ 3′ 5′3′CATREPORTER GENE:TEST ENHANCER-PROMOTERDRIVING TRANSCRIPTIONCAT GENETRANSFECT CELLS USING CaPO 4PRECIPITATED DNADivide and re-plateHARVEST 24 HOURS LATERASSAY FOR CAT ACTIVITYFigure 39–10. The use of reporter genes to defineDNA regulatory elements. A DNA fragment from thegene in question—in this example, approximately 2 kbof 5′-flanking DNA and cognate promoter—is ligatedinto a plasmid vector that contains a suitable reportergene—in this case, the bacterial enzyme chloramphenicoltransferase (CAT). The enzyme luciferase (abbreviatedLUC) is another popular reporter gene. NeitherLUC nor CAT is present in mammalian cells; hence, detectionof these activities in a cell extract means thatthe cell was successfully transfected by the plasmid. Anincrease of CAT activity over the basal level, eg, afteraddition of one or more hormones, means that the regionof DNA inserted into the reporter gene plasmidcontains functional hormone response elements (HRE).Progressively shorter pieces of DNA, regions with internaldeletions, or regions with point mutations can beconstructed and inserted to pinpoint the response element(see Figure 39–11 for deletion mapping of the relevantHREs).ner, but the process in most genes, especially in mammals,is much more complicated. Signals representing anumber of complex environmental stimuli may convergeon a single gene. The response of the gene tothese signals can have several physiologic characteristics.First, the response may extend over a considerablerange. This is accomplished by having additive and synergisticpositive responses counterbalanced by negativeor repressing effects. In some cases, either the positiveor the negative response can be dominant. Also requiredis a mechanism whereby an effector such as ahormone can activate some genes in a cell while repressingothers and leaving still others unaffected. When allof these processes are coupled with tissue-specific elementfactors, considerable flexibility is afforded. Thesephysiologic variables obviously require an arrangementmuch more complicated than an on-off switch. Thearray of DNA elements in a promoter specifies—withassociated factors—how a given gene will respond.Some simple examples are illustrated in Figure 39–12.Transcription Domains Can Be Defined byLocus Control Regions & InsulatorsThe large number of genes in eukaryotic cells and thecomplex arrays of transcription regulatory factors presentsan organizational problem. Why are some genesavailable for transcription in a given cell whereas othersare not? If enhancers can regulate several genes and arenot position- and orientation-dependent, how are theyprevented from triggering transcription randomly? Partof the solution to these problems is arrived at by havingthe chromatin arranged in functional units that restrictpatterns of gene expression. This may be achieved byhaving the chromatin form a structure with the nuclearmatrix or other physical entity, or compartmentswithin the nucleus. Alternatively, some regions are controlledby complex DNA elements called locus controlregions (LCRs). An LCR—with associated bound proteins—controlsthe expression of a cluster of genes. Thebest-defined LCR regulates expression of the globingene family over a large region of DNA. Another mechanismis provided by insulators. These DNA elements,also in association with one or more proteins, preventan enhancer from acting on a promoter on the otherside of an insulator in another transcription domain.Combinations of DNA Elements& Associated Proteins ProvideDiversity in ResponsesProkaryotic genes are often regulated in an on-off mannerin response to simple environmental cues. Some eukaryoticgenes are regulated in the simple on-off man-SEVERAL MOTIFS MEDIATE THE BINDINGOF REGULATORY PROTEINS TO DNAThe specificity involved in the control of transcriptionrequires that regulatory proteins bind with high affinityto the correct region of DNA. Three unique motifs—the helix-turn-helix, the zinc finger, and the leucine

388 / CHAPTER 395′HREAREPORTER GENE CONSTRUCTSWITH VARIABLE AMOUNTSOF 5'-FLANKING DNA2000 1000Nucleotide positionHREBHRECHORMONE-DEPENDENTTRANSCRIPTIONINDUCTION+1CATA+B+C++ ++ +++Figure 39–11. Location of hormone response elements(HREs) A, B, and C using the reportergene–transfection approach. A family of reportergenes, constructed as described in Figure 39–10, canbe transfected individually into a recipient cell. By analyzingwhen certain hormone responses are lost incomparison to the 5′ deletion, specific hormoneresponsiveelements can be located.zipper—account for many of these specific protein-DNA interactions. Examples of proteins containingthese motifs are given in Table 39–3.Comparison of the binding activities of the proteinsthat contain these motifs leads to several importantgeneralizations.(1) Binding must be of high affinity to the specificsite and of low affinity to other DNA.(2) Small regions of the protein make direct contactwith DNA; the rest of the protein, in addition to pro-GeneGeneAB111423233Table 39–3. Examples of transcription regulatoryproteins that contain the various binding motifs.Binding Motif Organism Regulatory ProteinHelix-turn-helix E coli lac repressorCAPPhage λcI, cro, and tryptophan and434 repressorsMammals homeo box proteinsPit-1, Oct1, Oct2Zinc finger E coli Gene 32 proteinYeast GaI4Drosophila Serendipity, HunchbackXenopus TFIIIAMammals steroid receptor family, Sp1Leucine zipper Yeast GCN4Mammals C/EBP, fos, Jun, Fra-1,CRE binding protein,c-myc, n-myc, I-mycGeneC5Figure 39–12. Combinations of DNA elements andproteins provide diversity in the response of a gene.Gene A is activated (the width of the arrow indicatesthe extent) by the combination of activators 1, 2, and 3(probably with coactivators, as shown in Figure 37–10).Gene B is activated, in this case more effectively, by thecombination of 1, 3, and 4; note that 4 does not contactDNA directly in this example. The activators could forma linear bridge that links the basal machinery to thepromoter, or this could be accomplished by loopingout of the DNA. In either case, the purpose is to directthe basal transcription machinery to the promoter.Gene C is inactivated by the combination of 1, 5, and 3;in this case, factor 5 is shown to preclude the essentialbinding of factor 2 to DNA, as occurs in example A. Ifactivator 1 helps repressor 5 bind and if activator 1binding requires a ligand (solid dot), it can be seen howthe ligand could activate one gene in a cell (gene A)and repress another (gene C).

REGULATION OF GENE EXPRESSION / 389viding the trans-activation domains, may be involved inthe dimerization of monomers of the binding protein,may provide a contact surface for the formation of heterodimers,may provide one or more ligand-bindingsites, or may provide surfaces for interaction with coactivatorsor corepressors.(3) The protein-DNA interactions are maintainedby hydrogen bonds and van der Waals forces.(4) The motifs found in these proteins are unique;their presence in a protein of unknown function suggeststhat the protein may bind to DNA.(5) Proteins with the helix-turn-helix or leucine zippermotifs form symmetric dimers, and their respectiveDNA binding sites are symmetric palindromes. In proteinswith the zinc finger motif, the binding site is repeatedtwo to nine times. These features allow for cooperativeinteractions between binding sites andenhance the degree and affinity of binding.The Helix-Turn-Helix MotifThe first motif described—and the one studied mostextensively—is the helix-turn-helix. Analysis of thethree-dimensional structure of the λ Cro transcriptionregulator has revealed that each monomer consists ofthree antiparallel β sheets and three α helices (Figure39–13). The dimer forms by association of the antiparallelβ 3 sheets. The α 3 helices form the DNA recognitionsurface, and the rest of the molecule appears to beinvolved in stabilizing these structures. The average diameterof an α helix is 1.2 nm, which is the approximatewidth of the major groove in the B form of DNA.The DNA recognition domain of each Cro monomerinteracts with 5 bp and the dimer binding sites span3.4 nm, allowing fit into successive half turns of themajor groove on the same surface (Figure 39–13). X-rayanalyses of the λ cI repressor, CAP (the cAMP receptorα 234 Åα 3α 1 N Cβ 1β 2β 3α 3Twofoldaxis ofβ 3symmetryβ 2α β 3 1α 2C Nα 2α 1α 3α 234 ÅFigure 39–13. A schematic representation of the three-dimensional structure of Cro protein and its binding toDNA by its helix-turn-helix motif. The Cro monomer consists of three antiparallel β sheets (β 1 –β 3 ) and threeα-helices (α 1 –α 3 ). The helix-turn-helix motif is formed because the α 3 and α 2 helices are held at about 90 degreesto each other by a turn of four amino acids. The α 3 helix of Cro is the DNA recognition surface (shaded). Twomonomers associate through the antiparallel β3 sheets to form a dimer that has a twofold axis of symmetry(right). A Cro dimer binds to DNA through its α 3 helices, each of which contacts about 5 bp on the same surface ofthe major groove. The distance between comparable points on the two DNA α-helices is 34 Å, which is the distancerequired for one complete turn of the double helix. (Courtesy of B Mathews.)

390 / CHAPTER 39protein of E coli), tryptophan repressor, and phage 434repressor all also display this dimeric helix-turn-helixstructure that is present in eukaryotic DNA proteins aswell (see Table 39–3).The Zinc Finger MotifThe zinc finger was the second DNA binding motifwhose atomic structure was elucidated. It was knownthat the protein TFIIIA, a positive regulator of 5S RNAtranscription, required zinc for activity. Structural andbiophysical analyses revealed that each TFIIIA moleculecontains nine zinc ions in a repeating coordinationcomplex formed by closely spaced cysteine-cysteineresidues followed 12–13 amino acids later by a histidine-histidinepair (Figure 39–14). In some instances—notably the steroid-thyroid receptor family—the His-His doublet is replaced by a second Cys-Cys pair. Theprotein containing zinc fingers appears to lie on oneface of the DNA helix, with successive fingers alternativelypositioned in one turn in the major groove. As isthe case with the recognition domain in the helix-turnhelixprotein, each TFIIIA zinc finger contacts about5 bp of DNA. The importance of this motif in the actionof steroid hormones is underscored by an “experimentof nature.” A single amino acid mutation in eitherof the two zinc fingers of the 1,25(OH) 2 -D 3 receptorprotein results in resistance to the action of this hormoneand the clinical syndrome of rickets.CCZnCCCys-Cys zinc fingerCCZnHHCys-His zinc fingerFigure 39–14. Zinc fingers are a series of repeateddomains (two to nine) in which each is centered on atetrahedral coordination with zinc. In the case of TFIIIA,the coordination is provided by a pair of cysteineresidues (C) separated by 12–13 amino acids from apair of histidine (H) residues. In other zinc finger proteins,the second pair also consists of C residues. Zincfingers bind in the major groove, with adjacent fingersmaking contact with 5 bp along the same face of thehelix.The Leucine Zipper MotifCareful analysis of a 30-amino-acid sequence in the carboxylterminal region of the enhancer binding proteinC/EBP revealed a novel structure. As illustrated in Figure39–15, this region of the protein forms an α helixin which there is a periodic repeat of leucine residues atevery seventh position. This occurs for eight helicalturns and four leucine repeats. Similar structures havebeen found in a number of other proteins associatedwith the regulation of transcription in mammalian andyeast cells. It is thought that this structure allows twoidentical monomers or heterodimers (eg, Fos-Jun orJun-Jun) to “zip together” in a coiled coil and form atight dimeric complex (Figure 39–15). This proteinproteininteraction may serve to enhance the associationof the separate DNA binding domains with theirtarget (Figure 39–15).THE DNA BINDING & TRANS-ACTIVATIONDOMAINS OF MOST REGULATORYPROTEINS ARE SEPARATE& NONINTERACTIVEDNA binding could result in a general conformationalchange that allows the bound protein to activate transcription,or these two functions could be served byseparate and independent domains. Domain swap experimentssuggest that the latter is the case.The GAL1 gene product is involved in galactose metabolismin yeast. Transcription of this gene is positivelyregulated by the GAL4 protein, which binds to an upstreamactivator sequence (UAS), or enhancer, throughan amino terminal domain. The amino terminal 73-amino-acid DNA-binding domain (DBD) of GAL4 wasremoved and replaced with the DBD of LexA, an E coliDNA-binding protein. This domain swap resulted in amolecule that did not bind to the GAL1 UAS and, ofcourse, did not activate the GAL1 gene (Figure 39–16).If, however, the lexA operator—the DNA sequence normallybound by the lexA DBD—was inserted into thepromoter region of the GAL gene, the hybrid proteinbound to this promoter (at the lexA operator) and it activatedtranscription of GAL1. This experiment, whichhas been repeated a number of times, affords solid evidencethat the carboxyl terminal region of GAL4 causestranscriptional activation. These data also demonstratethat the DNA-binding DBD and trans-activation domains(ADs) are independent and noninteractive. Thehierarchy involved in assembling gene transcription activatingcomplexes includes proteins that bind DNA andtrans-activate; others that form protein-protein complexeswhich bridge DNA-binding proteins to transactivatingproteins; and others that form protein-proteincomplexes with components of the basal transcription

REGULATION OF GENE EXPRESSION / 391ABIERD4LL22158L1L5N V L FNH 2COOHRQTQ72T R S RCOOHRGSK36DE D RNH 2Figure 39–15. The leucine zipper motif. A shows a helical wheel analysis of a carboxyl terminal portion of theDNA binding protein C/EBP. The amino acid sequence is displayed end-to-end down the axis of a schematicα-helix. The helical wheel consists of seven spokes that correspond to the seven amino acids that comprise everytwo turns of the α-helix. Note that leucine residues (L) occur at every seventh position. Other proteins with“leucine zippers” have a similar helical wheel pattern. B is a schematic model of the DNA binding domain of C/EBP.Two identical C/EBP polypeptide chains are held in dimer formation by the leucine zipper domain of eachpolypeptide (denoted by the rectangles and attached ovals). This association is apparently required to hold theDNA binding domains of each polypeptide (the shaded rectangles) in the proper conformation for DNA binding.(Courtesy of S McKnight.)apparatus. A given protein may thus have several surfacesor domains that serve different functions (see Figure39–17). As described in Chapter 37, the primarypurpose of these complex assemblies is to facilitate theassembly of the basal transcription apparatus on the cislinkedpromoter.GENE REGULATION IN PROKARYOTES& EUKARYOTES DIFFERS INIMPORTANT RESPECTSIn addition to transcription, eukaryotic cells employ avariety of mechanisms to regulate gene expression(Table 39–4). The nuclear membrane of eukaryoticcells physically segregates gene transcription from translation,since ribosomes exist only in the cytoplasm.Many more steps, especially in RNA processing, are involvedin the expression of eukaryotic genes than ofprokaryotic genes, and these steps provide additionalsites for regulatory influences that cannot exist inprokaryotes. These RNA processing steps in eukaryotes,described in detail in Chapter 37, include capping ofthe 5′ ends of the primary transcripts, addition of apolyadenylate tail to the 3′ ends of transcripts, and excisionof intron regions to generate spliced exons in themature mRNA molecule. To date, analyses of eukaryoticgene expression provide evidence that regulationoccurs at the level of transcription, nuclear RNA processing,and mRNA stability. In addition, gene amplificationand rearrangement influence gene expression.Owing to the advent of recombinant DNA technology,much progress has been made in recent years inthe understanding of eukaryotic gene expression. However,because most eukaryotic organisms contain somuch more genetic information than do prokaryotesand because manipulation of their genes is so muchmore limited, molecular aspects of eukaryotic gene regulationare less well understood than the examplesdiscussed earlier in this chapter. This section briefly describesa few different types of eukaryotic gene regulation.

392 / CHAPTER 39GAL4 +1ActiveAUASGAL1 geneLexA–GAL4+1InactiveBUASGAL1 geneLexA–GAL4 +1ActiveClexAoperatorGAL1 geneFigure 39–16. Domain-swap experiments demonstrate the independent nature of DNA binding and transcriptionactivation domains. The GAL1 gene promoter contains an upstream activating sequence (UAS) or enhancer thatbinds the regulatory protein GAL4 (A). This interaction results in a stimulation of GAL1 gene transcription. A chimericprotein, in which the amino terminal DNA binding domain of GAL4 is removed and replaced with the DNA bindingregion of the E coli protein LexA, fails to stimulate GAL1 transcription because the LexA domain cannot bind to theUAS (B). The LexA–GAL4 fusion protein does increase GAL1 transcription when the lexA operator (its natural target)is inserted into the GAL1 promoter region (C).Eukaryotic Genes Can Be Amplifiedor Rearranged During Developmentor in Response to DrugsDuring early development of metazoans, there is anabrupt increase in the need for specific molecules suchas ribosomal RNA and messenger RNA molecules forproteins that make up such organs as the eggshell. Oneway to increase the rate at which such molecules can beformed is to increase the number of genes available fortranscription of these specific molecules. Among therepetitive DNA sequences are hundreds of copies of ribosomalRNA genes and tRNA genes. These genes preexistrepetitively in the genomic material of the gametesLigand-binding domainDNA-binding domain2134Activationdomains1–4Figure 39–17. Proteins that regulate transcriptionhave several domains. This hypothetical transcriptionfactor has a DNA-binding domain (DBD) that is distinctfrom a ligand-binding domain (LBD) and several activationdomains (ADs) (1–4). Other proteins may lack theDBD or LBD and all may have variable numbers ofdomains that contact other proteins, includingco-regulators and those of the basal transcriptioncomplex (see also Chapters 42 and 43).Table 39–4. Gene expression is regulated bytranscription and in numerous other ways ineukaryotic cells.• Gene amplification• Gene rearrangement• RNA processing• Alternate mRNA splicing• Transport of mRNA from nucleus to cytoplasm• Regulation of mRNA stability

REGULATION OF GENE EXPRESSION / 393and thus are transmitted in high copy numbers fromgeneration to generation. In some specific organismssuch as the fruit fly (drosophila), there occurs duringoogenesis an amplification of a few preexisting genessuch as those for the chorion (eggshell) proteins. Subsequently,these amplified genes, presumably generatedby a process of repeated initiations during DNA synthesis,provide multiple sites for gene transcription(Figures 36–16 and 39–18).As noted in Chapter 37, the coding sequences responsiblefor the generation of specific protein moleculesare frequently not contiguous in the mammaliangenome. In the case of antibody encoding genes, this isparticularly true. As described in detail in Chapter 50,immunoglobulins are composed of two polypeptides,the so-called heavy (about 50 kDa) and light (about 25kDa) chains. The mRNAs encoding these two proteinsubunits are encoded by gene sequences that are subjectedto extensive DNA sequence-coding changes.These DNA coding changes are integral to generatingthe requisite recognition diversity central to appropriateimmune function.IgG heavy and light chain mRNAs are encoded byseveral different segments that are tandemly repeated inthe germline. Thus, for example, the IgG light chain iscomposed of variable (V L ), joining (J L ), and constant(C L ) domains or segments. For particular subsets ofIgG light chains, there are roughly 300 tandemly repeatedV L gene coding segments, five tandemlyarranged J L coding sequences, and roughly ten C L genecoding segments. All of these multiple, distinct codingregions are located in the same region of the same chromosome,and each type of coding segment (V L , J L , andC L ) is tandemly repeated in head-to-tail fashion withinthe segment repeat region. By having multiple V L , J L ,and C L segments to choose from, an immune cell has agreater repertoire of sequences to work with to developUnamplified s36 s38Amplifieds36s38Figure 39–18. Schematic representation of the amplificationof chorion protein genes s36 and s38. (Reproduced,with permission, from Chisholm R: Gene amplificationduring development. Trends Biochem Sci 1982;7:161.)both immunologic flexibility and specificity. However,a given functional IgG light chain transcription unit—like all other “normal” mammalian transcriptionunits—contains only the coding sequences for a singleprotein. Thus, before a particular IgG light chain canbe expressed, single V L , J L , and C L coding sequencesmust be recombined to generate a single, contiguoustranscription unit excluding the multiple nonutilizedsegments (ie, the other approximately 300 unused V Lsegments, the other four unused J L segments, and theother nine unused C L segments). This deletion of unusedgenetic information is accomplished by selectiveDNA recombination that removes the unwanted codingDNA while retaining the required coding sequences:one V L , one J L , and one C L sequence. (V L sequencesare subjected to additional point mutagenesisto generate even more variability—hence the name.)The newly recombined sequences thus form a singletranscription unit that is competent for RNA polymeraseII-mediated transcription. Although the IgGgenes represent one of the best-studied instances of directedDNA rearrangement modulating gene expression,other cases of gene regulatory DNA rearrangementhave been described in the literature. Indeed, asdetailed below, drug-induced gene amplification is animportant complication of cancer chemotherapy.In recent years, it has been possible to promote theamplification of specific genetic regions in culturedmammalian cells. In some cases, a several thousand-foldincrease in the copy number of specific genes can beachieved over a period of time involving increasing dosesof selective drugs. In fact, it has been demonstrated inpatients receiving methotrexate for cancer that malignantcells can develop drug resistance by increasing thenumber of genes for dihydrofolate reductase, the targetof methotrexate. Gene amplification events such as theseoccur spontaneously in vivo—ie, in the absence of exogenouslysupplied selective agents—and these unscheduledextra rounds of replication can become “frozen” inthe genome under appropriate selective pressures.Alternative RNA ProcessingIs Another Control MechanismIn addition to affecting the efficiency of promoter utilization,eukaryotic cells employ alternative RNA processingto control gene expression. This can result whenalternative promoters, intron-exon splice sites, orpolyadenylation sites are used. Occasionally, heterogeneitywithin a cell results, but more commonly thesame primary transcript is processed differently in differenttissues. A few examples of each of these types ofregulation are presented below.The use of alternative transcription start sites resultsin a different 5′ exon on mRNAs corresponding to

394 / CHAPTER 39mouse amylase and myosin light chain, rat glucokinase,and drosophila alcohol dehydrogenase and actin. Alternativepolyadenylation sites in the µ immunoglobulinheavy chain primary transcript result in mRNAs thatare either 2700 bases long (µ m ) or 2400 bases long (µ s ).This results in a different carboxyl terminal region ofthe encoded proteins such that the µ m protein remainsattached to the membrane of the B lymphocyte and theµ s immunoglobulin is secreted. Alternative splicingand processing results in the formation of sevenunique α-tropomyosin mRNAs in seven different tissues.It is not clear how these processing-splicing decisionsare made or whether these steps can be regulated.Regulation of Messenger RNA StabilityProvides Another Control MechanismAlthough most mRNAs in mammalian cells are verystable (half-lives measured in hours), some turn oververy rapidly (half-lives of 10–30 minutes). In certain instances,mRNA stability is subject to regulation. Thishas important implications since there is usually a directrelationship between mRNA amount and thetranslation of that mRNA into its cognate protein.Changes in the stability of a specific mRNA can thereforehave major effects on biologic processes.Messenger RNAs exist in the cytoplasm as ribonucleoproteinparticles (RNPs). Some of these proteinsprotect the mRNA from digestion by nucleases, whileothers may under certain conditions promote nucleaseattack. It is thought that mRNAs are stabilized or destabilizedby the interaction of proteins with these variousstructures or sequences. Certain effectors, such as hormones,may regulate mRNA stability by increasing ordecreasing the amount of these proteins.It appears that the ends of mRNA molecules areinvolved in mRNA stability (Figure 39–19). The 5′cap structure in eukaryotic mRNA prevents attack by 5′exonucleases, and the poly(A) tail prohibits the actionof 3′ exonucleases. In mRNA molecules with thosestructures, it is presumed that a single endonucleolyticcut allows exonucleases to attack and digest the entiremolecule. Other structures (sequences) in the 5′ noncodingsequence (5′ NCS), the coding region, and the3′ NCS are thought to promote or prevent this initialendonucleolytic action (Figure 39–19). A few illustrativeexamples will be cited.Deletion of the 5′ NCS results in a threefold to fivefoldprolongation of the half-life of c-myc mRNA. Shorteningthe coding region of histone mRNA results in aprolonged half-life. A form of autoregulation of mRNAstability indirectly involves the coding region. Free tubulinbinds to the first four amino acids of a nascent chainof tubulin as it emerges from the ribosome. This appearsto activate an RNase associated with the ribosome (RNP)which then digests the tubulin mRNA.Structures at the 3′ end, including the poly(A) tail,enhance or diminish the stability of specific mRNAs.The absence of a poly(A) tail is associated with rapiddegradation of mRNA, and the removal of poly(A)from some RNAs results in their destabilization. HistonemRNAs lack a poly(A) tail but have a sequencenear the 3′ terminal that can form a stem-loop structure,and this appears to provide resistance to exonucleolyticattack. Histone H4 mRNA, for example, is degradedin the 3′ to 5′ direction but only after a singleendonucleolytic cut occurs about nine nucleotides fromthe 3′ end in the region of the putative stem-loop structure.Stem-loop structures in the 3′ noncoding sequenceare also critical for the regulation, by iron, ofthe mRNA encoding the transferrin receptor. Stemloopstructures are also associated with mRNA stabilityin bacteria, suggesting that this mechanism may becommonly employed.Cap 5′ NCS Coding 3′ NCSA–A–A–A–A nAUUUAFigure 39–19. Structure of a typical eukaryotic mRNA showingelements that are involved in regulating mRNA stability. The typicaleukaryotic mRNA has a 5′ noncoding sequence (5′ NCS), a codingregion, and a 3′ NCS. All are capped at the 5′ end, and most have apolyadenylate sequence at the 3′ end. The 5′ cap and 3′ poly(A) tailprotect the mRNA against exonuclease attack. Stem-loop structuresin the 5′ and 3′ NCS, features in the coding sequence, and the AUrichregion in the 3′ NCS are thought to play roles in mRNA stability.

REGULATION OF GENE EXPRESSION / 395Other sequences in the 3′ ends of certain eukaryoticmRNAs appear to be involved in the destabilization ofthese molecules. Of particular interest are AU-rich regions,many of which contain the sequence AUUUA.This sequence appears in mRNAs that have a very shorthalf-life, including some encoding oncogene proteinsand cytokines. The importance of this region is underscoredby an experiment in which a sequence correspondingto the 3′ noncoding region of the short-halflifecolony-stimulating factor (CSF) mRNA, whichcontains the AUUUA motif, was added to the 3′ end ofthe β-globin mRNA. Instead of becoming very stable,this hybrid β-globin mRNA now had the short-half-lifecharacteristic of CSF mRNA.From the few examples cited, it is clear that a numberof mechanisms are used to regulate mRNA stability—justas several mechanisms are used to regulate thesynthesis of mRNA. Coordinate regulation of these twoprocesses confers on the cell remarkable adaptability.SUMMARY• The genetic constitutions of nearly all metazoan somaticcells are identical.• Phenotype (tissue or cell specificity) is dictated bydifferences in gene expression of this complement ofgenes.• Alterations in gene expression allow a cell to adapt toenvironmental changes.• Gene expression can be controlled at multiple levelsby changes in transcription, RNA processing, localization,and stability or utilization. Gene amplificationand rearrangements also influence gene expression.• Transcription controls operate at the level of protein-DNA and protein-protein interactions. These interactionsdisplay protein domain modularity and highspecificity.• Several different classes of DNA-binding domainshave been identified in transcription factors.• Chromatin modifications are important in eukaryotictranscription control.REFERENCESAlbright SR, Tjian R: TAFs revisited: more data reveal new twistsand confirm old ideas. Gene 2000;242:1.Bird AP, Wolffe AP: Methylation-induced repression—belts, bracesand chromatin. Cell 1999;99:451.Berger SL, Felsenfeld G: Chromatin goes global. Mol Cell 2001;8:263.Busby S, Ebright RH: Promoter structure, promoter recognition,and transcription activation in prokaryotes. Cell 1994;79:743.Busby S, Ebright RH: Transcription activation by catabolite activatorprotein (CAP). J Mol Biol 1999;293:199.Cowell IG: Repression versus activation in the control of gene transcription.Trends Biochem Sci 1994;1:38.Ebright RH: RNA polymerase: structural similarities between bacterialRNA polymerase and eukaryotic RNA polymerase II.J Mol Biol 2000;304:687.Fugman SD: RAG1 and RAG2 in V(D)J recombination and transposition.Immunol Res 2001;23:23.Jacob F, Monod J: Genetic regulatory mechanisms in protein synthesis.J Mol Biol 1961;3:318.Lemon B, Tjian R: Orchestrated response: a symphony of transcriptionfactors for gene control. Genes Dev 2000;14:2551.Letchman DS: Transcription factor mutations and disease. N EnglJ Med 1996;334:28.Merika M, Thanos D: Enhanceosomes. Curr Opin Genet Dev2001;11:205.Naar AM, Lemon BD, Tjian R: Transcriptional coactivator complexes.Annu Rev Biochem 2001;70:475.Narlikar GJ, Fan HY, Kingston RE: Cooperation between complexesthat regulate chromatin structure and transcription.Cell 2002;108:475.Oltz EM: Regulation of antigen receptor gene assembly in lymphocytes.Immunol Res 2001;23:121.Ptashne M: Control of gene transcription: an outline. Nat Med1997;3:1069.Ptashne M: A Genetic Switch, 2nd ed. Cell Press and Blackwell ScientificPublications, 1992.Sterner DE, Berger SL: Acetylation of histones and transcriptionrelatedfactors. Microbiol Mol Biol Rev 2000;64:435.Wu R, Bahl CP, Narang SA: Lactose operator-repressor interaction.Curr Top Cell Regul 1978;13:137.

Molecular Genetics, RecombinantDNA, & Genomic Technology40Daryl K. Granner, MD, & P. Anthony Weil, PhDBIOMEDICAL IMPORTANCE*The development of recombinant DNA, high-density,high-throughput screening, and other molecular geneticmethodologies has revolutionized biology and ishaving an increasing impact on clinical medicine.Much has been learned about human genetic diseasefrom pedigree analysis and study of affected proteins,but in many cases where the specific genetic defect isunknown, these approaches cannot be used. The newtechnologies circumvent these limitations by going directlyto the DNA molecule for information. Manipulationof a DNA sequence and the construction ofchimeric molecules—so-called genetic engineering—provides a means of studying how a specific segment ofDNA works. Novel molecular genetic tools allow investigatorsto query and manipulate genomic sequences aswell as to examine both cellular mRNA and proteinprofiles at the molecular level.Understanding this technology is important for severalreasons: (1) It offers a rational approach to understandingthe molecular basis of a number of diseases(eg, familial hypercholesterolemia, sickle cell disease,the thalassemias, cystic fibrosis, muscular dystrophy).(2) Human proteins can be produced in abundance fortherapy (eg, insulin, growth hormone, tissue plasminogenactivator). (3) Proteins for vaccines (eg, hepatitis B)and for diagnostic testing (eg, AIDS tests) can be obtained.(4) This technology is used to diagnose existingdiseases and predict the risk of developing a given disease.(5) Special techniques have led to remarkable advancesin forensic medicine. (6) Gene therapy for sicklecell disease, the thalassemias, adenosine deaminase deficiency,and other diseases may be devised.* See glossary of terms at the end of this chapter.396ELUCIDATION OF THE BASIC FEATURESOF DNA LED TO RECOMBINANTDNA TECHNOLOGYDNA Is a Complex BiopolymerOrganized as a Double HelixThe fundamental organizational element is the sequenceof purine (adenine [A] or guanine [G]) andpyrimidine (cytosine [C] or thymine [T]) bases. Thesebases are attached to the C-1′ position of the sugar deoxyribose,and the bases are linked together throughjoining of the sugar moieties at their 3′ and 5′ positionsvia a phosphodiester bond (Figure 35–1). The alternatingdeoxyribose and phosphate groups form the backboneof the double helix (Figure 35–2). These 3′–5′linkages also define the orientation of a given strand ofthe DNA molecule, and, since the two strands run inopposite directions, they are said to be antiparallel.Base Pairing Is a Fundamental Conceptof DNA Structure & FunctionAdenine and thymine always pair, by hydrogen bonding,as do guanine and cytosine (Figure 35–3). These basepairs are said to be complementary, and the guaninecontent of a fragment of double-stranded DNA will alwaysequal its cytosine content; likewise, the thymineand adenine contents are equal. Base pairing and hydrophobicbase-stacking interactions hold the two DNAstrands together. These interactions can be reduced byheating the DNA to denature it. The laws of base pairingpredict that two complementary DNA strands will reannealexactly in register upon renaturation, as happenswhen the temperature of the solution is slowly reduced tonormal. Indeed, the degree of base-pair matching (ormismatching) can be estimated from the temperature re-

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 397quired for denaturation-renaturation. Segments of DNAwith high degrees of base-pair matching require more energyinput (heat) to accomplish denaturation—or, to putit another way, a closely matched segment will withstandmore heat before the strands separate. This reaction isused to determine whether there are significant differencesbetween two DNA sequences, and it underlies theconcept of hybridization, which is fundamental to theprocesses described below.There are about 3 10 9 base pairs (bp) in eachhuman haploid genome. If an average gene length is3 × 10 3 bp (3 kilobases [kb]), the genome could consistof 10 6 genes, assuming that there is no overlap and thattranscription proceeds in only one direction. It isthought that there are < 10 5 genes in the human andthat only 1–2% of the DNA codes for proteins. Theexact function of the remaining ~98% of the humangenome has not yet been defined.The double-helical DNA is packaged into a morecompact structure by a number of proteins, mostnotably the basic proteins called histones. This condensationmay serve a regulatory role and certainly hasa practical purpose. The DNA present within the nucleusof a cell, if simply extended, would be about1 meter long. The chromosomal proteins compact thislong strand of DNA so that it can be packaged into anucleus with a volume of a few cubic micrometers.DNA Is Organized Into GenesIn general, prokaryotic genes consist of a small regulatoryregion (100–500 bp) and a large protein-codingsegment (500–10,000 bp). Several genes are often controlledby a single regulatory unit. Most mammaliangenes are more complicated in that the coding regionsare interrupted by noncoding regions that are eliminatedwhen the primary RNA transcript is processedinto mature messenger RNA (mRNA). The coding regions(those regions that appear in the mature RNAspecies) are called exons, and the noncoding regions,which interpose or intervene between the exons, arecalled introns (Figure 40–1). Introns are always removedfrom precursor RNA before transport into thecytoplasm occurs. The process by which introns are removedfrom precursor RNA and by which exons areligated together is called RNA splicing. Incorrect processingof the primary transcript into the maturemRNA can result in disease in humans (see below); thisunderscores the importance of these posttranscriptionalprocessing steps. The variation in size and complexityof some human genes is illustrated in Table 40–1. Althoughthere is a 300-fold difference in the sizes of thegenes illustrated, the mRNA sizes vary only about 20-fold. This is because most of the DNA in genes is presentas introns, and introns tend to be much larger thanexons. Regulatory regions for specific eukaryotic genesare usually located in the DNA that flanks the transcriptioninitiation site at its 5′ end (5′ flankingsequenceDNA). Occasionally, such sequences arefound within the gene itself or in the region that flanksthe 3′ end of the gene. In mammalian cells, each genehas its own regulatory region. Many eukaryotic genes(and some viruses that replicate in mammalian cells)have special regions, called enhancers, that increase therate of transcription. Some genes also have DNA sequences,known as silencers, that repress transcription.Mammalian genes are obviously complicated, multicomponentstructures.Genes Are Transcribed Into RNAInformation generally flows from DNA to mRNA toprotein, as illustrated in Figure 40–1 and discussed inmore detail in Chapter 39. This is a rigidly controlledprocess involving a number of complex steps, each ofwhich no doubt is regulated by one or more enzymes orfactors; faulty function at any of these steps can causedisease.RECOMBINANT DNA TECHNOLOGYINVOLVES ISOLATION & MANIPULATIONOF DNA TO MAKE CHIMERIC MOLECULESIsolation and manipulation of DNA, including end-toendjoining of sequences from very different sources tomake chimeric molecules (eg, molecules containingboth human and bacterial DNA sequences in a sequence-independentfashion), is the essence of recombinantDNA research. This involves several uniquetechniques and reagents.Restriction Enzymes Cut DNAChains at Specific LocationsCertain endonucleases—enzymes that cut DNA at specificDNA sequences within the molecule (as opposedto exonucleases, which digest from the ends of DNAmolecules)—are a key tool in recombinant DNA research.These enzymes were called restriction enzymesbecause their presence in a given bacterium restrictedthe growth of certain bacterial viruses called bacteriophages.Restriction enzymes cut DNA of any sourceinto short pieces in a sequence-specific manner—incontrast to most other enzymatic, chemical, or physicalmethods, which break DNA randomly. These defensiveenzymes (hundreds have been discovered) protect thehost bacterial DNA from DNA from foreign organisms(primarily infective phages). However, they are presentonly in cells that also have a companion enzyme whichmethylates the host DNA, rendering it an unsuitablesubstrate for digestion by the restriction enzyme. Thus,

398 / CHAPTER 40DNA 5′RegulatoryregionBasal TranscriptionPoly(A)promoter start siteadditionregionsiteExon ExonCAAT TATA AATAAA3′5′NoncodingregionIntron 3′NoncodingregionNUCLEUSTranscriptionPrimary RNA transcriptPPPModification of5′ and 3′ endsModified transcriptProcessed nuclear mRNACapAAA---APoly(A) tailRemoval of intronsand splicing of exonsAAA---ACYTOPLASMmRNATransmembranetransportTranslationAAA---AProteinNH 2COOHFigure 40–1. Organization of a eukaryotic transcription unit and the pathway of eukaryotic gene expression.Eukaryotic genes have structural and regulatory regions. The structural region consists of the codingDNA and 5′ and 3′ noncoding DNA sequences. The coding regions are divided into two parts: (1) exons, whicheventually are ligated together to become mature RNA, and (2) introns, which are processed out of the primarytranscript. The structural region is bounded at its 5′ end by the transcription initiation site and at its3′ end by the polyadenylate addition or termination site. The promoter region, which contains specific DNAsequences that interact with various protein factors to regulate transcription, is discussed in detail in Chapters37 and 39. The primary transcript has a special structure, a cap, at the 5′ end and a stretch of As at the 3′end. This transcript is processed to remove the introns; and the mature mRNA is then transported to the cytoplasm,where it is translated into protein.site-specific DNA methylases and restriction enzymesalways exist in pairs in a bacterium.Restriction enzymes are named after the bacteriumfrom which they are isolated. For example,EcoRI is from Escherichia coli, and BamHI is from Bacillusamyloliquefaciens (Table 40–2). The first three lettersin the restriction enzyme name consist of the first letterof the genus (E) and the first two letters of the species(co). These may be followed by a strain designation (R)and a roman numeral (I) to indicate the order of discovery(eg, EcoRI, EcoRII). Each enzyme recognizes andcleaves a specific double-stranded DNA sequence that is4–7 bp long. These DNA cuts result in blunt ends (eg,HpaI) or overlapping (sticky) ends (eg, BamHI) (Figure40–2), depending on the mechanism used by the enzyme.Sticky ends are particularly useful in constructinghybrid or chimeric DNA molecules (see below). If thefour nucleotides are distributed randomly in a givenDNA molecule, one can calculate how frequently agiven enzyme will cut a length of DNA. For each positionin the DNA molecule, there are four possibilities(A, C, G, and T); therefore, a restriction enzyme thatrecognizes a 4-bp sequence cuts, on average, once every256 bp (4 4 ), whereas another enzyme that recognizes a6-bp sequence cuts once every 4096 bp (4 6 ). A givenpiece of DNA has a characteristic linear array of sites for

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 399Table 40–1. Variations in the size and complexityof some human genes and mRNAs. 1mRNAGene Size Number SizeGene (kb) of Introns (kb)β-Globin 1.5 2 0.6Insulin 1.7 2 0.4β-Adrenergic receptor 3 0 2.2Albumin 25 14 2.1LDL receptor 45 17 5.5Factor VIII 186 25 9.0Thyroglobulin 300 36 8.71 The sizes are given in kilobases (kb). The sizes of the genes includesome proximal promoter and regulatory region sequences;these are generally about the same size for all genes. Genes varyin size from about 1500 base pairs (bp) to over 2 × 10 6 bp. There isalso great variation in the number of introns and exons. Theβ-adrenergic receptor gene is intronless, and the thyroglobulingene has 36 introns. As noted by the smaller difference in mRNAsizes, introns comprise most of the gene sequence.the various enzymes dictated by the linear sequence ofits bases; hence, a restriction map can be constructed.When DNA is digested with a given enzyme, the endsof all the fragments have the same DNA sequence. Thefragments produced can be isolated by electrophoresison agarose or polyacrylamide gels (see the discussion ofblot transfer, below); this is an essential step in cloningand a major use of these enzymes.A number of other enzymes that act on DNA andRNA are an important part of recombinant DNA technology.Many of these are referred to in this and subsequentchapters (Table 40–3).Restriction Enzymes & DNA Ligase AreUsed to Prepare Chimeric DNA MoleculesSticky-end ligation is technically easy, but some specialtechniques are often required to overcome problems inherentin this approach. Sticky ends of a vector may reconnectwith themselves, with no net gain of DNA.Sticky ends of fragments can also anneal, so that tandemheterogeneous inserts form. Also, sticky-end sites maynot be available or in a convenient position. To circumventthese problems, an enzyme that generates bluntends is used, and new ends are added using the enzymeterminal transferase. If poly d(G) is added to the 3′ endsof the vector and poly d(C) is added to the 3′ ends ofthe foreign DNA, the two molecules can only anneal toeach other, thus circumventing the problems listedabove. This procedure is called homopolymer tailing.Sometimes, synthetic blunt-ended duplex oligonucleotidelinkers with a convenient restriction enzyme se-Table 40–2. Selected restriction endonucleasesand their sequence specificities. 1Sequence Recognized BacterialEndonuclease Cleavage Sites Shown Source↓BamHI GGATCC Bacillus amylo-CCTAGGliquefaciens H↑↓BgIII AGATCT Bacillus glolbigiiTCTAGA↑↓EcoRI GAATTC Escherichia coliCTTAAGRY13↑↓EcoRII CCTGG Escherichia coliGGACCR245↑↓HindIII AAGCTT HaemophilusTTCGAAinfluenzae R d↑↓Hhal GCGC HaemophilusCGCGhaemolyticus↑↓Hpal GTTAAC HaemophilusCAATTGparainfluenzae↑↓MstII CCTNAGG MicrocoleusGGANTCCstrain↑↓PstI CTGCAG ProvidenciaGACGTC stuartii 164↑↓Taql TCGA ThermusAGCTaquaticus YTI↑1 A, adenine; C, cytosine; G, guanine, T, thymine. Arrows show the siteof cleavage; depending on the site, sticky ends (BamHI) or blunt ends(Hpal) may result. The length of the recognition sequence can be 4 bp(Taql), 5 bp (EcoRII), 6 bp (EcoRI), or 7 bp (MstII) or longer. By convention,these are written in the 5′ or 3′ direction for the upper strand ofeach recognition sequence, and the lower strand is shown with theopposite (ie, 3′ or 5′) polarity. Note that most recognition sequencesare palindromes (ie, the sequence reads the same in opposite directionson the two strands). A residue designated N means that any nucleotideis permitted.

400 / CHAPTER 40A. Sticky or staggered ends5’ G G A T C C 3’BamHI3’ C C T A G G 5’B. Blunt ends5’ G T T A A C 3’HpaI3’ C A A T T G 5’GC CG TC ATTAAG A+G+TATCATCGCGFigure 40–2. Results of restriction endonucleasedigestion. Digestion with a restrictionendonuclease can result in the formationof DNA fragments with sticky, orcohesive, ends (A) or blunt ends (B). This isan important consideration in devisingcloning strategies.quence are ligated to the blunt-ended DNA. Directblunt-end ligation is accomplished using the enzymebacteriophage T4 DNA ligase. This technique, thoughless efficient than sticky-end ligation, has the advantageof joining together any pairs of ends. The disadvantagesare that there is no control over the orientation of insertionor the number of molecules annealed together, andthere is no easy way to retrieve the insert.Cloning Amplifies DNAA clone is a large population of identical molecules, bacteria,or cells that arise from a common ancestor. Molecularcloning allows for the production of a large numberof identical DNA molecules, which can then be characterizedor used for other purposes. This technique isbased on the fact that chimeric or hybrid DNA moleculescan be constructed in cloning vectors—typically bacterialplasmids, phages, or cosmids—which then continueto replicate in a host cell under their own control systems.In this way, the chimeric DNA is amplified. The generalprocedure is illustrated in Figure 40–3.Bacterial plasmids are small, circular, duplex DNAmolecules whose natural function is to confer antibioticresistance to the host cell. Plasmids have several propertiesthat make them extremely useful as cloning vectors.They exist as single or multiple copies within the bacteriumand replicate independently from the bacterialDNA. The complete DNA sequence of many plasmids isknown; hence, the precise location of restriction enzymeTable 40–3. Some of the enzymes used in recombinant DNA research. 1Enzyme Reaction Primary UseAlkaline phosphatase Dephosphorylates 5′ ends of RNA and DNA. Removal of 5′-PO 4 groups prior to kinase labeling to preventself-ligation.BAL 31 nuclease Degrades both the 3′ and 5′ ends of DNA. Progressive shortening of DNA molecules.DNA ligase Catalyzes bonds between DNA molecules. Joining of DNA molecules.DNA polymerase I Synthesizes double-stranded DNA from Synthesis of double-stranded cDNA; nick translation; genersingle-strandedDNA.ation of blunt ends from sticky ends.DNase I Under appropriate conditions, produces Nick translation; mapping of hypersensitive sites; mappingsingle-stranded nicks in DNA.protein-DNA interactions.Exonuclease III Removes nucleotides from 3′ ends of DNA. DNA sequencing; mapping of DNA-protein interactions.λ exonuclease Removes nucleotides from 5′ ends of DNA. DNA sequencing.Polynucleotide kinase Transfers terminal phosphate (γ position)32 P labeling of DNA or RNA.from ATP to 5′-OH groups of DNA or RNA.Reverse transcriptase Synthesizes DNA from RNA template. Synthesis of cDNA from mRNA; RNA (5′ end) mappingstudies.S1 nuclease Degrades single-stranded DNA. Removal of “hairpin” in synthesis of cDNA; RNA mappingstudies (both 5′ and 3′ ends).Terminal transferase Adds nucleotides to the 3′ ends of DNA. Homopolymer tailing.1 Adapted and reproduced, with permission, from Emery AEH: Page 41 in: An Introduction to Recombinant DNA. Wiley, 1984.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 401EcoRIrestrictionendonucleaseTTAAAATTHuman DNACircular plasmid DNALinear plasmid DNAwith sticky endsEcoRI restrictionendonucleaseAATT T T AADNAligaseAATT T T AAAnnealAATTTTAAPiece of human DNA cut withsame restriction nuclease andcontaining same sticky endsAA ATTATTPlasmid DNA molecule with human DNA insert(recombinant DNA molecule)AA ATTATTFigure 40–3. Use of restriction nucleases to make new recombinant or chimeric DNA molecules. When insertedback into a bacterial cell (by the process called transformation), typically only a single plasmid is taken upby a single cell, and the plasmid DNA replicates not only itself but also the physically linked new DNA insert. Sincerecombining the sticky ends, as indicated, regenerates the same DNA sequence recognized by the original restrictionenzyme, the cloned DNA insert can be cleanly cut back out of the recombinant plasmid circle with this endonuclease.If a mixture of all of the DNA pieces created by treatment of total human DNA with a single restrictionnuclease is used as the source of human DNA, a million or so different types of recombinant DNA molecules canbe obtained, each pure in its own bacterial clone. (Modified and reproduced, with permission, from Cohen SN: Themanipulation of genes. Sci Am [July] 1975;233:34.)cleavage sites for inserting the foreign DNA is available.Plasmids are smaller than the host chromosome and aretherefore easily separated from the latter, and the desiredplasmid-inserted DNA is readily removed by cutting theplasmid with the enzyme specific for the restriction siteinto which the original piece of DNA was inserted.Phages usually have linear DNA molecules intowhich foreign DNA can be inserted at several restrictionenzyme sites. The chimeric DNA is collected afterthe phage proceeds through its lytic cycle and producesmature, infective phage particles. A major advantage ofphage vectors is that while plasmids accept DNA piecesabout 6–10 kb long, phages can accept DNA fragments10–20 kb long, a limitation imposed by the amount ofDNA that can be packed into the phage head.Larger fragments of DNA can be cloned in cosmids,which combine the best features of plasmids andphages. Cosmids are plasmids that contain the DNA sequences,so-called cos sites, required for packaginglambda DNA into the phage particle. These vectorsgrow in the plasmid form in bacteria, but since much ofthe unnecessary lambda DNA has been removed, morechimeric DNA can be packaged into the particle head.It is not unusual for cosmids to carry inserts of chimericDNA that are 35–50 kb long. Even larger pieces ofDNA can be incorporated into bacterial artificial chromosome(BAC), yeast artificial chromosome (YAC), orE. coli bacteriophage P1-based (PAC) vectors. Thesevectors will accept and propagate DNA inserts of severalhundred kilobases or more and have largely re-

402 / CHAPTER 40Table 40–4. Cloning capacities of commoncloning vectors.VectorPlasmid pBR322Lambda charon 4ACosmidsBAC, P1YACDNA Insert Size0.01–10 kb10–20 kb35–50 kb50–250 kb500–3000 kbplaced the plasmid, phage, and cosmid vectors for somecloning and gene mapping applications. A comparisonof these vectors is shown in Table 40–4.Because insertion of DNA into a functional regionof the vector will interfere with the action of this region,care must be taken not to interrupt an essentialfunction of the vector. This concept can be exploited,however, to provide a selection technique. For example,the common plasmid vector pBR322 has both tetracycline(tet) and ampicillin (amp) resistance genes. Asingle PstI restriction enzyme site within the amp resistancegene is commonly used as the insertion site for apiece of foreign DNA. In addition to having sticky ends(Table 40–2 and Figure 40–2), the DNA inserted atthis site disrupts the amp resistance gene and makes thebacterium carrying this plasmid amp-sensitive (Figure40–4). Thus, the parental plasmid, which provides resistanceto both antibiotics, can be readily separatedfrom the chimeric plasmid, which is resistant only totetracycline. YACs contain replication and segregationfunctions that work in both bacteria and yeast cells andtherefore can be propagated in either organism.In addition to the vectors described in Table 40–4that are designed primarily for propagation in bacterialcells, vectors for mammalian cell propagation and insertgene (cDNA)/protein expression have also been developed.These vectors are all based upon various eukaryoticviruses that are composed of RNA or DNAgenomes. Notable examples of such viral vectors arethose utilizing adenoviral (DNA-based) and retroviral(RNA-based) genomes. Though somewhat limited inthe size of DNA sequences that can be inserted, suchmammalian viral cloning vectors make up for thisshortcoming because they will efficiently infect a widerange of different cell types. For this reason, variousmammalian viral vectors are being investigated for usein gene therapy experiments.A Library Is a Collectionof Recombinant ClonesThe combination of restriction enzymes and variouscloning vectors allows the entire genome of an organismto be packed into a vector. A collection of these differentrecombinant clones is called a library. A genomiclibrary is prepared from the total DNA of a cell line ortissue. A cDNA library comprises complementaryDNA copies of the population of mRNAs in a tissue.Genomic DNA libraries are often prepared by performingpartial digestion of total DNA with a restriction enzymethat cuts DNA frequently (eg, a four base cuttersuch as TaqI ). The idea is to generate rather large fragmentsso that most genes will be left intact. The BAC,YAC, and P1 vectors are preferred since they can acceptvery large fragments of DNA and thus offer a betterchance of isolating an intact gene on a single DNAfragment.A vector in which the protein coded by the gene introducedby recombinant DNA technology is actuallysynthesized is known as an expression vector. Suchvectors are now commonly used to detect specificcDNA molecules in libraries and to produce proteinsby genetic engineering techniques. These vectors arespecially constructed to contain very active induciblepromoters, proper in-phase translation initiationcodons, both transcription and translation terminationsignals, and appropriate protein processing signals, ifneeded. Some expression vectors even contain genesthat code for protease inhibitors, so that the final yieldof product is enhanced.Probes Search Libraries for SpecificGenes or cDNA MoleculesA variety of molecules can be used to “probe” libraries insearch of a specific gene or cDNA molecule or to defineand quantitate DNA or RNA separated by electrophoresisthrough various gels. Probes are generally pieces ofDNA or RNA labeled with a 32 P-containing nucleotide—orfluorescently labeled nucleotides (morecommonly now). Importantly, neither modification ( 32 Por fluorescent-label) affects the hybridization propertiesof the resulting labeled nucleic acid probes. The probemust recognize a complementary sequence to be effective.A cDNA synthesized from a specific mRNA can beused to screen either a cDNA library for a longer cDNAor a genomic library for a complementary sequence inthe coding region of a gene. A popular technique forfinding specific genes entails taking a short amino acidsequence and, employing the codon usage for thatspecies (see Chapter 38), making an oligonucleotideprobe that will detect the corresponding DNA fragmentin a genomic library. If the sequences match exactly,probes 15–20 nucleotides long will hybridize. cDNAprobes are used to detect DNA fragments on Southernblot transfers and to detect and quantitate RNA onNorthern blot transfers. Specific antibodies can also beused as probes provided that the vector used synthesizesprotein molecules that are recognized by them.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 403Ampicillinresistance geneTetracyclineresistance geneEcoRIEcoRIHindIIIBamHITetracyclineresistance genePstIHindIIIBamHIPstISalICut open withPstIPstISalIAmp rTet rThen insertPstI-cut DNAAmp sTet rHost pBR322Chimeric pBR322Figure 40–4. A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, apiece of DNA is inserted into the unique PstI site. This insertion disrupts the gene coding for a protein that providesampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when platedon a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin cantherefore be used to distinguish clones of plasmid that contain an insert. A similar scheme relying upon productionof an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing aninactive, deleted form of the enzyme β-galactosidase allows for blue-white colony formation on agar plates containinga dye hydrolyzable by β-galactoside. β-Galactosidase-positive colonies are blue.Blotting & Hybridization Techniques AllowVisualization of Specific FragmentsVisualization of a specific DNA or RNA fragmentamong the many thousands of “contaminating” moleculesrequires the convergence of a number of techniques,collectively termed blot transfer. Figure 40–5illustrates the Southern (DNA), Northern (RNA), andWestern (protein) blot transfer procedures. (The first isnamed for the person who devised the technique, andthe other names began as laboratory jargon but are nowaccepted terms.) These procedures are useful in determininghow many copies of a gene are in a given tissueor whether there are any gross alterations in a gene(deletions, insertions, or rearrangements). Occasionally,if a specific base is changed and a restriction site is altered,these procedures can detect a point mutation.The Northern and Western blot transfer techniques areused to size and quantitate specific RNA and proteinmolecules, respectively. A fourth hybridization technique,the Southwestern blot, examines protein•DNAinteractions. Proteins are separated by electrophoresis,renatured, and analyzed for an interaction by hybridizationwith a specific labeled DNA probe.Colony or plaque hybridization is the method bywhich specific clones are identified and purified. Bacteriaare grown on colonies on an agar plate and overlaidwith nitrocellulose filter paper. Cells from each colonystick to the filter and are permanently fixed thereto byheat, which with NaOH treatment also lyses the cellsand denatures the DNA so that it will hybridize withthe probe. A radioactive probe is added to the filter,and (after washing) the hybrid complex is localized byexposing the filter to x-ray film. By matching the spoton the autoradiograph to a colony, the latter can bepicked from the plate. A similar strategy is used to identifyfragments in phage libraries. Successive rounds ofthis procedure result in a clonal isolate (bacterialcolony) or individual phage plaque.All of the hybridization procedures discussed in thissection depend on the specific base-pairing propertiesof complementary nucleic acid strands described above.Perfect matches hybridize readily and withstand hightemperatures in the hybridization and washing reac-

404 / CHAPTER 40Southern Northern WesternDNARNA ProteinGelelectrophoresisTransfer to papertions. Specific complexes also form in the presence oflow salt concentrations. Less than perfect matches donot tolerate these stringent conditions (ie, elevatedtemperatures and low salt concentrations); thus, hybridizationeither never occurs or is disrupted duringthe washing step. Gene families, in which there is somedegree of homology, can be detected by varying thestringency of the hybridization and washing steps.Cross-species comparisons of a given gene can also bemade using this approach. Hybridization conditions capableof detecting just a single base pair mismatch betweenprobe and target have been devised.cDNA*cDNA*Antibody*Add probeAutoradiographFigure 40–5. The blot transfer procedure. In aSouthern, or DNA, blot transfer, DNA isolated from acell line or tissue is digested with one or more restrictionenzymes. This mixture is pipetted into a well in anagarose or polyacrylamide gel and exposed to a directelectrical current. DNA, being negatively charged, migratestoward the anode; the smaller fragments movethe most rapidly. After a suitable time, the DNA is denaturedby exposure to mild alkali and transferred to nitrocelluloseor nylon paper, in an exact replica of thepattern on the gel, by the blotting technique devisedby Southern. The DNA is bound to the paper by exposureto heat, and the paper is then exposed to thelabeled cDNA probe, which hybridizes to complementaryfragments on the filter. After thorough washing,the paper is exposed to x-ray film, which is developedto reveal several specific bands corresponding to theDNA fragment that recognized the sequences in thecDNA probe. The RNA, or Northern, blot is conceptuallysimilar. RNA is subjected to electrophoresis before blottransfer. This requires some different steps from thoseof DNA transfer, primarily to ensure that the RNA remainsintact, and is generally somewhat more difficult.In the protein, or Western, blot, proteins are electrophoresedand transferred to nitrocellulose and thenprobed with a specific antibody or other probe molecule.(Asterisks signify labeling, either radioactive orfluorescent.)Manual & Automatic TechniquesAre Available to Determinethe Sequence of DNAThe segments of specific DNA molecules obtained byrecombinant DNA technology can be analyzed to determinetheir nucleotide sequence. This method dependsupon having a large number of identical DNAmolecules. This requirement can be satisfied by cloningthe fragment of interest, using the techniques describedabove. The manual enzymatic method (Sanger) employsspecific dideoxynucleotides that terminate DNAstrand synthesis at specific nucleotides as the strand issynthesized on purified template nucleic acid. The reactionsare adjusted so that a population of DNA fragmentsrepresenting termination at every nucleotide isobtained. By having a radioactive label incorporated atthe end opposite the termination site, one can separatethe fragments according to size using polyacrylamidegel electrophoresis. An autoradiograph is made, andeach of the fragments produces an image (band) on anx-ray film. These are read in order to give the DNA sequence(Figure 40–6). Another manual method, that ofMaxam and Gilbert, employs chemical methods tocleave the DNA molecules where they contain the specificnucleotides. Techniques that do not require theuse of radioisotopes are commonly employed in automatedDNA sequencing. Most commonly employed isan automated procedure in which four different fluorescentlabels—one representing each nucleotide—areused. Each emits a specific signal upon excitation by alaser beam, and this can be recorded by a computer.Oligonucleotide Synthesis Is Now RoutineThe automated chemical synthesis of moderately longoligonucleotides (about 100 nucleotides) of precise sequenceis now a routine laboratory procedure. Eachsynthetic cycle takes but a few minutes, so an entiremolecule can be made by synthesizing relatively shortsegments that can then be ligated to one another.Oligonucleotides are now indispensable for DNA se-

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 405Reaction containing radiolabel:ddGTP ddATP ddTTP ddCTPSequence of original strand:– A – G – T – C – T – T – G – G – A – G – C – T– 3′Slab gelElectrophoresisGA TBases terminatedCA G T C T T G G A G C TFigure 40–6. Sequencing of DNA by the method devised by Sanger. The ladder-like arrays represent from bottomto top all of the successively longer fragments of the original DNA strand. Knowing which specific dideoxynucleotidereaction was conducted to produce each mixture of fragments, one can determine the sequence of nucleotidesfrom the labeled end (asterisk) toward the unlabeled end by reading up the gel. Automated sequencinginvolves the reading of chemically modified deoxynucleotides. The base-pairing rules of Watson and Crick (A–T,G–C) dictate the sequence of the other (complementary) strand. (Asterisks signify radiolabeling.)quencing, library screening, protein-DNA binding,DNA mobility shift assays, the polymerase chain reaction(see below), site-directed mutagenesis, and numerousother applications.The Polymerase Chain Reaction(PCR) Amplifies DNA SequencesThe polymerase chain reaction (PCR) is a method ofamplifying a target sequence of DNA. PCR provides asensitive, selective, and extremely rapid means of amplifyinga desired sequence of DNA. Specificity is basedon the use of two oligonucleotide primers that hybridizeto complementary sequences on oppositestrands of DNA and flank the target sequence (Figure40–7). The DNA sample is first heated to separate thetwo strands; the primers are allowed to bind to theDNA; and each strand is copied by a DNA polymerase,starting at the primer site. The two DNA strands eachserve as a template for the synthesis of new DNA fromthe two primers. Repeated cycles of heat denaturation,annealing of the primers to their complementary sequences,and extension of the annealed primers withDNA polymerase result in the exponential amplificationof DNA segments of defined length. Early PCR reactionsused an E coli DNA polymerase that was destroyedby each heat denaturation cycle. Substitution ofa heat-stable DNA polymerase from Thermus aquaticus(or the corresponding DNA polymerase from otherthermophilic bacteria), an organism that lives and replicatesat 70–80 °C, obviates this problem and has madepossible automation of the reaction, since the polymerasereactions can be run at 70 °C. This has also improvedthe specificity and the yield of DNA.DNA sequences as short as 50–100 bp and as longas 10 kb can be amplified. Twenty cycles provide anamplification of 10 6 and 30 cycles of 10 9 . The PCR allowsthe DNA in a single cell, hair follicle, or spermatozoonto be amplified and analyzed. Thus, the applicationsof PCR to forensic medicine are obvious. ThePCR is also used (1) to detect infectious agents, especiallylatent viruses; (2) to make prenatal genetic diagnoses;(3) to detect allelic polymorphisms; (4) to establishprecise tissue types for transplants; and (5) to study

406 / CHAPTER 40STARTCYCLE 1CYCLE 2CYCLE 3Targeted sequenceevolution, using DNA from archeological samples afterRNA copying and mRNA quantitation by the so-calledRT-PCR method (cDNA copies of mRNA generatedby a retroviral reverse transcriptase). There are an equalnumber of applications of PCR to problems in basicscience, and new uses are developed every year.PRACTICAL APPLICATIONSOF RECOMBINANT DNA TECHNOLOGYARE NUMEROUSThe isolation of a specific gene from an entire genomerequires a technique that will discriminate one part in amillion. The identification of a regulatory region thatmay be only 10 bp in length requires a sensitivity ofone part in 3 × 10 8 ; a disease such as sickle cell anemiais caused by a single base change, or one part in 3 × 10 9 .Recombinant DNA technology is powerful enough toaccomplish all these things.Gene Mapping Localizes SpecificGenes to Distinct ChromosomesGene localizing thus can define a map of the humangenome. This is already yielding useful information inthe definition of human disease. Somatic cell hybridizationand in situ hybridization are two techniques usedto accomplish this. In in situ hybridization, the simplerand more direct procedure, a radioactive probe isadded to a metaphase spread of chromosomes on a glassslide. The exact area of hybridization is localized by layeringphotographic emulsion over the slide and, afterexposure, lining up the grains with some histologicidentification of the chromosome. Fluorescence in situhybridization (FISH) is a very sensitive technique thatis also used for this purpose. This often places the geneat a location on a given band or region on the chromosome.Some of the human genes localized using thesetechniques are listed in Table 40–5. This table representsonly a sampling, since thousands of genes havebeen mapped as a result of the recent sequencing of theCYCLES 4–nFigure 40–7. The polymerase chain reaction is used toamplify specific gene sequences. Double-stranded DNA isheated to separate it into individual strands. These bind twodistinct primers that are directed at specific sequences onopposite strands and that define the segment to be amplified.DNA polymerase extends the primers in each directionand synthesizes two strands complementary to the originaltwo. This cycle is repeated several times, giving an amplifiedproduct of defined length and sequence. Note that the twoprimers are present in excess.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 407Table 40–5. Localization of human genes. 1Gene Chromosome DiseaseInsulin11p15Prolactin6p23-q12Growth hormone 17q21-qter Growth hormone deficiencyα-Globin 16p12-pter α-Thalassemiaβ-Globin 11p12 β-Thalassemia, sickle cellAdenosine deaminase 20q13-qter Adenosine deaminase deficiencyPhenylalanine hydroxylase 12q24 PhenylketonuriaHypoxanthine-guanine Xq26-q27 Lesch-Nyhan syndromephosphoribosyltransferaseDNA segment G8 4p Huntington’s chorea1 This table indicates the chromosomal location of several genes and the diseases associatedwith deficient or abnormal production of the gene products. The chromosomeinvolved is indicated by the first number or letter. The other numbers and letters referto precise localizations, as defined in McKusick VA: Mendelian Inheritance in Man, 6thed. John Hopkins Univ Press, 1983.human genome. Once the defect is localized to a regionof DNA that has the characteristic structure of a gene(Figure 40–1), a synthetic gene can be constructed andexpressed in an appropriate vector and its function canbe assessed—or the putative peptide, deduced from theopen reading frame in the coding region, can be synthesized.Antibodies directed against this peptide can beused to assess whether this peptide is expressed in normalpersons and whether it is absent in those with thegenetic syndrome.Proteins Can Be Producedfor Research & DiagnosisA practical goal of recombinant DNA research is theproduction of materials for biomedical applications.This technology has two distinct merits: (1) It can supplylarge amounts of material that could not be obtainedby conventional purification methods (eg, interferon,tissue plasminogen activating factor). (2) It canprovide human material (eg, insulin, growth hormone).The advantages in both cases are obvious. Although theprimary aim is to supply products—generally proteins—fortreatment (insulin) and diagnosis (AIDStesting) of human and other animal diseases and fordisease prevention (hepatitis B vaccine), there are otherpotential commercial applications, especially in agriculture.An example of the latter is the attempt to engineerplants that are more resistant to drought or temperatureextremes, more efficient at fixing nitrogen, or that produceseeds containing the complete complement of essentialamino acids (rice, wheat, corn, etc).Recombinant DNA Technology Is Usedin the Molecular Analysis of DiseaseA. NORMAL GENE VARIATIONSThere is a normal variation of DNA sequence just as istrue of more obvious aspects of human structure. Variationsof DNA sequence, polymorphisms, occur approximatelyonce in every 500 nucleotides, or about10 7 times per genome. There are without doubt deletionsand insertions of DNA as well as single-base substitutions.In healthy people, these alterations obviouslyoccur in noncoding regions of DNA or at sites thatcause no change in function of the encoded protein.This heritable polymorphism of DNA structure can beassociated with certain diseases within a large kindredand can be used to search for the specific gene involved,as is illustrated below. It can also be used in a variety ofapplications in forensic medicine.B. GENE VARIATIONS CAUSING DISEASEClassic genetics taught that most genetic diseases weredue to point mutations which resulted in an impairedprotein. This may still be true, but if on reading theinitial sections of this chapter one predicted that geneticdisease could result from derangement of any ofthe steps illustrated in Figure 40–1, one would havemade a proper assessment. This point is nicely illustratedby examination of the β-globin gene. This geneis located in a cluster on chromosome 11 (Figure40–8), and an expanded version of the gene is illustratedin Figure 40–9. Defective production of β-globinresults in a variety of diseases and is due to many

408 / CHAPTER 40∋Gγ Aγ Ψβ δ β5′ LCR 3′10 kbHemoglobinopathyβ 0 -Thalassemiaβ 0 -ThalassemiaInvertedHemoglobin Lepore(Aγδβ) 0 -ThalassemiaFigure 40–8. Schematic representation of the β-globin gene cluster and of the lesions in some geneticdisorders. The β-globin gene is located on chromosome 11 in close association with the two γ-globingenes and the δ-globin gene. The β-gene family is arranged in the order 5′-ε-Gγ-Aγ-ψβ-δ-β-3′. Theε locus is expressed in early embryonic life (as a 2 ε 2 ). The γ genes are expressed in fetal life, making fetalhemoglobin (HbF, α 2 γ 2 ). Adult hemoglobin consists of HbA (α 2 β 2 ) or HbA 2 (α 2 δ 2 ). The Ψβ is a pseudogenethat has sequence homology with β but contains mutations that prevent its expression. A locuscontrol region (LCR) located upstream (5′) from the ε gene controls the rate of transcription of the entireβ-globin gene cluster. Deletions (solid bar) of the β locus cause β-thalassemia (deficiency or absence[β 0 ] of β-globin). A deletion of δ and β causes hemoglobin Lepore (only hemoglobin α is present).An inversion (Aγδβ) 0 in this region (colored bar) disrupts gene function and also results in thalassemia(type III). Each type of thalassemia tends to be found in a certain group of people, eg, the (Aγδβ) 0 deletioninversion occurs in persons from India. Many more deletions in this region have been mapped, andeach causes some type of thalassemia.different lesions in and around the β-globin gene(Table 40–6).C. POINT MUTATIONSThe classic example is sickle cell disease, which iscaused by mutation of a single base out of the 3 × 10 9in the genome, a T-to-A DNA substitution, which inturn results in an A-to-U change in the mRNA correspondingto the sixth codon of the β-globin gene. Thealtered codon specifies a different amino acid (valinerather than glutamic acid), and this causes a structuralabnormality of the β-globin molecule. Other point mutationsin and around the β-globin gene result in decreasedproduction or, in some instances, no produc-5′ I1 I23′Figure 40–9. Mutations in the β-globin gene causing β-thalassemia. The β-globin gene is shown in the 5′to 3′ orientation. The cross-hatched areas indicate the 5′ and 3′ nontranslated regions. Reading from the 5′ to3′ direction, the shaded areas are exons 1–3 and the clear spaces are introns 1 (I 1 ) and 2 (I 2 ). Mutations that affecttranscription control (•) are located in the 5′ flanking-region DNA. Examples of nonsense mutations (),mutations in RNA processing (), and RNA cleavage mutations () have been identified and are indicated. Insome regions, many mutations have been found. These are indicated by the brackets.

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 409Table 40–6. Structural alterations of the β-globingene.Alteration Function Affected DiseasePoint mutations Protein folding Sickle cell diseaseTranscriptional control β-ThalassemiaFrameshift and non- β-Thalassemiasense mutationsRNA processing β-ThalassemiaDeletion mRNA production β 0 -ThalassemiaHemoglobinLeporeRearrangement mRNA production β-Thalassemiatype IIItion of β-globin; β-thalassemia is the result of thesemutations. (The thalassemias are characterized by defectsin the synthesis of hemoglobin subunits, and soβ-thalassemia results when there is insufficient productionof β-globin.) Figure 40–9 illustrates that pointmutations affecting each of the many processes involvedin generating a normal mRNA (and therefore anormal protein) have been implicated as a cause ofβ-thalassemia.D. DELETIONS, INSERTIONS, &REARRANGEMENTS OF DNAStudies of bacteria, viruses, yeasts, and fruit flies showthat pieces of DNA can move from one place to anotherwithin a genome. The deletion of a critical pieceof DNA, the rearrangement of DNA within a gene, orthe insertion of a piece of DNA within a coding or regulatoryregion can all cause changes in gene expressionresulting in disease. Again, a molecular analysis ofβ-thalassemia produces numerous examples of theseprocesses—particularly deletions—as causes of disease(Figure 40–8). The globin gene clusters seem particularlyprone to this lesion. Deletions in the α-globincluster, located on chromosome 16, cause α-thalassemia.There is a strong ethnic association for manyof these deletions, so that northern Europeans, Filipinos,blacks, and Mediterranean peoples have differentlesions all resulting in the absence of hemoglobin Aand α-thalassemia.A similar analysis could be made for a number ofother diseases. Point mutations are usually defined bysequencing the gene in question, though occasionally, ifthe mutation destroys or creates a restriction enzymesite, the technique of restriction fragment analysis canbe used to pinpoint the lesion. Deletions or insertionsof DNA larger than 50 bp can often be detected by theSouthern blotting procedure.E. PEDIGREE ANALYSISSickle cell disease again provides an excellent exampleof how recombinant DNA technology can be appliedto the study of human disease. The substitution of Tfor A in the template strand of DNA in the β-globingene changes the sequence in the region that correspondsto the sixth codon fromto↓CCTGAGGGGACT CC↑CCTGTGGGGAC A CCCoding strandTemplate strandCoding strandTemplate strandand destroys a recognition site for the restriction enzymeMstII (CCTNAGG; denoted by the small verticalarrows; Table 40–2). Other MstII sites 5′ and 3′ fromthis site (Figure 40–10) are not affected and so will becut. Therefore, incubation of DNA from normal (AA),heterozygous (AS), and homozygous (SS) individualsresults in three different patterns on Southern blottransfer (Figure 40–10). This illustrates how a DNApedigree can be established using the principles discussedin this chapter. Pedigree analysis has been appliedto a number of genetic diseases and is most usefulin those caused by deletions and insertions or the rarerinstances in which a restriction endonuclease cleavagesite is affected, as in the example cited in this paragraph.The analysis is facilitated by the PCR reaction,which can provide sufficient DNA for analysis fromjust a few nucleated red blood cells.F. PRENATAL DIAGNOSISIf the genetic lesion is understood and a specific probeis available, prenatal diagnosis is possible. DNA fromcells collected from as little as 10 mL of amniotic fluid(or by chorionic villus biopsy) can be analyzed bySouthern blot transfer. A fetus with the restriction patternAA in Figure 40–10 does not have sickle cell disease,nor is it a carrier. A fetus with the SS pattern willdevelop the disease. Probes are now available for thistype of analysis of many genetic diseases.G. RESTRICTION FRAGMENT LENGTHPOLYMORPHISM (RFLP)The differences in DNA sequence cited above can resultin variations of restriction sites and thus in thelength of restriction fragments. An inherited differencein the pattern of restriction (eg, a DNA variation occurringin more than 1% of the general population) isknown as a restriction fragment length polymorphism,

410 / CHAPTER 40A. MstII restriction sites around and in the β-globin geneNormal (A) 5′3′1.15 kb 0.2kbSickle (S) 5′3′1.35 kbB. Pedigree analysisFragmentsize1.35 kb1.15 kbAS AS SS AA AS ASPhenotypeFigure 40–10. Pedigree analysis of sickle cell disease. The top part of the figure(A) shows the first part of the β-globin gene and the MstII restriction enzymesites in the normal (A) and sickle cell (S) β-globin genes. Digestion withthe restriction enzyme MstII results in DNA fragments 1.15 kb and 0.2 kb long innormal individuals. The T-to-A change in individuals with sickle cell diseaseabolishes one of the three MstII sites around the β-globin gene; hence, a singlerestriction fragment 1.35 kb in length is generated in response to MstII. This sizedifference is easily detected on a Southern blot. (The 0.2-kb fragment wouldrun off the gel in this illustration.) (B) Pedigree analysis shows three possibilities:AA = normal (open circle); AS = heterozygous (half-solid circles, half-solidsquare); SS = homozygous (solid square). This approach allows for prenatal diagnosisof sickle cell disease (dash-sided square).

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 411or RFLP. An extensive RFLP map of the humangenome has been constructed. This is proving useful inthe human genome sequencing project and is an importantcomponent of the effort to understand various single-geneand multigenic diseases. RFLPs result fromsingle-base changes (eg, sickle cell disease) or from deletionsor insertions of DNA into a restriction fragment(eg, the thalassemias) and have proved to be useful diagnostictools. They have been found at known geneloci and in sequences that have no known function;thus, RFLPs may disrupt the function of the gene ormay have no biologic consequences.RFLPs are inherited, and they segregate in amendelian fashion. A major use of RFLPs (thousandsare now known) is in the definition of inherited diseasesin which the functional deficit is unknown.RFLPs can be used to establish linkage groups, whichin turn, by the process of chromosome walking, willeventually define the disease locus. In chromosomewalking (Figure 40–11), a fragment representing oneend of a long piece of DNA is used to isolate anotherthat overlaps but extends the first. The direction of extensionis determined by restriction mapping, and theprocedure is repeated sequentially until the desired sequenceis obtained. The X chromosome-linked disordersare particularly amenable to this approach, sinceonly a single allele is expressed. Hence, 20% of the definedRFLPs are on the X chromosome, and a reasonablycomplete linkage map of this chromosome exists.The gene for the X-linked disorder, Duchenne-typemuscular dystrophy, was found using RFLPs. Likewise,the defect in Huntington’s disease was localized to theterminal region of the short arm of chromosome 4, andthe defect that causes polycystic kidney disease is linkedto the α-globin locus on chromosome 16.H. MICROSATELLITE DNA POLYMORPHISMSShort (2–6 bp), inherited, tandem repeat units of DNAoccur about 50,000–100,000 times in the humangenome (Chapter 36). Because they occur more frequently—andin view of the routine application of sensitivePCR methods—they are replacing RFLPs as themarker loci for various genome searches.I. RFLPS & VNTRS INFORENSIC MEDICINEVariable numbers of tandemly repeated (VNTR) unitsare one common type of “insertion” that results in anRFLP. The VNTRs can be inherited, in which casethey are useful in establishing genetic association with adisease in a family or kindred; or they can be unique toan individual and thus serve as a molecular fingerprintof that person.J. GENE THERAPYDiseases caused by deficiency of a gene product (Table40–5) are amenable to replacement therapy. The strategyis to clone a gene (eg, the gene that codes foradenosine deaminase) into a vector that will readily betaken up and incorporated into the genome of a hostcell. Bone marrow precursor cells are being investigatedfor this purpose because they presumably will resettle inthe marrow and replicate there. The introduced genewould begin to direct the expression of its protein product,and this would correct the deficiency in the hostcell.K. TRANSGENIC ANIMALSThe somatic cell gene replacement described abovewould obviously not be passed on to offspring. Otherstrategies to alter germ cell lines have been devised buthave been tested only in experimental animals. A certainIntact DNA 5′Gene X3′FragmentsInitialprobe1*23Figure 40–11. The technique of chromosome walking. Gene X is to be isolated from a large pieceof DNA. The exact location of this gene is not known, but a probe (*——) directed against a fragmentof DNA (shown at the 5′ end in this representation) is available, as is a library containing a seriesof overlapping DNA fragments. For the sake of simplicity, only five of these are shown. The initialprobe will hybridize only with clones containing fragment 1, which can then be isolated and used asa probe to detect fragment 2. This procedure is repeated until fragment 4 hybridizes with fragment5, which contains the entire sequence of gene X.45

412 / CHAPTER 40percentage of genes injected into a fertilized mouse ovumwill be incorporated into the genome and found in bothsomatic and germ cells. Hundreds of transgenic animalshave been established, and these are useful for analysis oftissue-specific effects on gene expression and effects ofoverproduction of gene products (eg, those from thegrowth hormone gene or oncogenes) and in discoveringgenes involved in development—a process that heretoforehas been difficult to study. The transgenic approachhas recently been used to correct a genetic deficiency inmice. Fertilized ova obtained from mice with genetic hypogonadismwere injected with DNA containing thecoding sequence for the gonadotropin-releasing hormone(GnRH) precursor protein. This gene was expressed andregulated normally in the hypothalamus of a certainnumber of the resultant mice, and these animals were inall respects normal. Their offspring also showed no evidenceof GnRH deficiency. This is, therefore, evidenceof somatic cell expression of the transgene and of itsmaintenance in germ cells.Targeted Gene Disruption or KnockoutIn transgenic animals, one is adding one or more copiesof a gene to the genome, and there is no way to controlwhere that gene eventually resides. A complementary—and much more difficult—approach involves the selectiveremoval of a gene from the genome. Gene knockoutanimals (usually mice) are made by creating amutation that totally disrupts the function of a gene.This is then used to replace one of the two genes in anembryonic stem cell that can be used to create a heterozygoustransgenic animal. The mating of two suchanimals will, by mendelian genetics, result in a homozygousmutation in 25% of offspring. Several hundredstrains of mice with knockouts of specific geneshave been developed.RNA Transcript & Protein ProfilingThe “-omic” revolution of the last several years has culminatedin the determination of the nucleotide sequencesof entire genomes, including those of buddingand fission yeasts, various bacteria, the fruit fly, the wormCaenorhabditis elegans, the mouse and, most notably, humans.Additional genomes are being sequenced at an acceleratingpace. The availability of all of this DNA sequenceinformation, coupled with engineering advances,has lead to the development of several revolutionarymethodologies, most of which are based upon high-densitymicroarray technology. We now have the ability todeposit thousands of specific, known, definable DNA sequences(more typically now synthetic oligonucleotides)on a glass microscope-style slide in the space of a fewsquare centimeters. By coupling such DNA microarrayswith highly sensitive detection of hybridized fluorescentlylabeled nucleic acid probes derived from mRNA,investigators can rapidly and accurately generate profilesof gene expression (eg, specific cellular mRNA content)from cell and tissue samples as small as 1 gram or less.Thus entire transcriptome information (the entire collectionof cellular mRNAs) for such cell or tissue sourcescan readily be obtained in only a few days. Transcriptomeinformation allows one to predict the collection ofproteins that might be expressed in a particular cell, tissue,or organ in normal and disease states based upon themRNAs present in those cells. Complementing this highthroughput,transcript-profiling method is the recent developmentof high-sensitivity, high-throughput massspectrometry of complex protein samples. Newer massspectrometry methods allow one to identify hundreds tothousands of proteins in proteins extracted from verysmall numbers of cells (< 1 g). This critical informationtells investigators which of the many mRNAs detected intranscript microarray mapping studies are actually translatedinto protein, generally the ultimate dictator of phenotype.Microarray techniques and mass spectrometricprotein identification experiments both lead to the generationof huge amounts of data. Appropriate data managementand interpretation of the deluge of informationforthcoming from such studies has relied upon statisticalmethods; and this new technology, coupled with theflood of DNA sequence information, has led to the developmentof the field of bioinformatics, a new disciplinewhose goal is to help manage, analyze, and integratethis flood of biologically important information.Future work at the intersection of bioinformatics andtranscript-protein profiling will revolutionize our understandingof biology and medicine.SUMMARY• A variety of very sensitive techniques can now be appliedto the isolation and characterization of genesand to the quantitation of gene products.• In DNA cloning, a particular segment of DNA is removedfrom its normal environment using one ofmany restriction endonucleases. This is then ligatedinto one of several vectors in which the DNA segmentcan be amplified and produced in abundance.• The cloned DNA can be used as a probe in one ofseveral types of hybridization reactions to detectother related or adjacent pieces of DNA, or it can beused to quantitate gene products such as mRNA.• Manipulation of the DNA to change its structure, socalledgenetic engineering, is a key element in cloning(eg, the construction of chimeric molecules) and can

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 413also be used to study the function of a certain fragmentof DNA and to analyze how genes are regulated.• Chimeric DNA molecules are introduced into cellsto make transfected cells or into the fertilized oocyteto make transgenic animals.• Techniques involving cloned DNA are used to locategenes to specific regions of chromosomes, to identifythe genes responsible for diseases, to study how faultygene regulation causes disease, to diagnose geneticdiseases, and increasingly to treat genetic diseases.GLOSSARYARS: Autonomously replicating sequence; the originof replication in yeast.Autoradiography: The detection of radioactivemolecules (eg, DNA, RNA, protein) by visualizationof their effects on photographic film.Bacteriophage: A virus that infects a bacterium.Blunt-ended DNA: Two strands of a DNA duplexhaving ends that are flush with each other.cDNA: A single-stranded DNA molecule that iscomplementary to an mRNA molecule and is synthesizedfrom it by the action of reverse transcriptase.Chimeric molecule: A molecule (eg, DNA, RNA,protein) containing sequences derived from twodifferent species.Clone: A large number of organisms, cells or moleculesthat are identical with a single parental organismcell or molecule.Cosmid: A plasmid into which the DNA sequencesfrom bacteriophage lambda that are necessary forthe packaging of DNA (cos sites) have been inserted;this permits the plasmid DNA to be packagedin vitro.Endonuclease: An enzyme that cleaves internalbonds in DNA or RNA.Excinuclease: The excision nuclease involved in nucleotideexchange repair of DNA.Exon: The sequence of a gene that is represented(expressed) as mRNA.Exonuclease: An enzyme that cleaves nucleotidesfrom either the 3′ or 5′ ends of DNA or RNA.Fingerprinting: The use of RFLPs or repeat sequenceDNA to establish a unique pattern ofDNA fragments for an individual.Footprinting: DNA with protein bound is resistantto digestion by DNase enzymes. When a sequencingreaction is performed using such DNA, a protectedarea, representing the “footprint” of thebound protein, will be detected.Hairpin: A double-helical stretch formed by basepairing between neighboring complementary sequencesof a single strand of DNA or RNA.Hybridization: The specific reassociation of complementarystrands of nucleic acids (DNA withDNA, DNA with RNA, or RNA with RNA).Insert: An additional length of base pairs in DNA,generally introduced by the techniques of recombinantDNA technology.Intron: The sequence of a gene that is transcribedbut excised before translation.Library: A collection of cloned fragments that representsthe entire genome. Libraries may be eithergenomic DNA (in which both introns and exonsare represented) or cDNA (in which only exonsare represented).Ligation: The enzyme-catalyzed joining in phosphodiesterlinkage of two stretches of DNA orRNA into one; the respective enzymes are DNAand RNA ligases.Lines: Long interspersed repeat sequences.Microsatellite polymorphism: Heterozygosity of acertain microsatellite repeat in an individual.Microsatellite repeat sequences: Dispersed orgroup repeat sequences of 2–5 bp repeated up to50 times. May occur at 50–100 thousand locationsin the genome.Nick translation: A technique for labeling DNAbased on the ability of the DNA polymerase fromE coli to degrade a strand of DNA that has beennicked and then to resynthesize the strand; if a radioactivenucleoside triphosphate is employed, therebuilt strand becomes labeled and can be used asa radioactive probe.Northern blot: A method for transferring RNAfrom an agarose gel to a nitrocellulose filter, onwhich the RNA can be detected by a suitableprobe.Oligonucleotide: A short, defined sequence of nucleotidesjoined together in the typical phosphodiesterlinkage.Ori: The origin of DNA replication.PAC: A high capacity (70–95 kb) cloning vectorbased upon the lytic E. coli bacteriophage P1 thatreplicates in bacteria as an extrachromosomal element.Palindrome: A sequence of duplex DNA that is thesame when the two strands are read in opposite directions.Plasmid: A small, extrachromosomal, circular moleculeof DNA that replicates independently of thehost DNA.Polymerase chain reaction (PCR): An enzymaticmethod for the repeated copying (and thus amplification)of the two strands of DNA that make upa particular gene sequence.

414 / CHAPTER 40Primosome: The mobile complex of helicase andprimase that is involved in DNA replication.Probe: A molecule used to detect the presence of aspecific fragment of DNA or RNA in, for instance,a bacterial colony that is formed from a geneticlibrary or during analysis by blot transfertechniques; common probes are cDNA molecules,synthetic oligodeoxynucleotides of defined sequence,or antibodies to specific proteins.Proteome: The entire collection of expressed proteinsin an organism.Pseudogene: An inactive segment of DNA arisingby mutation of a parental active gene.Recombinant DNA: The altered DNA that resultsfrom the insertion of a sequence of deoxynucleotidesnot previously present into an existingmolecule of DNA by enzymatic or chemicalmeans.Restriction enzyme: An endodeoxynuclease thatcauses cleavage of both strands of DNA at highlyspecific sites dictated by the base sequence.Reverse transcription: RNA-directed synthesis ofDNA, catalyzed by reverse transcriptase.RT-PCR: A method used to quantitate mRNA levelsthat relies upon a first step of cDNA copying ofmRNAs prior to PCR amplification and quantitation.Signal: The end product observed when a specificsequence of DNA or RNA is detected by autoradiographyor some other method. Hybridizationwith a complementary radioactive polynucleotide(eg, by Southern or Northern blotting) is commonlyused to generate the signal.Sines: Short interspersed repeat sequences.SNP: Single nucleotide polymorphism. Refers tothe fact that single nucleotide genetic variation ingenome sequence exists at discrete loci throughoutthe chromosomes. Measurement of allelic SNPdifferences is useful for gene mapping studies.snRNA: Small nuclear RNA. This family of RNAsis best known for its role in mRNA processing.Southern blot: A method for transferring DNAfrom an agarose gel to nitrocellulose filter, onwhich the DNA can be detected by a suitableprobe (eg, complementary DNA or RNA).Southwestern blot: A method for detecting protein-DNAinteractions by applying a labeled DNAprobe to a transfer membrane that contains a renaturedprotein.Spliceosome: The macromolecular complex responsiblefor precursor mRNA splicing. The spliceosomeconsists of at least five small nuclear RNAs(snRNA; U1, U2, U4, U5, and U6) and manyproteins.Splicing: The removal of introns from RNA accompaniedby the joining of its exons.Sticky-ended DNA: Complementary single strandsof DNA that protrude from opposite ends of aDNA duplex or from the ends of different duplexmolecules (see also Blunt-ended DNA, above).Tandem: Used to describe multiple copies of thesame sequence (eg, DNA) that lie adjacent to oneanother.Terminal transferase: An enzyme that adds nucleotidesof one type (eg, deoxyadenonucleotidylresidues) to the 3′ end of DNA strands.Transcription: Template DNA-directed synthesisof nucleic acids; typically DNA-directed synthesisof RNA.Transcriptome: The entire collection of expressedmRNAs in an organism.Transgenic: Describing the introduction of newDNA into germ cells by its injection into the nucleusof the ovum.Translation: Synthesis of protein using mRNA astemplate.Vector: A plasmid or bacteriophage into which foreignDNA can be introduced for the purposes ofcloning.Western blot: A method for transferring protein toa nitrocellulose filter, on which the protein can bedetected by a suitable probe (eg, an antibody).REFERENCESLewin B: Genes VII. Oxford Univ Press, 1999.Martin JB, Gusella JF: Huntington’s disease: pathogenesis andmanagement. N Engl J Med 1986:315:1267.Sambrook J, Fritsch EF, Maniatis T: Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press, 1989.Spector DL, Goldman RD, Leinwand LA: Cells: A LaboratoryManual. Cold Spring Harbor Laboratory Press, 1998.Watson JD et al: Recombinant DNA, 2nd ed. Scientific AmericanBooks. Freeman, 1992.Weatherall DJ: The New Genetics and Clinical Practice, 3rd ed. OxfordUniv Press, 1991.

SECTION VBiochemistry of Extracellular& Intracellular CommunicationMembranes: Structure & Function 41Robert K. Murray, MD, PhD, & Daryl K. Granner, MDBIOMEDICAL IMPORTANCEMembranes are highly viscous, plastic structures.Plasma membranes form closed compartments aroundcellular protoplasm to separate one cell from anotherand thus permit cellular individuality. The plasmamembrane has selective permeabilities and acts as abarrier, thereby maintaining differences in compositionbetween the inside and outside of the cell. The selectivepermeabilities are provided mainly by channels andpumps for ions and substrates. The plasma membranealso exchanges material with the extracellular environmentby exocytosis and endocytosis, and there are specialareas of membrane structure—the gap junctions—through which adjacent cells exchange material. Inaddition, the plasma membrane plays key roles in cellcellinteractions and in transmembrane signaling.Membranes also form specialized compartmentswithin the cell. Such intracellular membranes helpshape many of the morphologically distinguishablestructures (organelles), eg, mitochondria, ER, sarcoplasmicreticulum, Golgi complexes, secretory granules,lysosomes, and the nuclear membrane. Membranes localizeenzymes, function as integral elements in excitation-responsecoupling, and provide sites of energytransduction, such as in photosynthesis and oxidativephosphorylation.Changes in membrane structure (eg caused by ischemia)can affect water balance and ion flux and thereforeevery process within the cell. Specific deficienciesor alterations of certain membrane components lead toa variety of diseases (see Table 41–5). In short, normalcellular function depends on normal membranes.415MAINTENANCE OF A NORMAL INTRA-& EXTRACELLULAR ENVIRONMENTIS FUNDAMENTAL TO LIFELife originated in an aqueous environment; enzyme reactions,cellular and subcellular processes, and so forthhave therefore evolved to work in this milieu. Sincemammals live in a gaseous environment, how is theaqueous state maintained? Membranes accomplish thisby internalizing and compartmentalizing body water.The Body’s Internal WaterIs CompartmentalizedWater makes up about 60% of the lean body mass ofthe human body and is distributed in two large compartments.A. INTRACELLULAR FLUID (ICF)This compartment constitutes two-thirds of total bodywater and provides the environment for the cell (1) tomake, store, and utilize energy; (2) to repair itself;(3) to replicate; and (4) to perform special functions.B. EXTRACELLULAR FLUID (ECF)This compartment contains about one-third of totalbody water and is distributed between the plasma andinterstitial compartments. The extracellular fluid is adelivery system. It brings to the cells nutrients (eg, glucose,fatty acids, amino acids), oxygen, various ions andtrace minerals, and a variety of regulatory molecules(hormones) that coordinate the functions of widely separatedcells. Extracellular fluid removes CO 2 , waste

416 / CHAPTER 41products, and toxic or detoxified materials from the immediatecellular environment.Myelin0.23The Ionic Compositions of Intracellular& Extracellular Fluids Differ GreatlyAs illustrated in Table 41–1, the internal environmentis rich in K + and Mg 2+ , and phosphate is its majoranion. Extracellular fluid is characterized by high Na +and Ca 2+ content, and Cl − is the major anion. Notealso that the concentration of glucose is higher in extracellularfluid than in the cell, whereas the opposite istrue for proteins. Why is there such a difference? It isthought that the primordial sea in which life originatedwas rich in K + and Mg 2+ . It therefore follows that enzymereactions and other biologic processes evolved tofunction best in that environment—hence the highconcentration of these ions within cells. Cells werefaced with strong selection pressure as the sea graduallychanged to a composition rich in Na + and Ca 2+ . Vastchanges would have been required for evolution of acompletely new set of biochemical and physiologic machinery;instead, as it happened, cells developed barriers—membraneswith associated “pumps”—to maintainthe internal microenvironment.MEMBRANES ARE COMPLEXSTRUCTURES COMPOSED OF LIPIDS,PROTEINS, & CARBOHYDRATESWe shall mainly discuss the membranes present in eukaryoticcells, although many of the principles describedalso apply to the membranes of prokaryotes.The various cellular membranes have different compositions,as reflected in the ratio of protein to lipid (Figure41–1). This is not surprising, given their divergentfunctions. Membranes are asymmetric sheet-like enclosedstructures with distinct inner and outer surfaces.Table 41–1. Comparison of the meanconcentrations of various molecules outside andinside a mammalian cell.Substance Extracellular Fluid Intracellular FluidNa + 140 mmol/L 10 mmol/LK + 4 mmol/L 140 mmol/LCa 2+ (free) 2.5 mmol/L 0.1 µmol/LMg 2+ 1.5 mmol/L 30 mmol/LCI − 100 mmol/L 4 mmol/L−HCO 3 27 mmol/L 10 mmol/L3−PO 4 2 mmol/L 60 mmol/LGlucose 5.5 mmol/L 0–1 mmol/LProtein 2 g/dL 16 g/dLPlasma membraneMouseliver cellsRetinal rods(bovine)HumanerythrocyteAmebaHeLa cellsMitochondrialouter membraneSarcoplasmicreticulumMitochondrialinner membrane0.851.01.11.31.11.52.0Ratio of protein to lipid3.20 1 2 3 4Figure 41–1. Ratio of protein to lipid in differentmembranes. Proteins equal or exceed the quantity oflipid in nearly all membranes. The outstanding exceptionis myelin, an electrical insulator found on manynerve fibers.These sheet-like structures are noncovalent assembliesthat are thermodynamically stable and metabolically active.Numerous proteins are located in membranes,where they carry out specific functions of the organelle,the cell, or the organism.The Major Lipids in MammalianMembranes Are Phospholipids,Glycosphingolipids, & CholesterolA. PHOSPHOLIPIDSOf the two major phospholipid classes present in membranes,phosphoglycerides are the more common andconsist of a glycerol backbone to which are attachedtwo fatty acids in ester linkage and a phosphorylated alcohol(Figure 41–2). The fatty acid constituents areusually even-numbered carbon molecules, most commonlycontaining 16 or 18 carbons. They are unbranchedand can be saturated or unsaturated. The simplestphosphoglyceride is phosphatidic acid, which is

MEMBRANES: STRUCTURE & FUNCTION / 417Fatty acidsR 1R 2OC O 1 CH 2C O 2 CHOO –3 CH 2 O P O R 3GlycerolOAlcoholFigure 41–2. A phosphoglyceride showing the fattyacids (R 1 and R 2 ), glycerol, and phosphorylated alcoholcomponents. In phosphatidic acid, R 3 is hydrogen.1,2-diacylglycerol 3-phosphate, a key intermediate inthe formation of all other phosphoglycerides (Chapter24). In other phosphoglycerides, the 3-phosphate is esterifiedto an alcohol such as ethanolamine, choline,serine, glycerol, or inositol (Chapter 14).The second major class of phospholipids is composedof sphingomyelin, which contains a sphingosinebackbone rather than glycerol. A fatty acid is attachedby an amide linkage to the amino group of sphingosine,forming ceramide. The primary hydroxyl group ofsphingosine is esterified to phosphorylcholine. Sphingomyelin,as the name implies, is prominent in myelinsheaths.The amounts and fatty acid compositions of the variousphospholipids vary among the different cellularmembranes.B. GLYCOSPHINGOLIPIDSThe glycosphingolipids (GSLs) are sugar-containinglipids built on a backbone of ceramide; they includegalactosyl- and glucosylceramide (cerebrosides) and thegangliosides. Their structures are described in Chapter14. They are mainly located in the plasma membranesof cells.C. STEROLSThe most common sterol in membranes is cholesterol(Chapter 14), which resides mainly in the plasma membranesof mammalian cells but can also be found inlesser quantities in mitochondria, Golgi complexes, andnuclear membranes. Cholesterol intercalates among thephospholipids of the membrane, with its hydroxylgroup at the aqueous interface and the remainder of themolecule within the leaflet. Its effect on the fluidity ofmembranes is discussed subsequently.All of the above lipids can be separated from one anotherby techniques such as column, thin layer, andgas-liquid chromatography and their structures establishedby mass spectrometry.Each eukaryotic cell membrane has a somewhat differentlipid composition, though phospholipids are themajor class in all.Membrane Lipids Are AmphipathicAll major lipids in membranes contain both hydrophobicand hydrophilic regions and are therefore termed“amphipathic.” Membranes themselves are thus amphipathic.If the hydrophobic regions were separatedfrom the rest of the molecule, it would be insoluble inwater but soluble in oil. Conversely, if the hydrophilicregion were separated from the rest of the molecule, itwould be insoluble in oil but soluble in water. The amphipathicnature of a phospholipid is represented inFigure 41–3. Thus, the polar head groups of the phospholipidsand the hydroxyl group of cholesterol interfacewith the aqueous environment; a similar situationapplies to the sugar moieties of the GSLs (see below).Saturated fatty acids have straight tails, whereasunsaturated fatty acids, which generally exist in the cisform in membranes, make kinked tails (Figure 41–3).As more kinks are inserted in the tails, the membranebecomes less tightly packed and therefore more fluid.Detergents are amphipathic molecules that are importantin biochemistry as well as in the household. Themolecular structure of a detergent is not unlike that of aphospholipid. Certain detergents are widely used to solubilizemembrane proteins as a first step in their purification.The hydrophobic end of the detergent binds toS U S SPolar head groupApolar, hydrocarbon tailsFigure 41–3. Diagrammatic representation of aphospholipid or other membrane lipid. The polar headgroup is hydrophilic, and the hydrocarbon tails are hydrophobicor lipophilic. The fatty acids in the tails aresaturated (S) or unsaturated (U); the former are usuallyattached to carbon 1 of glycerol and the latter to carbon2. Note the kink in the tail of the unsaturated fattyacid (U), which is important in conferring increasedmembrane fluidity.

418 / CHAPTER 41hydrophobic regions of the proteins, displacing most oftheir bound lipids. The polar end of the detergent isfree, bringing the proteins into solution as detergentproteincomplexes, usually also containing some residuallipids.Figure 41–4. Diagrammatic cross-section of a micelle.The polar head groups are bathed in water,whereas the hydrophobic hydrocarbon tails are surroundedby other hydrocarbons and thereby protectedfrom water. Micelles are relatively small (comparedwith lipid bilayers) spherical structures.AqueousAqueousHydrophilicHydrophobicHydrophilicFigure 41–5. Diagram of a section of a bilayer membraneformed from phospholipid molecules. The unsaturatedfatty acid tails are kinked and lead to more spacingbetween the polar head groups, hence to moreroom for movement. This in turn results in increasedmembrane fluidity. (Slightly modified and reproduced,with permission, from Stryer L: Biochemistry, 2nd ed. Freeman,1981.)Membrane Lipids Form BilayersThe amphipathic character of phospholipids suggeststhat the two regions of the molecule have incompatiblesolubilities; however, in a solvent such as water, phospholipidsorganize themselves into a form that thermodynamicallyserves the solubility requirements of bothregions. A micelle (Figure 41–4) is such a structure;the hydrophobic regions are shielded from water, whilethe hydrophilic polar groups are immersed in the aqueousenvironment. However, micelles are usually relativelysmall in size (eg, approximately 200 nm) andthus are limited in their potential to form membranes.As was recognized in 1925 by Gorter and Grendel, abimolecular layer, or lipid bilayer, can also satisfy thethermodynamic requirements of amphipathic moleculesin an aqueous environment. Bilayers, not micelles,are indeed the key structures in biologic membranes.A bilayer exists as a sheet in which thehydrophobic regions of the phospholipids are protectedfrom the aqueous environment, while the hydrophilicregions are immersed in water (Figure 41–5). Only theends or edges of the bilayer sheet are exposed to an unfavorableenvironment, but even these exposed edgescan be eliminated by folding the sheet back upon itselfto form an enclosed vesicle with no edges. A bilayer canextend over relatively large distances (eg, 1 mm). Theclosed bilayer provides one of the most essential propertiesof membranes. It is impermeable to most watersolublemolecules, since they would be insoluble in thehydrophobic core of the bilayer.Lipid bilayers are formed by self-assembly, drivenby the hydrophobic effect. When lipid moleculescome together in a bilayer, the entropy of the surroundingsolvent molecules increases.Two questions arise from consideration of theabove. First, how many biologic materials are lipidsolubleand can therefore readily enter the cell? Gasessuch as oxygen, CO 2 , and nitrogen—small moleculeswith little interaction with solvents—readily diffusethrough the hydrophobic regions of the membrane.The permeability coefficients of several ions and of anumber of other molecules in a lipid bilayer are shownin Figure 41–6. The three electrolytes shown (Na + , K + ,and Cl − ) cross the bilayer much more slowly thanwater. In general, the permeability coefficients of smallmolecules in a lipid bilayer correlate with their solubilitiesin nonpolar solvents. For instance, steroids morereadily traverse the lipid bilayer compared with electrolytes.The high permeability coefficient of water itselfis surprising but is partly explained by its small sizeand relative lack of charge.The second question concerns molecules that arenot lipid-soluble: How are the transmembrane concentrationgradients for non-lipid-soluble molecules maintained?The answer is that membranes contain proteins,

MEMBRANES: STRUCTURE & FUNCTION / 419K + TryptophanNa + Cl – Glucose Urea,glycerolIndoleH 2 O10 –14 10 –12 10 –10 10 –8 10 –6 10 –4 10 –2Permeability coefficient (cm/s)LowHighPermeabilityFigure 41–6. Permeability coefficients of water,some ions, and other small molecules in lipid bilayermembranes. Molecules that move rapidly through agiven membrane are said to have a high permeabilitycoefficient. (Slightly modified and reproduced, with permission,from Stryer L: Biochemistry, 2nd ed. Freeman,1981.)and proteins are also amphipathic molecules that can beinserted into the correspondingly amphipathic lipid bilayer.Proteins form channels for the movement of ionsand small molecules and serve as transporters for largermolecules that otherwise could not pass the bilayer.These processes are described below.Membrane Proteins Are AssociatedWith the Lipid BilayerMembrane phospholipids act as a solvent for membraneproteins, creating an environment in which thelatter can function. Of the 20 amino acids contributingto the primary structure of proteins, the functionalgroups attached to the α carbon are strongly hydrophobicin six, weakly hydrophobic in a few, and hydrophilicin the remainder. As described in Chapter 5,the α-helical structure of proteins minimizes the hydrophiliccharacter of the peptide bonds themselves.Thus, proteins can be amphipathic and form an integralpart of the membrane by having hydrophilic regionsprotruding at the inside and outside faces of themembrane but connected by a hydrophobic region traversingthe hydrophobic core of the bilayer. In fact,those portions of membrane proteins that traversemembranes do contain substantial numbers of hydrophobicamino acids and almost invariably have eithera high α-helical or β-pleated sheet content. Formany membranes, a stretch of approximately 20 aminoacids in an α helix will span the bilayer.It is possible to calculate whether a particular sequenceof amino acids present in a protein is consistentwith a transmembrane location. This can be done byconsulting a table that lists the hydrophobicities of eachof the 20 common amino acids and the free energy valuesfor their transfer from the interior of a membraneto water. Hydrophobic amino acids have positive values;polar amino acids have negative values. The totalfree energy values for transferring successive sequencesof 20 amino acids in the protein are plotted, yielding aso-called hydropathy plot. Values of over 20 kcal⋅mol −1are consistent with—but do not prove—a transmembranelocation.Another aspect of the interaction of lipids and proteinsis that some proteins are anchored to one leaflet oranother of the bilayer by covalent linkages to certainlipids. Palmitate and myristate are fatty acids involvedin such linkages to specific proteins. A number of otherproteins (see Chapter 47) are linked to glycophosphatidylinositol(GPI) structures.Different Membranes Have DifferentProtein CompositionsThe number of different proteins in a membranevaries from less than a dozen in the sarcoplasmic reticulumto over 100 in the plasma membrane. Most membraneproteins can be separated from one another usingsodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE), a technique that has revolutionizedtheir study. In the absence of SDS, few membraneproteins would remain soluble during electrophoresis.Proteins are the major functional molecules of membranesand consist of enzymes, pumps and channels,structural components, antigens (eg, for histocompatibility),and receptors for various molecules. Becauseevery membrane possesses a different complement ofproteins, there is no such thing as a typical membranestructure. The enzymatic properties of several differentmembranes are shown in Table 41–2.Membranes Are Dynamic StructuresMembranes and their components are dynamic structures.The lipids and proteins in membranes undergoturnover there just as they do in other compartments ofthe cell. Different lipids have different turnover rates,and the turnover rates of individual species of membraneproteins may vary widely. The membrane itselfcan turn over even more rapidly than any of its constituents.This is discussed in more detail in the sectionon endocytosis.Membranes Are Asymmetric StructuresThis asymmetry can be partially attributed to the irregulardistribution of proteins within the membranes. Aninside-outside asymmetry is also provided by the externallocation of the carbohydrates attached to membraneproteins. In addition, specific enzymes are lo-

420 / CHAPTER 41Table 41–2. Enzymatic markers of differentmembranes. 1MembraneEnzymePlasma5’-NucleotidaseAdenylyl cyclaseNa + -K + ATPaseEndoplasmic reticulumGlucose-6-phosphataseGolgi apparatusCisGlcNAc transferase IMedialGolgi mannosidase IITransGalactosyl transferaseTGNSialyl transferaseInner mitochondrial membrane ATP synthase1 Membranes contain many proteins, some of which have enzymaticactivity. Some of these enzymes are located only in certainmembranes and can therefore be used as markers to follow thepurification of these membranes.TGN, trans golgi network.cated exclusively on the outside or inside of membranes,as in the mitochondrial and plasma membranes.There are regional asymmetries in membranes.Some, such as occur at the villous borders of mucosalcells, are almost macroscopically visible. Others, such asthose at gap junctions, tight junctions, and synapses,occupy much smaller regions of the membrane andgenerate correspondingly smaller local asymmetries.There is also inside-outside (transverse) asymmetryof the phospholipids. The choline-containing phospholipids(phosphatidylcholine and sphingomyelin)are located mainly in the outer molecular layer; theaminophospholipids (phosphatidylserine and phosphatidylethanolamine)are preferentially located in theinner leaflet. Obviously, if this asymmetry is to exist atall, there must be limited transverse mobility (flip-flop)of the membrane phospholipids. In fact, phospholipidsin synthetic bilayers exhibit an extraordinarily slowrate of flip-flop; the half-life of the asymmetry can bemeasured in several weeks. However, when certainmembrane proteins such as the erythrocyte protein glycophorinare inserted artificially into synthetic bilayers,the frequency of phospholipid flip-flop may increase asmuch as 100-fold.The mechanisms involved in the establishment oflipid asymmetry are not well understood. The enzymesinvolved in the synthesis of phospholipids are locatedon the cytoplasmic side of microsomal membrane vesicles.Translocases (flippases) exist that transfer certainphospholipids (eg, phosphatidylcholine) from the innerto the outer leaflet. Specific proteins that preferentiallybind individual phospholipids also appear to bepresent in the two leaflets, contributing to the asymmetricdistribution of these lipid molecules. In addition,phospholipid exchange proteins recognize specificphospholipids and transfer them from one membrane(eg, the endoplasmic reticulum [ER]) to others (eg, mitochondrialand peroxisomal). There is further asymmetrywith regard to GSLs and also glycoproteins; thesugar moieties of these molecules all protrude outwardfrom the plasma membrane and are absent from itsinner face.Membranes Contain Integral& Peripheral Proteins(Figure 41–7)It is useful to classify membrane proteins into twotypes: integral and peripheral. Most membrane proteinsfall into the integral class, meaning that they interactextensively with the phospholipids and require theuse of detergents for their solubilization. Also, they generallyspan the bilayer. Integral proteins are usuallyglobular and are themselves amphipathic. They consistof two hydrophilic ends separated by an intervening hydrophobicregion that traverses the hydrophobic core ofthe bilayer. As the structures of integral membrane proteinswere being elucidated, it became apparent thatcertain ones (eg, transporter molecules, various receptors,and G proteins) span the bilayer many times (seeFigure 46–5). Integral proteins are also asymmetricallydistributed across the membrane bilayer. This asymmetricorientation is conferred at the time of their insertionin the lipid bilayer. The hydrophilic external regionof an amphipathic protein, which is synthesizedon polyribosomes, must traverse the hydrophobic coreof its target membrane and eventually be found on theoutside of that membrane. The molecular mechanismsinvolved in insertion of proteins into membranes andthe topic of membrane assembly are discussed in Chapter46.Peripheral proteins do not interact directly withthe phospholipids in the bilayer and thus do not requireuse of detergents for their release. They are weaklybound to the hydrophilic regions of specific integralproteins and can be released from them by treatmentwith salt solutions of high ionic strength. For example,ankyrin, a peripheral protein, is bound to the integralprotein “band 3” of erythrocyte membrane. Spectrin, acytoskeletal structure within the erythrocyte, is in turnbound to ankyrin and thereby plays an important rolein maintenance of the biconcave shape of the erythrocyte.Many hormone receptor molecules are integralproteins, and the specific polypeptide hormones thatbind to these receptor molecules may therefore be consideredperipheral proteins. Peripheral proteins, such aspolypeptide hormones, may help organize the distribu-

MEMBRANES: STRUCTURE & FUNCTION / 421Figure 41–7. The fluid mosaic model of membrane structure. The membrane consists of a bimolecularlipid layer with proteins inserted in it or bound to either surface. Integral membrane proteins arefirmly embedded in the lipid layers. Some of these proteins completely span the bilayer and are calledtransmembrane proteins, while others are embedded in either the outer or inner leaflet of the lipid bilayer.Loosely bound to the outer or inner surface of the membrane are the peripheral proteins. Many ofthe proteins and lipids have externally exposed oligosaccharide chains. (Reproduced, with permission,from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas, 10th ed. McGraw-Hill, 2003.)tion of integral proteins, such as their receptors, withinthe plane of the bilayer (see below).ARTIFICIAL MEMBRANES MODELMEMBRANE FUNCTIONArtificial membrane systems can be prepared by appropriatetechniques. These systems generally consist ofmixtures of one or more phospholipids of natural orsynthetic origin that can be treated (eg, by using mildsonication) to form spherical vesicles in which the lipidsform a bilayer. Such vesicles, surrounded by a lipid bilayer,are termed liposomes.Some of the advantages and uses of artificial membranesystems in the study of membrane function canbe briefly explained.(1) The lipid content of the membranes can be varied,allowing systematic examination of the effects ofvarying lipid composition on certain functions. For instance,vesicles can be made that are composed solely ofphosphatidylcholine or, alternatively, of known mixturesof different phospholipids, glycolipids, and cholesterol.The fatty acid moieties of the lipids used canalso be varied by employing synthetic lipids of knowncomposition to permit systematic examination of theeffects of fatty acid composition on certain membranefunctions (eg, transport).(2) Purified membrane proteins or enzymes can beincorporated into these vesicles in order to assess whatfactors (eg, specific lipids or ancillary proteins) the proteinsrequire to reconstitute their function. Investigationsof purified proteins, eg, the Ca 2+ ATPase of thesarcoplasmic reticulum, have in certain cases suggestedthat only a single protein and a single lipid are requiredto reconstitute an ion pump.(3) The environment of these systems can be rigidlycontrolled and systematically varied (eg, ion concentrations).The systems can also be exposed to known ligandsif, for example, the liposomes contain specific receptorproteins.(4) When liposomes are formed, they can be madeto entrap certain compounds inside themselves, eg,drugs and isolated genes. There is interest in using liposomesto distribute drugs to certain tissues, and if components(eg, antibodies to certain cell surface molecules)could be incorporated into liposomes so that theywould be targeted to specific tissues or tumors, thetherapeutic impact would be considerable. DNA entrappedinside liposomes appears to be less sensitive to

422 / CHAPTER 41attack by nucleases; this approach may prove useful inattempts at gene therapy.THE FLUID MOSAIC MODELOF MEMBRANE STRUCTUREIS WIDELY ACCEPTEDThe fluid mosaic model of membrane structure proposedin 1972 by Singer and Nicolson (Figure 41–7) isnow widely accepted. The model is often likened to icebergs(membrane proteins) floating in a sea of predominantlyphospholipid molecules. Early evidence for themodel was the finding that certain species-specific integralproteins (detected by fluorescent labeling techniques)rapidly and randomly redistributed in theplasma membrane of an interspecies hybrid cell formedby the artificially induced fusion of two different parentcells. It has subsequently been demonstrated that phospholipidsalso undergo rapid redistribution in the planeof the membrane. This diffusion within the plane ofthe membrane, termed translational diffusion, can bequite rapid for a phospholipid; in fact, within the planeof the membrane, one molecule of phospholipid canmove several micrometers per second.The phase changes—and thus the fluidity of membranes—arelargely dependent upon the lipid compositionof the membrane. In a lipid bilayer, the hydrophobicchains of the fatty acids can be highly aligned orordered to provide a rather stiff structure. As the temperatureincreases, the hydrophobic side chains undergoa transition from the ordered state (more gel-like orcrystalline phase) to a disordered one, taking on a moreliquid-like or fluid arrangement. The temperature atwhich the structure undergoes the transition from orderedto disordered (ie, melts) is called the “transitiontemperature” (T m ). The longer and more saturatedfatty acid chains interact more strongly with each othervia their longer hydrocarbon chains and thus causehigher values of T m —ie, higher temperatures are requiredto increase the fluidity of the bilayer. On theother hand, unsaturated bonds that exist in the cis configurationtend to increase the fluidity of a bilayer by decreasingthe compactness of the side chain packing withoutdiminishing hydrophobicity (Figure 41–3). Thephospholipids of cellular membranes generally containat least one unsaturated fatty acid with at least one cisdouble bond.Cholesterol modifies the fluidity of membranes. Attemperatures below the T m , it interferes with the interactionof the hydrocarbon tails of fatty acids and thusincreases fluidity. At temperatures above the T m , it limitsdisorder because it is more rigid than the hydrocarbontails of the fatty acids and cannot move in themembrane to the same extent, thus limiting fluidity. Athigh cholesterol:phospholipid ratios, transition temperaturesare altogether indistinguishable.The fluidity of a membrane significantly affects itsfunctions. As membrane fluidity increases, so does itspermeability to water and other small hydrophilic molecules.The lateral mobility of integral proteins increasesas the fluidity of the membrane increases. If theactive site of an integral protein involved in a givenfunction is exclusively in its hydrophilic regions, changinglipid fluidity will probably have little effect on theactivity of the protein; however, if the protein is involvedin a transport function in which transport componentsspan the membrane, lipid phase effects maysignificantly alter the transport rate. The insulin receptoris an excellent example of altered function withchanges in fluidity. As the concentration of unsaturatedfatty acids in the membrane is increased (by growingcultured cells in a medium rich in such molecules), fluidityincreases. This alters the receptor so that it bindsmore insulin.A state of fluidity and thus of translational mobilityin a membrane may be confined to certain regions ofmembranes under certain conditions. For example,protein-protein interactions may take place within theplane of the membrane, such that the integral proteinsform a rigid matrix—in contrast to the more usual situation,where the lipid acts as the matrix. Such regionsof rigid protein matrix can exist side by side in the samemembrane with the usual lipid matrix. Gap junctionsand tight junctions are clear examples of such side-bysidecoexistence of different matrices.Lipid Rafts & Caveolae Are SpecialFeatures of Some MembranesWhile the fluid mosaic model of membrane structurehas stood up well to detailed scrutiny, additional featuresof membrane structure and function are constantlyemerging. Two structures of particular currentinterest, located in surface membranes, are lipid raftsand caveolae. The former are dynamic areas of the exoplasmicleaflet of the lipid bilayer enriched in cholesteroland sphingolipids; they are involved in signaltransduction and possibly other processes. Caveolaemay derive from lipid rafts. Many if not all of themcontain the protein caveolin-1, which may be involvedin their formation from rafts. Caveolae are observableby electron microscopy as flask-shaped indentations ofthe cell membrane. Proteins detected in caveolae includevarious components of the signal-transductionsystem (eg, the insulin receptor and some G proteins),the folate receptor, and endothelial nitric oxide synthase(eNOS). Caveolae and lipid rafts are active areasof research, and ideas concerning them and their possibleroles in various diseases are rapidly evolving.

MEMBRANES: STRUCTURE & FUNCTION / 423MEMBRANE SELECTIVITY ALLOWSSPECIALIZED FUNCTIONSIf the plasma membrane is relatively impermeable, howdo most molecules enter a cell? How is selectivity ofthis movement established? Answers to such questionsare important in understanding how cells adjust to aconstantly changing extracellular environment. Metazoanorganisms also must have means of communicatingbetween adjacent and distant cells, so that complexbiologic processes can be coordinated. These signalsmust arrive at and be transmitted by the membrane, orthey must be generated as a consequence of some interactionwith the membrane. Some of the major mechanismsused to accomplish these different objectives arelisted in Table 41–3.Passive Mechanisms Move Some SmallMolecules Across MembranesMolecules can passively traverse the bilayer down electrochemicalgradients by simple diffusion or by facilitateddiffusion. This spontaneous movement towardequilibrium contrasts with active transport, which requiresenergy because it constitutes movement againstan electrochemical gradient. Figure 41–8 provides aschematic representation of these mechanisms.As described above, some solutes such as gases canenter the cell by diffusing down an electrochemical gradientacross the membrane and do not require metabolicenergy. The simple passive diffusion of a soluteacross the membrane is limited by the thermal agitationof that specific molecule, by the concentration gradientacross the membrane, and by the solubility of thatsolute (the permeability coefficient, Figure 41–6) in thehydrophobic core of the membrane bilayer. Solubility isTable 41–3. Transfer of material and informationacross membranes.Cross-membrane movement of small moleculesDiffusion (passive and facilitated)Active transportCross-membrane movement of large moleculesEndocytosisExocytosisSignal transmission across membranesCell surface receptors1. Signal transduction (eg, glucagon → cAMP)2. Signal internalization (coupled with endocytosis, eg,the LDL receptor)Movement to intracellular receptors (steroid hormones; aform of diffusion)Intercellular contact and communicationinversely proportionate to the number of hydrogenbonds that must be broken in order for a solute in theexternal aqueous phase to become incorporated in thehydrophobic bilayer. Electrolytes, poorly soluble inlipid, do not form hydrogen bonds with water, but theydo acquire a shell of water from hydration by electrostaticinteraction. The size of the shell is directly proportionateto the charge density of the electrolyte. Electrolyteswith a large charge density have a larger shell ofhydration and thus a slower diffusion rate. Na + , for example,has a higher charge density than K + . HydratedNa + is therefore larger than hydrated K + ; hence, the lattertends to move more easily through the membrane.The following factors affect net diffusion of a substance:(1) Its concentration gradient across the membrane.Solutes move from high to low concentration.(2) The electrical potential across the membrane.Solutes move toward the solution that has the oppositecharge. The inside of the cell usually has a negativecharge. (3) The permeability coefficient of the substancefor the membrane. (4) The hydrostatic pressuregradient across the membrane. Increased pressure willincrease the rate and force of the collision between themolecules and the membrane. (5) Temperature. Increasedtemperature will increase particle motion andthus increase the frequency of collisions between externalparticles and the membrane. In addition, a multitudeof channels exist in membranes that route theentry of ions into cells.Ion Channels Are TransmembraneProteins That Allow the SelectiveEntry of Various IonsIn natural membranes, as opposed to synthetic membranebilayers, there are transmembrane channels, porelikestructures composed of proteins that constitute selectiveion channels. Cation-conductive channels havean average diameter of about 5–8 nm and are negativelycharged within the channel. The permeability of a channeldepends upon the size, the extent of hydration, andthe extent of charge density on the ion. Specific channelsfor Na + , K + , Ca 2+ , and Cl − have been identified; twosuch channels are illustrated in Figure 41–9. Both areseen to consist of four subunits. Each subunit consists ofsix α-helical transmembrane domains. The amino andcarboxyl terminals of both ion channels are located inthe cytoplasm, with both extracellular and intracellularloops being present. The actual pores in the channelsthrough which the ions pass are not shown in the figure.They form the center (diameter about 5–8 nm) of astructure formed by apposition of the subunits. Thechannels are very selective, in most cases permitting thepassage of only one type of ion (Na + , Ca 2+ , etc). Manyvariations on the above structural themes are found, but

424 / CHAPTER 41TransportedmoleculeChannelproteinCarrierproteinLipidbilayerElectrochemicalgradientEnergySimplediffusionFacilitateddiffusionPassive transportActive transportFigure 41–8. Many small uncharged molecules pass freely through the lipidbilayer. Charged molecules, larger uncharged molecules, and some small unchargedmolecules are transferred through channels or pores or by specific carrierproteins. Passive transport is always down an electrochemical gradient, towardequilibrium. Active transport is against an electrochemical gradient and requiresan input of energy, whereas passive transport does not. (Redrawn and reproduced,with permission, from Alberts B et al: Molecular Biology of the Cell. Garland, 1983.)all ion channels are basically made up of transmembranesubunits that come together to form a central porethrough which ions pass selectively. The combination ofx-ray crystallography (where possible) and site-directedmutagenesis affords a powerful approach to delineatingthe structure-function relationships of ion channels.The membranes of nerve cells contain well-studiedion channels that are responsible for the action potentialsgenerated across the membrane. The activity ofsome of these channels is controlled by neurotransmitters;hence, channel activity can be regulated. One ioncan regulate the activity of the channel of another ion.For example, a decrease of Ca 2+ concentration in theextracellular fluid increases membrane permeability andincreases the diffusion of Na + . This depolarizes themembrane and triggers nerve discharge, which may explainthe numbness, tingling, and muscle cramps symptomaticof a low level of plasma Ca 2+ .Channels are open transiently and thus are “gated.”Gates can be controlled by opening or closing. In ligand-gatedchannels, a specific molecule binds to a receptorand opens the channel.Voltage-gated channelsopen (or close) in response to changes in membrane potential.Some properties of ion channels are listed inTable 41–4; other aspects of ion channels are discussedbriefly in Chapter 49.Ionophores Are Molecules That Act asMembrane Shuttles for Various IonsCertain microbes synthesize small organic molecules,ionophores, that function as shuttles for the movementof ions across membranes. These ionophores contain hydrophiliccenters that bind specific ions and are surroundedby peripheral hydrophobic regions; this arrangementallows the molecules to dissolve effectively in themembrane and diffuse transversely therein. Others, likethe well-studied polypeptide gramicidin, form channels.Microbial toxins such as diphtheria toxin and activatedserum complement components can produce largepores in cellular membranes and thereby provide macromoleculeswith direct access to the internal milieu.Aquaporins Are Proteins That Form WaterChannels in Certain MembranesIn certain cells (eg, red cells, cells of the collecting ductulesof the kidney), the movement of water by simple

MEMBRANES: STRUCTURE & FUNCTION / 425Rat brainNa + channelOutsideI II III IV1 2 3 4 5 6 1 2 3 4 5 61 2 3 4 5 6 1 2 3 4 5 6InsideCNRabbit skeletal muscleCa 2+ channelI II III IV1 2 3 4 5 6 1 2 3 4 5 61 2 3 4 5 6 1 2 3 4 5 6Figure 41–9. Diagrammatic representation of the structures of two ion channels. The Roman numeralsindicate the four subunits of each channel and the Arabic numerals the α-helical transmembrane domainsof each subunit. The actual pores through which the ions pass are not shown but are formed byapposition of the various subunits. The specific areas of the subunits involved in the opening and closingof the channels are also not indicated. (After WK Catterall. Modified and reproduced from Hall ZW: AnIntroduction to Molecular Neurobiology. Sinauer, 1992.)

426 / CHAPTER 41Table 41–4. Some properties of ion channels.• They are composed of transmembrane protein subunits.• Most are highly selective for one ion; a few are nonselective.• They allow impermeable ions to cross membranes at ratesapproaching diffusion limits.• They can permit ion fluxes of 10 6 –10 7 /s.• Their activities are regulated.• The two main types are voltage-gated and ligand-gated.• They are usually highly conserved across species.• Most cells have a variety of Na + , K + , Ca 2+ , and CI − channels.• Mutations in genes encoding them can cause specificdiseases. 1• Their activities are affected by certain drugs.1 Some diseases caused by mutations of ion channels are brieflydiscussed in Chapter 49.Uniport Symport AntiportCotransportLipidbilayerFigure 41–10. Schematic representation of types oftransport systems. Transporters can be classified withregard to the direction of movement and whether oneor more unique molecules are moved. (Redrawn and reproduced,with permission, from Alberts B et al: MolecularBiology of the Cell. Garland, 1983.)diffusion is augmented by movement through waterchannels. These channels are composed of tetramerictransmembrane proteins named aquaporins. At leastfive distinct aquaporins (AP-1 to AP-5) have been identified.Mutations in the gene encoding AP-2 have beenshown to be the cause of one type of nephrogenic diabetesinsipidus.PLASMA MEMBRANES ARE INVOLVEDIN FACILITATED DIFFUSION, ACTIVETRANSPORT, & OTHER PROCESSESTransport systems can be described in a functionalsense according to the number of molecules moved andthe direction of movement (Figure 41–10) or accordingto whether movement is toward or away from equilibrium.A uniport system moves one type of moleculebidirectionally. In cotransport systems, the transfer ofone solute depends upon the stoichiometric simultaneousor sequential transfer of another solute. A symportmoves these solutes in the same direction. Examples arethe proton-sugar transporter in bacteria and the Na+ -sugar transporters (for glucose and certain other sugars)and Na + -amino acid transporters in mammalian cells.Antiport systems move two molecules in opposite directions(eg, Na + in and Ca 2+ out).Molecules that cannot pass freely through the lipidbilayer membrane by themselves do so in associationwith carrier proteins. This involves two processes—facilitated diffusion and active transport—and highlyspecific transport systems.Facilitated diffusion and active transport share manyfeatures. Both appear to involve carrier proteins, andboth show specificity for ions, sugars, and amino acids.Mutations in bacteria and mammalian cells (includingsome that result in human disease) have supportedthese conclusions. Facilitated diffusion and active transportresemble a substrate-enzyme reaction exceptthat no covalent interaction occurs. These points of resemblanceare as follows: (1) There is a specific bindingsite for the solute. (2) The carrier is saturable, so it has amaximum rate of transport (V max ; Figure 41–11).(3) There is a binding constant (K m ) for the solute, andRateK mPassivediffusionCarrier-mediateddiffusionV maxSolute concentrationFigure 41–11. A comparison of the kinetics of carrier-mediated(facilitated) diffusion with passive diffusion.The rate of movement in the latter is directly proportionateto solute concentration, whereas theprocess is saturable when carriers are involved. Theconcentration at half-maximal velocity is equal to thebinding constant (K m ) of the carrier for the solute. (V max ,maximal rate.)

MEMBRANES: STRUCTURE & FUNCTION / 427so the whole system has a K m (Figure 41–11). (4) Structurallysimilar competitive inhibitors block transport.Major differences are the following: (1) Facilitateddiffusion can operate bidirectionally, whereas activetransport is usually unidirectional. (2) Active transportalways occurs against an electrical or chemical gradient,and so it requires energy.Facilitated DiffusionSome specific solutes diffuse down electrochemical gradientsacross membranes more rapidly than might beexpected from their size, charge, or partition coefficients.This facilitated diffusion exhibits propertiesdistinct from those of simple diffusion. The rate of facilitateddiffusion, a uniport system, can be saturated;ie, the number of sites involved in diffusion of the specificsolutes appears finite. Many facilitated diffusionsystems are stereospecific but, like simple diffusion, requireno metabolic energy.As described earlier, the inside-outside asymmetry ofmembrane proteins is stable, and mobility of proteinsacross (rather than in) the membrane is rare; therefore,transverse mobility of specific carrier proteins is notlikely to account for facilitated diffusion processes exceptin a few unusual cases.A “Ping-Pong” mechanism (Figure 41–12) explainsfacilitated diffusion. In this model, the carrierprotein exists in two principal conformations. In the“pong” state, it is exposed to high concentrations ofsolute, and molecules of the solute bind to specific siteson the carrier protein. Transport occurs when a conformationalchange exposes the carrier to a lower concentrationof solute (“ping” state). This process is completelyreversible, and net flux across the membranedepends upon the concentration gradient. The rate atwhich solutes enter a cell by facilitated diffusion is determinedby the following factors: (1) The concentrationgradient across the membrane. (2) The amount ofcarrier available (this is a key control step). (3) The rapidityof the solute-carrier interaction. (4) The rapidityof the conformational change for both the loaded andthe unloaded carrier.Hormones regulate facilitated diffusion by changingthe number of transporters available. Insulin increasesglucose transport in fat and muscle by recruiting transportersfrom an intracellular reservoir. Insulin also enhancesamino acid transport in liver and other tissues.One of the coordinated actions of glucocorticoid hormonesis to enhance transport of amino acids into liver,where the amino acids then serve as a substrate for gluconeogenesis.Growth hormone increases amino acidtransport in all cells, and estrogens do this in the uterus.There are at least five different carrier systems foramino acids in animal cells. Each is specific for a groupof closely related amino acids, and most operate as Na + -symport systems (Figure 41–10).Active TransportThe process of active transport differs from diffusion inthat molecules are transported away from thermodynamicequilibrium; hence, energy is required. This energycan come from the hydrolysis of ATP, from electronmovement, or from light. The maintenance ofelectrochemical gradients in biologic systems is so importantthat it consumes perhaps 30–40% of the totalenergy expenditure in a cell.In general, cells maintain a low intracellular Na +concentration and a high intracellular K + concentration(Table 41–1), along with a net negative electrical potentialinside. The pump that maintains these gradientsis an ATPase that is activated by Na + and K + (Na + -K +ATPase; see Figure 41–13). The ATPase is an integralPongPingFigure 41–12. The “Ping-Pong” model of facilitated diffusion. A protein carrier (gray structure) in the lipid bilayerassociates with a solute in high concentration on one side of the membrane. A conformational change ensues(“pong” to “ping”), and the solute is discharged on the side favoring the new equilibrium. The empty carrierthen reverts to the original conformation (“ping” to “pong”) to complete the cycle.

428 / CHAPTER 41INSIDEATPADP+P i3 Na +Mg 2+Membrane2 K + 2 K +OUTSIDE3 Na +Figure 41–13. Stoichiometry of the Na + -K + ATPasepump. This pump moves three Na + ions from inside thecell to the outside and brings two K + ions from the outsideto the inside for every molecule of ATP hydrolyzedto ADP by the membrane-associated ATPase. Ouabainand other cardiac glycosides inhibit this pump by actingon the extracellular surface of the membrane.(Courtesy of R Post.)membrane protein and requires phospholipids for activity.The ATPase has catalytic centers for both ATPand Na + on the cytoplasmic side of the membrane, butthe K + binding site is located on the extracellular side ofthe membrane. Ouabain or digitalis inhibits this ATPaseby binding to the extracellular domain. Inhibitionof the ATPase by ouabain can be antagonized by extracellularK + .Nerve Impulses Are TransmittedUp & Down MembranesThe membrane forming the surface of neuronal cellsmaintains an asymmetry of inside-outside voltage (electricalpotential) and is electrically excitable. When appropriatelystimulated by a chemical signal mediated bya specific synaptic membrane receptor (see discussion ofthe transmission of biochemical signals, below), gates inthe membrane are opened to allow the rapid influx ofNa + or Ca 2+ (with or without the efflux of K + ), so thatthe voltage difference rapidly collapses and that segmentof the membrane is depolarized. However, as a resultof the action of the ion pumps in the membrane,the gradient is quickly restored.When large areas of the membrane are depolarizedin this manner, the electrochemical disturbance propagatesin wave-like form down the membrane, generatinga nerve impulse. Myelin sheets, formed bySchwann cells, wrap around nerve fibers and provide anelectrical insulator that surrounds most of the nerve andgreatly speeds up the propagation of the wave (signal)by allowing ions to flow in and out of the membraneonly where the membrane is free of the insulation. Themyelin membrane is composed of phospholipids, cholesterol,proteins, and GSLs. Relatively few proteins arefound in the myelin membrane; those present appear tohold together multiple membrane bilayers to form thehydrophobic insulating structure that is impermeableto ions and water. Certain diseases, eg, multiple sclerosisand the Guillain-Barré syndrome, are characterizedby demyelination and impaired nerve conduction.Glucose Transport InvolvesSeveral MechanismsA discussion of the transport of glucose summarizesmany of the points made in this chapter. Glucose mustenter cells as the first step in energy utilization. Inadipocytes and muscle, glucose enters by a specifictransport system that is enhanced by insulin. Changesin transport are primarily due to alterations of V max(presumably from more or fewer active transporters),but changes in K m may also be involved. Glucose transportinvolves different aspects of the principles of transportdiscussed above. Glucose and Na + bind to differentsites on the glucose transporter. Na + moves into the celldown its electrochemical gradient and “drags” glucosewith it (Figure 41–14). Therefore, the greater the Na +gradient, the more glucose enters; and if Na + in extracellularfluid is low, glucose transport stops. To maintaina steep Na + gradient, this Na + -glucose symport isdependent on gradients generated by an Na + -K + pumpthat maintains a low intracellular Na + concentration.Similar mechanisms are used to transport other sugarsas well as amino acids.The transcellular movement of sugars involves oneadditional component: a uniport that allows the glucoseaccumulated within the cell to move across a differentsurface toward a new equilibrium; this occurs in intestinaland renal cells, for example.Cells Transport Certain MacromoleculesAcross the Plasma MembraneThe process by which cells take up large molecules iscalled “endocytosis.” Some of these molecules (eg,polysaccharides, proteins, and polynucleotides), whenhydrolyzed inside the cell, yield nutrients. Endocytosisprovides a mechanism for regulating the content of certainmembrane components, hormone receptors beinga case in point. Endocytosis can be used to learn moreabout how cells function. DNA from one cell type canbe used to transfect a different cell and alter the latter’sfunction or phenotype. A specific gene is often employedin these experiments, and this provides a uniqueway to study and analyze the regulation of that gene.DNA transfection depends upon endocytosis; endocy-

MEMBRANES: STRUCTURE & FUNCTION / 429GlucoseNa +LUMENAB(Symport)CYTOSOLGlucoseNa +K +Na +K +CPGlucoseEXTRACELLULAR FLUIDFigure 41–14. The transcellular movement of glucosein an intestinal cell. Glucose follows Na + across theluminal epithelial membrane. The Na + gradient thatdrives this symport is established by Na + -K + exchange,which occurs at the basal membrane facing the extracellularfluid compartment. Glucose at high concentrationwithin the cell moves “downhill” into the extracellularfluid by facilitated diffusion (a uniport mechanism).tosis is responsible for the entry of DNA into the cell.Such experiments commonly use calcium phosphate,since Ca 2+ stimulates endocytosis and precipitatesDNA, which makes the DNA a better object for endocytosis.Cells also release macromolecules by exocytosis.Endocytosis and exocytosis both involve vesicle formationwith or from the plasma membrane.A. ENDOCYTOSISAll eukaryotic cells are continuously ingesting parts oftheir plasma membranes. Endocytotic vesicles are generatedwhen segments of the plasma membrane invaginate,enclosing a minute volume of extracellular fluidand its contents. The vesicle then pinches off as the fusionof plasma membranes seals the neck of the vesicleat the original site of invagination (Figure 41–15). Thisvesicle fuses with other membrane structures and thusachieves the transport of its contents to other cellularcompartments or even back to the cell exterior. Mostendocytotic vesicles fuse with primary lysosomes toform secondary lysosomes, which contain hydrolyticenzymes and are therefore specialized organelles for intracellulardisposal. The macromolecular contents aredigested to yield amino acids, simple sugars, or nucleotides,and they diffuse out of the vesicles to beVFigure 41–15. Two types of endocytosis. An endocytoticvesicle (V) forms as a result of invagination of aportion of the plasma membrane. Fluid-phase endocytosis(A) is random and nondirected. Receptor-mediatedendocytosis (B) is selective and occurs in coatedpits (CP) lined with the protein clathrin (the fuzzy material).Targeting is provided by receptors (black symbols)specific for a variety of molecules. This results in the formationof a coated vesicle (CV).reused in the cytoplasm. Endocytosis requires (1) energy,usually from the hydrolysis of ATP; (2) Ca 2+ inextracellular fluid; and (3) contractile elements in thecell (probably the microfilament system) (Chapter 49).There are two general types of endocytosis. Phagocytosisoccurs only in specialized cells such asmacrophages and granulocytes. Phagocytosis involvesthe ingestion of large particles such as viruses, bacteria,cells, or debris. Macrophages are extremely active inthis regard and may ingest 25% of their volume perhour. In so doing, a macrophage may internalize 3% ofits plasma membrane each minute or the entire membraneevery 30 minutes.Pinocytosis is a property of all cells and leads to thecellular uptake of fluid and fluid contents. There aretwo types. Fluid-phase pinocytosis is a nonselectiveprocess in which the uptake of a solute by formation ofsmall vesicles is simply proportionate to its concentrationin the surrounding extracellular fluid. The formationof these vesicles is an extremely active process. Fi-CV

430 / CHAPTER 41broblasts, for example, internalize their plasma membraneat about one-third the rate of macrophages. Thisprocess occurs more rapidly than membranes are made.The surface area and volume of a cell do not changemuch, so membranes must be replaced by exocytosis orby being recycled as fast as they are removed by endocytosis.The other type of pinocytosis, absorptive pinocytosis,is a receptor-mediated selective process primarily responsiblefor the uptake of macromolecules for whichthere are a finite number of binding sites on the plasmamembrane. These high-affinity receptors permit the selectiveconcentration of ligands from the medium, minimizethe uptake of fluid or soluble unbound macromolecules,and markedly increase the rate at whichspecific molecules enter the cell. The vesicles formedduring absorptive pinocytosis are derived from invaginations(pits) that are coated on the cytoplasmic sidewith a filamentous material. In many systems, the proteinclathrin is the filamentous material. It has a threelimbedstructure (called a triskelion), with each limbbeing made up of one light and one heavy chain ofclathrin. The polymerization of clathrin into a vesicle isdirected by assembly particles, composed of fouradapter proteins. These interact with certain aminoacid sequences in the receptors that become cargo, ensuringselectivity of uptake. The lipid PIP 2 also plays animportant role in vesicle assembly. In addition, the proteindynamin, which both binds and hydrolyzes GTP,is necessary for the pinching off of clathrin-coated vesiclesfrom the cell surface. Coated pits may constitute asmuch as 2% of the surface of some cells.As an example, the low-density lipoprotein (LDL)molecule and its receptor (Chapter 25) are internalizedby means of coated pits containing the LDL receptor.These endocytotic vesicles containing LDL and its receptorfuse to lysosomes in the cell. The receptor is releasedand recycled back to the cell surface membrane,but the apoprotein of LDL is degraded and the cholesterylesters metabolized. Synthesis of the LDL receptoris regulated by secondary or tertiary consequences ofpinocytosis, eg, by metabolic products—such as cholesterol—releasedduring the degradation of LDL. Disordersof the LDL receptor and its internalization aremedically important and are discussed in Chapter 25.Absorptive pinocytosis of extracellular glycoproteinsrequires that the glycoproteins carry specific carbohydraterecognition signals. These recognition signalsare bound by membrane receptor molecules, whichplay a role analogous to that of the LDL receptor. Agalactosyl receptor on the surface of hepatocytes is instrumentalin the absorptive pinocytosis of asialoglycoproteinsfrom the circulation (Chapter 47). Acid hydrolasestaken up by absorptive pinocytosis in fibroblastsare recognized by their mannose 6-phosphate moieties.Interestingly, the mannose 6-phosphate moiety alsoseems to play an important role in the intracellular targetingof the acid hydrolases to the lysosomes of thecells in which they are synthesized (Chapter 47).There is a dark side to receptor-mediated endocytosisin that viruses which cause such diseases as hepatitis(affecting liver cells), poliomyelitis (affecting motorneurons), and AIDS (affecting T cells) initiate theirdamage by this mechanism. Iron toxicity also beginswith excessive uptake due to endocytosis.B. EXOCYTOSISMost cells release macromolecules to the exterior by exocytosis.This process is also involved in membrane remodeling,when the components synthesized in theGolgi apparatus are carried in vesicles to the plasmamembrane. The signal for exocytosis is often a hormonewhich, when it binds to a cell-surface receptor,induces a local and transient change in Ca 2+ concentration.Ca 2+ triggers exocytosis. Figure 41–16 provides acomparison of the mechanisms of exocytosis and endocytosis.Molecules released by exocytosis fall into three categories:(1) They can attach to the cell surface and becomeperipheral proteins, eg, antigens. (2) They can becomepart of the extracellular matrix, eg, collagen andglycosaminoglycans. (3) They can enter extracellularfluid and signal other cells. Insulin, parathyroid hormone,and the catecholamines are all packaged in gran-ExocytosisEndocytosisFigure 41–16. A comparison of the mechanisms of endocytosis and exocytosis.Exocytosis involves the contact of two inside surface (cytoplasmic side) monolayers,whereas endocytosis results from the contact of two outer surface monolayers.

MEMBRANES: STRUCTURE & FUNCTION / 431ules and processed within cells, to be released upon appropriatestimulation.Some Signals Are TransmittedAcross MembranesSpecific biochemical signals such as neurotransmitters,hormones, and immunoglobulins bind to specific receptors(integral proteins) exposed to the outside ofcellular membranes and transmit information throughthese membranes to the cytoplasm. This process, calledtransmembrane signaling, involves the generation of anumber of signals, including cyclic nucleotides, calcium,phosphoinositides, and diacylglycerol. It is discussedin detail in Chapter 43.Information Can Be Communicatedby Intercellular ContactThere are many areas of intercellular contact in a metazoanorganism. This necessitates contact between theplasma membranes of the individual cells. Cells havedeveloped specialized regions on their membranes forintercellular communication in close proximity. Gapjunctions mediate and regulate the passage of ions andsmall molecules (up to 1000–2000 MW) through anarrow hydrophilic core connecting the cytosol of adjacentcells. These structures are primarily composed ofthe protein connexin, which contains four membranespanningα helices. About a dozen genes encoding differentconnexins have been cloned. An assembly of 12connexin molecules forms a structure (a connexon)with a central channel that forms bridges between adjacentcells. Ions and small molecules pass from the cytosolof one cell to that of another through the channels,which open and close in a regulated fashion.MUTATIONS AFFECTING MEMBRANEPROTEINS CAUSE DISEASESIn view of the fact that membranes are located in somany organelles and are involved in so many processes,it is not surprising that mutations affecting their proteinconstituents should result in many diseases or disorders.Proteins in membranes can be classified as receptors,transporters, ion channels, enzymes, andstructural components. Members of all of these classesare often glycosylated, so that mutations affecting thisprocess may alter their function. Examples of diseasesor disorders due to abnormalities in membrane proteinsare listed in Table 41–5; these mainly reflect mutationsin proteins of the plasma membrane, with one affectinglysosomal function (I-cell disease). Over 30 geneticdiseases or disorders have been ascribed to mutationsaffecting various proteins involved in the transport ofamino acids, sugars, lipids, urate, anions, cations, water,and vitamins across the plasma membrane. Mutationsin genes encoding proteins in other membranes canalso have harmful consequences. For example, mutationsin genes encoding mitochondrial membraneproteins involved in oxidative phosphorylation cancause neurologic and other problems (eg, Leber’s hereditaryoptic neuropathy; LHON). Membrane proteinscan also be affected by conditions other than mutations.Formation of autoantibodies to the acetylcholinereceptor in skeletal muscle causes myastheniagravis. Ischemia can quickly affect the integrity of variousion channels in membranes. Abnormalities ofmembrane constituents other than proteins can also beharmful. With regard to lipids, excess of cholesterol(eg, in familial hypercholesterolemia), of lysophospholipid(eg, after bites by certain snakes, whose venomcontains phospholipases), or of glycosphingolipids (eg,in a sphingolipidosis) can all affect membrane function.Cystic Fibrosis Is Due to Mutations in theGene Encoding a Chloride ChannelCystic fibrosis (CF) is a recessive genetic disorder prevalentamong whites in North America and certain partsof northern Europe. It is characterized by chronic bacterialinfections of the airways and sinuses, fat maldigestiondue to pancreatic exocrine insufficiency, infertilityin males due to abnormal development of the vas deferens,and elevated levels of chloride in sweat (> 60mmol/L).After a Herculean landmark endeavor, the gene forCF was identified in 1989 on chromosome 7. It wasfound to encode a protein of 1480 amino acids, namedcystic fibrosis transmembrane regulator (CFTR), acyclic AMP-regulated Cl − channel (see Figure 41–17).An abnormality of membrane Cl − permeability is believedto result in the increased viscosity of many bodilysecretions, though the precise mechanisms are stillunder investigation. The commonest mutation (~70%in certain Caucasian populations) is deletion of threebases, resulting in loss of residue 508, a phenylalanine(∆F 508 ). However, more than 900 other mutations havebeen identified. These mutations affect CFTR in atleast four ways: (1) its amount is reduced; (2) dependingupon the particular mutation, it may be susceptibleto misfolding and retention within the ER or Golgi apparatus;(3) mutations in the nucleotide-binding domainsmay affect the ability of the Cl − channel to open,an event affected by ATP; (4) the mutations may alsoreduce the rate of ion flow through a channel, generatingless of a Cl − current.The most serious and life-threatening complicationis recurrent pulmonary infections due to overgrowth ofvarious pathogens in the viscous secretions of the respi-

432 / CHAPTER 41Table 41–5. Some diseases or pathologic states resulting from or attributed to abnormalitiesof membranes. 1DiseaseAbnormalityAchondroplasia Mutations in the gene encoding the fibroblast growth factor receptor 3(MIM 100800)Familial hypercholester- Mutations in the gene encoding the LDL receptorolemia (MIM 143890)Cystic fibrosisMutations in the gene encoding the CFTR protein, a Cl − transporter(MIM 219700)Congenital long QT syn- Mutations in genes encoding ion channels in the heartdrome (MIM 192500)Wilson diseaseMutations in the gene encoding a copper-dependent ATPase(MIM 277900)I-cell disease Mutations in the gene encoding GIcNAc phosphotransferase, resulting in absence of the Man 6-P(MIM 252500) signal for lysosomal localization of certain hydrolasesHereditary spherocytosis Mutations in the genes encoding spectrin or other structural proteins in the red cell membrane(MIM 182900)MetastasisAbnormalities in the oligosaccharide chains of membrane glycoproteins and glycolipids are thoughtto be of importanceParoxysmal nocturnal Mutation resulting in deficient attachment of the GPI anchor to certain proteins of the red cellhemoglobinuria membrane(MIM 311770)1 The disorders listed are discussed further in other chapters. The table lists examples of mutations affecting receptors, a transporter, anion channel, an enzyme, and a structural protein. Examples of altered or defective glycosylation of glycoproteins are also presented. Mostof the conditions listed affect the plasma membrane.AminoterminalNBF1R domainNBF2CarboxylterminalFigure 41–17. Diagram of the structure of the CFTRprotein (not to scale). The protein contains twelvetransmembrane segments (probably helical), two nucleotide-bindingfolds or domains (NBF1 and NBF2),and one regulatory (R) domain. NBF1 and NBF2 probablybind ATP and couple its hydrolysis to transport ofCl − . Phe 508, the major locus of mutations in cystic fibrosis,is located in NBF1.ratory tract. Poor nutrition as a result of pancreatic insufficiencyworsens the situation. The treatment of CFthus requires a comprehensive effort to maintain nutritionalstatus, to prevent and combat pulmonary infections,and to maintain physical and psychologic health.Advances in molecular genetics mean that mutationanalysis can be performed for prenatal diagnosis and forcarrier testing in families in which one child already hasthe condition. Efforts are in progress to use gene therapyto restore the activity of CFTR. An aerosolizedpreparation of human DNase that digests the DNA ofmicroorganisms in the respiratory tract has provedhelpful in therapy.SUMMARY• Membranes are complex structures composed oflipids, carbohydrates, and proteins.• The basic structure of all membranes is the lipid bilayer.This bilayer is formed by two sheets of phospholipidsin which the hydrophilic polar head groups

MEMBRANES: STRUCTURE & FUNCTION / 433are directed away from each other and are exposed tothe aqueous environment on the outer and inner surfacesof the membrane. The hydrophobic nonpolartails of these molecules are oriented toward eachother, in the direction of the center of the membrane.• Membrane proteins are classified as integral if theyare firmly embedded in the bilayer and as peripheralif they are loosely attached to the outer or inner surface.• The 20 or so different membranes in a mammaliancell have intrinsic functions (eg, enzymatic activity),and they define compartments, or specialized environments,within the cell that have specific functions(eg, lysosomes).• Certain molecules freely diffuse across membranes,but the movement of others is restricted because ofsize, charge, or solubility.• Various passive and active mechanisms are employedto maintain gradients of such molecules across differentmembranes.• Certain solutes, eg, glucose, enter cells by facilitateddiffusion, along a downhill gradient from high to lowconcentration. Specific carrier molecules, or transporters,are involved in such processes.• Ligand- or voltage-gated ion channels are often employedto move charged molecules (Na + , K + , Ca 2+ ,etc) across membranes.• Large molecules can enter or leave cells throughmechanisms such as endocytosis or exocytosis. Theseprocesses often require binding of the molecule to areceptor, which affords specificity to the process.• Receptors may be integral components of membranes(particularly the plasma membrane). The interactionof a ligand with its receptor may not involvethe movement of either into the cell, but theinteraction results in the generation of a signal thatinfluences intracellular processes (transmembranesignaling).• Mutations that affect the structure of membrane proteins(receptors, transporters, ion channels, enzymes,and structural proteins) may cause diseases; examplesinclude cystic fibrosis and familial hypercholesterolemia.REFERENCESDoyle DA et al: The structure of the potassium channel: molecularbasis of K + conductance and selectivity. Science 1998;280:69.Felix R: Channelopathies: ion channel defects linked to heritableclinical disorders. J Med Genet 2000;37:729.Garavito RM, Ferguson-Miller S: Detergents as tools in membranebiochemistry. J Biol Chem 2001;276:32403.Gillooly DJ, Stenmark H: A lipid oils the endocytosis machine. Science2001;291;993.Knowles MR, Durie PR: What is cystic fibrosis? N Engl J Med2002;347:439.Longo N: Inherited defects of membrane transport. In: Harrison’sPrinciples of Internal Medicine, 15th ed. Braunwald E et al(editors). McGraw-Hill, 2001.Marx J: Caveolae: a once-elusive structure gets some respect. Science2001;294;1862.White SH et al: How membranes shape protein structure. J BiolChem 2001:276:32395.

The Diversity of theEndocrine System 42Daryl K. Granner, MDACTH Adrenocorticotropic hormoneANF Atrial natriuretic factorcAMP Cyclic adenosine monophosphateCBG Corticosteroid-binding globulinCG Chorionic gonadotropincGMP Cyclic guanosine monophosphateCLIP Corticotropin-like intermediate lobepeptideDBH Dopamine β-hydroxylaseDHEA DehydroepiandrosteroneDHT DihydrotestosteroneDIT DiiodotyrosineDOC DeoxycorticosteroneEGF Epidermal growth factorFSH Follicle-stimulating hormoneGH Growth hormoneIGF-I Insulin-like growth factor-ILH Luteotropic hormoneLPH LipotropinMIT MonoiodotyrosineMSH Melanocyte-stimulating hormoneOHSD Hydroxysteroid dehydrogenasePNMT Phenylethanolamine-N-methyltransferasePOMC Pro-opiomelanocortinSHBG Sex hormone-binding globulinStAR Steroidogenic acute regulatory (protein)TBG Thyroxine-binding globulinTEBG Testosterone-estrogen-binding globulinTRH Thyrotropin-releasing hormoneTSH Thyrotropin-stimulating hormoneBIOMEDICAL IMPORTANCEtive because hormones can act on adjacent cells(paracrine action) and on the cell in which they wereThe survival of multicellular organisms depends on theirsynthesized (autocrine action) without entering the systemiccirculation. A diverse array of hormones—eachability to adapt to a constantly changing environment.Intercellular communication mechanisms are necessarywith distinctive mechanisms of action and properties ofrequirements for this adaptation. The nervous systembiosynthesis, storage, secretion, transport, and metabolism—hasevolved to provide homeostatic responses.and the endocrine system provide this intercellular, organism-widecommunication. The nervous system wasThis biochemical diversity is the topic of this chapter.originally viewed as providing a fixed communicationsystem, whereas the endocrine system supplied hormones,which are mobile messages. In fact, there is a remarkableTHE TARGET CELL CONCEPTconvergence of these regulatory systems. For There are about 200 types of differentiated cells in hu-example, neural regulation of the endocrine system is mans. Only a few produce hormones, but virtually all ofimportant in the production and secretion of some hormones;the 75 trillion cells in a human are targets of one ormany neurotransmitters resemble hormones in more of the over 50 known hormones. The concept oftheir synthesis, transport, and mechanism of action; and the target cell is a useful way of looking at hormone action.many hormones are synthesized in the nervous system.It was thought that hormones affected a single cellThe word “hormone” is derived from a Greek term that type—or only a few kinds of cells—and that a hormonemeans to arouse to activity. As classically defined, a hormoneelicited a unique biochemical or physiologic action. Weis a substance that is synthesized in one organ and now know that a given hormone can affect several dif-transported by the circulatory system to act on another ferent cell types; that more than one hormone can affecttissue. However, this original description is too restric-a given cell type; and that hormones can exert many dif-434

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 435ferent effects in one cell or in different cells. With thediscovery of specific cell-surface and intracellular hormonereceptors, the definition of a target has been expandedto include any cell in which the hormone (ligand)binds to its receptor, whether or not a biochemicalor physiologic response has yet been determined.Several factors determine the response of a target cellto a hormone. These can be thought of in two generalways: (1) as factors that affect the concentration of thehormone at the target cell (see Table 42–1) and (2) asfactors that affect the actual response of the target cellto the hormone (see Table 42–2).Table 42–1. Determinants of the concentrationof a hormone at the target cell.The rate of synthesis and secretion of the hormones.The proximity of the target cell to the hormone source (dilutioneffect).The dissociation constants of the hormone with specificplasma transport proteins (if any).The conversion of inactive or suboptimally active forms of thehormone into the fully active form.The rate of clearance from plasma by other tissues or bydigestion, metabolism, or excretion.Table 42–2. Determinants of the targetcell response.The number, relative activity, and state of occupancy of thespecific receptors on the plasma membrane or in thecytoplasm or nucleus.The metabolism (activation or inactivation) of the hormone inthe target cell.The presence of other factors within the cell that are necessaryfor the hormone response.Up- or down-regulation of the receptor consequent to theinteraction with the ligand.Postreceptor desensitzation of the cell, including downregulationof the receptor.HORMONE RECEPTORS AREOF CENTRAL IMPORTANCEReceptors Discriminate PreciselyOne of the major challenges faced in making the hormone-basedcommunication system work is illustratedin Figure 42–1. Hormones are present at very low concentrationsin the extracellular fluid, generally in therange of 10 –15 to 10 –9 mol/L. This concentration ismuch lower than that of the many structurally similarmolecules (sterols, amino acids, peptides, proteins) andother molecules that circulate at concentrations in the10 –5 to 10 –3 mol/L range. Target cells, therefore, mustdistinguish not only between different hormones presentin small amounts but also between a given hormoneand the 10 6 - to 10 9 -fold excess of other similarmolecules. This high degree of discrimination is providedby cell-associated recognition molecules called receptors.Hormones initiate their biologic effects bybinding to specific receptors, and since any effectivecontrol system also must provide a means of stopping aresponse, hormone-induced actions generally terminatewhen the effector dissociates from the receptor.A target cell is defined by its ability to selectivelybind a given hormone to its cognate receptor. Severalbiochemical features of this interaction are important inorder for hormone-receptor interactions to be physiologicallyrelevant: (1) binding should be specific, ie, displaceableby agonist or antagonist; (2) binding shouldbe saturable; and (3) binding should occur within theconcentration range of the expected biologic response.Both Recognition & CouplingDomains Occur on ReceptorsAll receptors have at least two functional domains. Arecognition domain binds the hormone ligand and asecond region generates a signal that couples hormonerecognition to some intracellular function. Coupling(signal transduction) occurs in two general ways.Polypeptide and protein hormones and the catecholaminesbind to receptors located in the plasmamembrane and thereby generate a signal that regulatesvarious intracellular functions, often by changing theactivity of an enzyme. In contrast, steroid, retinoid, andthyroid hormones interact with intracellular receptors,and it is this ligand-receptor complex that directly providesthe signal, generally to specific genes whose rate oftranscription is thereby affected.The domains responsible for hormone recognitionand signal generation have been identified in the proteinpolypeptide and catecholamine hormone receptors.Steroid, thyroid, and retinoid hormone receptors haveseveral functional domains: one site binds the hormone;another binds to specific DNA regions; a third is involvedin the interaction with other coregulator proteinsthat result in the activation (or repression) of genetranscription; and a fourth may specify binding to oneor more other proteins that influence the intracellulartrafficking of the receptor.The dual functions of binding and coupling ultimatelydefine a receptor, and it is the coupling of hormonebinding to signal transduction—so-called receptor-effectorcoupling—that provides the first step inamplification of the hormonal response. This dual purposealso distinguishes the target cell receptor from the

436 / CHAPTER 42◆❁❁✪✴ ❖❁◗❉ Figure 42–1. Specificity and selectivity of✧❙◆✪hormone receptors. Many different molecules✴❙ ECF circulate in the extracellular fluid (ECF), but✧❃❃content❍only a few are recognized by hormone receptors.Receptors must select these molecules❍◗✧❉❖✪❉❁❁from among high concentrations of the otherHormone molecules. This simplified drawing shows thatReceptor a cell may have no hormone receptors (1),have one receptor (2+5+6), have receptors for1 2 3 4 5 6 Cell types several hormones (3), or have a receptor butno hormone in the vicinity (4).plasma carrier proteins that bind hormone but do notgenerate a signal (see Table 42–6).Receptors Are ProteinsSeveral classes of peptide hormone receptors have beendefined. For example, the insulin receptor is a heterotetramer(α 2 β 2 ) linked by multiple disulfide bondsin which the extracellular α subunit binds insulin andthe membrane-spanning β subunit transduces the signalthrough the tyrosine protein kinase domain locatedin the cytoplasmic portion of this polypeptide. The receptorsfor insulin-like growth factor I (IGF-I) andepidermal growth factor (EGF) are generally similar instructure to the insulin receptor. The growth hormoneand prolactin receptors also span the plasma membraneof target cells but do not contain intrinsic proteinkinase activity. Ligand binding to these receptors,however, results in the association and activation of acompletely different protein kinase pathway, the Jak-Stat pathway. Polypeptide hormone and catecholaminereceptors, which transduce signals by alteringthe rate of production of cAMP through G-proteins,are characterized by the presence of seven domains thatspan the plasma membrane. Protein kinase activationand the generation of cyclic AMP, (cAMP, 3′5′-adenylic acid; see Figure 20–5) is a downstream actionof this class of receptor (see Chapter 43 for further details).A comparison of several different steroid receptorswith thyroid hormone receptors revealed a remarkableconservation of the amino acid sequence in certain regions,particularly in the DNA-binding domains. Thisled to the realization that receptors of the steroid orthyroid type are members of a large superfamily of nuclearreceptors. Many related members of this familyhave no known ligand at present and thus are called orphanreceptors. The nuclear receptor superfamily playsa critical role in the regulation of gene transcription byhormones, as described in Chapter 43.HORMONES CAN BE CLASSIFIEDIN SEVERAL WAYSHormones can be classified according to chemical composition,solubility properties, location of receptors,and the nature of the signal used to mediate hormonalaction within the cell. A classification based on the lasttwo properties is illustrated in Table 42–3, and generalfeatures of each group are illustrated in Table 42–4.The hormones in group I are lipophilic. After secretion,these hormones associate with plasma transport orcarrier proteins, a process that circumvents the problemof solubility while prolonging the plasma half-life of thehormone. The relative percentages of bound and freehormone are determined by the binding affinity andbinding capacity of the transport protein. The free hormone,which is the biologically active form, readily traversesthe lipophilic plasma membrane of all cells andencounters receptors in either the cytosol or nucleus oftarget cells. The ligand-receptor complex is assumed tobe the intracellular messenger in this group.The second major group consists of water-solublehormones that bind to the plasma membrane of the targetcell. Hormones that bind to the surfaces of cellscommunicate with intracellular metabolic processesthrough intermediary molecules called second messengers(the hormone itself is the first messenger), whichare generated as a consequence of the ligand-receptorinteraction. The second messenger concept arose froman observation that epinephrine binds to the plasmamembrane of certain cells and increases intracellularcAMP. This was followed by a series of experiments inwhich cAMP was found to mediate the effects of manyhormones. Hormones that clearly employ this mechanismare shown in group II.A of Table 42–3. To date,only one hormone, atrial natriuretic factor (ANF), usescGMP as its second messenger, but other hormoneswill probably be added to group II.B. Several hormones,many of which were previously thought to affectcAMP, appear to use ionic calcium (Ca 2+ ) or

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 437Table 42–3. Classification of hormones bymechanism of action.I. Hormones that bind to intracellular receptorsAndrogensCalcitriol (1,25[OH] 2 -D 3 )EstrogensGlucocorticoidsMineralocorticoidsProgestinsRetinoic acidThyroid hormones (T 3 and T 4 )II. Hormones that bind to cell surface receptorsA. The second messenger is cAMP:α 2 -Adrenergic catecholaminesβ-Adrenergic catecholaminesAdrenocorticotropic hormoneAntidiuretic hormoneCalcitoninChorionic gonadotropin, humanCorticotropin-releasing hormoneFollicle-stimulating hormoneGlucagonLipotropinLuteinizing hormoneMelanocyte-stimulating hormoneParathyroid hormoneSomatostatinThyroid-stimulating hormoneB. The second messenger is cGMP:Atrial natriuretic factorNitric oxideC. The second messenger is calcium or phosphatidylinositols(or both):Acetylcholine (muscarinic)α 1 -Adrenergic catecholaminesAngiotensin IIAntidiuretic hormone (vasopressin)CholecystokininGastrinGonadotropin-releasing hormoneOxytocinPlatelet-derived growth factorSubstance PThyrotropin-releasing hormoneD. The second messenger is a kinase or phosphatasecascade:Chorionic somatomammotropinEpidermal growth factorErythropoietinFibroblast growth factorGrowth hormoneInsulinInsulin-like growth factors I and IINerve growth factorPlatelet-derived growth factorProlactinTable 42–4. General features of hormone classes.Group IGroup IITypes Steroids, iodothyro- Polypeptides, proteins,nines, calcitriol, glycoproteins, cateretinoidscholaminesSolubility Lipophilic HydrophilicTransport Yes NoproteinsPlasma half- Long (hours to Short (minutes)life days)Receptor Intracellular Plasma membraneMediator Receptor-hormone cAMP, cGMP, Ca 2+ ,complexmetabolites of complexphosphoinositols,kinase cascadesmetabolites of complex phosphoinositides (or both) asthe intracellular signal. These are shown in group II.Cof the table. The intracellular messenger for group II.Dis a protein kinase-phosphatase cascade. Several of thesehave been identified, and a given hormone may usemore than one kinase cascade. A few hormones fit intomore than one category, and assignments change asnew information is brought forward.DIVERSITY OF THE ENDOCRINE SYSTEMHormones Are Synthesized in aVariety of Cellular ArrangementsHormones are synthesized in discrete organs designedsolely for this specific purpose, such as the thyroid (triiodothyronine),adrenal (glucocorticoids and mineralocorticoids),and the pituitary (TSH, FSH, LH, growthhormone, prolactin, ACTH). Some organs are designedto perform two distinct but closely related functions.For example, the ovaries produce mature oocytes andthe reproductive hormones estradiol and progesterone.The testes produce mature spermatozoa and testosterone.Hormones are also produced in specialized cellswithin other organs such as the small intestine(glucagon-like peptide), thyroid (calcitonin), and kidney(angiotensin II). Finally, the synthesis of some hormonesrequires the parenchymal cells of more than oneorgan—eg, the skin, liver, and kidney are required forthe production of 1,25(OH) 2 -D 3 (calcitriol). Examplesof this diversity in the approach to hormone synthesis,each of which has evolved to fulfill a specific purpose,are discussed below.

438 / CHAPTER 42Hormones Are Chemically DiverseHormones are synthesized from a wide variety of chemicalbuilding blocks. A large series is derived from cholesterol.These include the glucocorticoids, mineralocorticoids,estrogens, progestins, and 1,25(OH) 2 -D 3(see Figure 42–2). In some cases, a steroid hormone isthe precursor molecule for another hormone. For example,progesterone is a hormone in its own right butis also a precursor in the formation of glucocorticoids,mineralocorticoids, testosterone, and estrogens. Testosteroneis an obligatory intermediate in the biosynthesisof estradiol and in the formation of dihydrotestosterone(DHT). In these examples, described in detail below,the final product is determined by the cell type and theassociated set of enzymes in which the precursor exists.The amino acid tyrosine is the starting point in thesynthesis of the catecholamines and of the thyroid hormonestetraiodothyronine (thyroxine; T 4 ) and triiodothyronine(T 3 ) (Figure 42–2). T 3 and T 4 are unique inthat they require the addition of iodine (as I − ) for bioactivity.Because dietary iodine is very scarce in manyparts of the world, an intricate mechanism for accumulatingand retaining I − has evolved.Many hormones are polypeptides or glycoproteins.These range in size from thyrotropin-releasing hormone(TRH), a tripeptide, to single-chain polypeptideslike adrenocorticotropic hormone (ACTH; 39 aminoacids), parathyroid hormone (PTH; 84 amino acids),and growth hormone (GH; 191 amino acids) (Figure42–2). Insulin is an AB chain heterodimer of 21 and 30amino acids, respectively. Follicle-stimulating hormone(FSH), luteinizing hormone (LH), thyroid-stimulatinghormone (TSH), and chorionic gonadotropin (CG) areglycoprotein hormones of αβ heterodimeric structure.The α chain is identical in all of these hormones, anddistinct β chains impart hormone uniqueness. Thesehormones have a molecular mass in the range of 25–30kDa depending on the degree of glycosylation and thelength of the β chain.Hormones Are Synthesized & ModifiedFor Full Activity in a Variety of WaysSome hormones are synthesized in final form and secretedimmediately. Included in this class are the hormonesderived from cholesterol. Others such as the catecholaminesare synthesized in final form and stored inthe producing cells. Others are synthesized from precursormolecules in the producing cell, then areprocessed and secreted upon a physiologic cue (insulin).Finally, still others are converted to active forms fromprecursor molecules in the periphery (T 3 and DHT).All of these examples are discussed in more detailbelow.MANY HORMONES ARE MADEFROM CHOLESTEROLAdrenal SteroidogenesisThe adrenal steroid hormones are synthesized fromcholesterol. Cholesterol is mostly derived from theplasma, but a small portion is synthesized in situ fromacetyl-CoA via mevalonate and squalene. Much of thecholesterol in the adrenal is esterified and stored in cytoplasmiclipid droplets. Upon stimulation of theadrenal by ACTH, an esterase is activated, and the freecholesterol formed is transported into the mitochondrion,where a cytochrome P450 side chain cleavageenzyme (P450scc) converts cholesterol to pregnenolone.Cleavage of the side chain involves sequentialhydroxylations, first at C 22 and then at C 20 , followed byside chain cleavage (removal of the six-carbon fragmentisocaproaldehyde) to give the 21-carbon steroid (Figure42–3, top). An ACTH-dependent steroidogenic acuteregulatory (StAR) protein is essential for the transportof cholesterol to P450scc in the inner mitochondrialmembrane.All mammalian steroid hormones are formed fromcholesterol via pregnenolone through a series of reactionsthat occur in either the mitochondria or endoplasmicreticulum of the adrenal cell. Hydroxylases that requiremolecular oxygen and NADPH are essential, anddehydrogenases, an isomerase, and a lyase reaction arealso necessary for certain steps. There is cellular specificityin adrenal steroidogenesis. For instance, 18-hydroxylase and 19-hydroxysteroid dehydrogenase,which are required for aldosterone synthesis, are foundonly in the zona glomerulosa cells (the outer region ofthe adrenal cortex), so that the biosynthesis of this mineralocorticoidis confined to this region. A schematicrepresentation of the pathways involved in the synthesisof the three major classes of adrenal steroids is presentedin Figure 42–4. The enzymes are shown in therectangular boxes, and the modifications at each stepare shaded.A. MINERALOCORTICOID SYNTHESISSynthesis of aldosterone follows the mineralocorticoidpathway and occurs in the zona glomerulosa. Pregnenoloneis converted to progesterone by the action oftwo smooth endoplasmic reticulum enzymes, 3hydroxysteroiddehydrogenase (3-OHSD) and 5,4 -isomerase. Progesterone is hydroxylated at the C 21 positionto form 11-deoxycorticosterone (DOC), which is anactive (Na + -retaining) mineralocorticoid. The next hydroxylation,at C 11 , produces corticosterone, which hasglucocorticoid activity and is a weak mineralocorticoid (ithas less than 5% of the potency of aldosterone). In somespecies (eg, rodents), it is the most potent glucocorticoid.

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 439A. CHOLESTEROL DERIVATIVESOHOHOHCH 2 OHC OOHCH 3C OCH 2HO17ß-EstradiolOTestosteroneOCortisolHOHOOHProgesterone 1,25(OH) 2 -D 3B. TYROSINE DERIVATIVESIIOH O CH 2 CH COOHI NH 2T 3IIOH O CH 2 CH COOHIIT 4HOHOHOHOHOCHHCHNorepinephrineHOCHHEpinephrineNH 2H CH 3C NHNH 2NH 2C. PEPTIDES OF VARIOUS SIZES(pyro)1 2 3Glu Hls Pro1Ser ser2Tyr ser3Ser ser4Met ser5Glu ser6Hls ser7Phe ser8Arg ser9Trp ser10Gly ser11Lys serConserved region; required for full biologic activity24 23 22 21 20 19 18 17 1625 Pro Tyr Val Lys Val ser Pro ser Arg ser Arg ser Lys serAsp26AlaLys ser12Pro ser1513ser ValGly ser14TRHD. GLYCOPROTEINS (TSH, FSH, LH)27Gly28GluVariable region; not required for biologic activityAsp ser29Gln ser30Ser ser31Ala ser32Glu ser33Ala ser34Phe ser35Structure of human ACTH.ACTHPro ser36Leu ser37Glu ser38Phe ser39commonuniqueαβsubunitssubunitsFigure 42–2. Chemical diversity of hormones. A. Cholesterol derivatives. B. Tyrosine derivatives.C. Peptides of various sizes D. Glycoproteins (TSH, FSH, LH) with common α subunits and unique βsubunits.

440 / CHAPTER 42HOABCCCDC C CC CCCholesterol side chain cleavageACTH(cAMP)P450sccHO2314191052120CH 3C1812 1711 13 1615149876O+HCC C C COCCholesterolPregnenolone + isocaproaldehydeBasic steroid hormone structuresCH 2 OHOHOHHOCOOHCH 3O CHOOOO17β—EstradiolTestosteroneCortisolProgesteroneEstrane group (C18)Androstane group (C19)Pregnane group (C21)Figure 42–3. Cholesterol side-chain cleavage and basic steroid hormone structures. The basic sterol rings are identifiedby the letters A–D. The carbon atoms are numbered 1–21 starting with the A ring. Note that the estrane grouphas 18 carbons (C18), etc.C 21 hydroxylation is necessary for both mineralocorticoidand glucocorticoid activity, but most steroids with a C 17hydroxyl group have more glucocorticoid and less mineralocorticoidaction. In the zona glomerulosa, which doesnot have the smooth endoplasmic reticulum enzyme17α-hydroxylase, a mitochondrial 18-hydroxylase is present.The 18-hydroxylase (aldosterone synthase) acts oncorticosterone to form 18-hydroxycorticosterone, whichis changed to aldosterone by conversion of the 18-alcoholto an aldehyde. This unique distribution of enzymes andthe special regulation of the zona glomerulosa by K + andangiotensin II have led some investigators to suggest that,in addition to the adrenal being two glands, the adrenalcortex is actually two separate organs.B. GLUCOCORTICOID SYNTHESISCortisol synthesis requires three hydroxylases located inthe fasciculata and reticularis zones of the adrenal cortexthat act sequentially on the C 17 , C 21 , and C 11 positions.The first two reactions are rapid, while C 11 hydroxylationis relatively slow. If the C 11 position is hydroxylatedfirst, the action of 17-hydroxylase is impeded and themineralocorticoid pathway is followed (forming corticosteroneor aldosterone, depending on the cell type).17α-Hydroxylase is a smooth endoplasmic reticulumenzyme that acts upon either progesterone or, morecommonly, pregnenolone. 17α-Hydroxyprogesterone ishydroxylated at C 21 to form 11-deoxycortisol, which isthen hydroxylated at C 11 to form cortisol, the most potentnatural glucocorticoid hormone in humans. 21-Hydroxylaseis a smooth endoplasmic reticulum enzyme,whereas 11β-hydroxylase is a mitochondrial enzyme.Steroidogenesis thus involves the repeated shuttling ofsubstrates into and out of the mitochondria.C. ANDROGEN SYNTHESISThe major androgen or androgen precursor produced bythe adrenal cortex is dehydroepiandrosterone (DHEA).Most 17-hydroxypregnenolone follows the glucocorticoidpathway, but a small fraction is subjected to oxidative fissionand removal of the two-carbon side chain throughthe action of 17,20-lyase. The lyase activity is actuallypart of the same enzyme (P450c17) that catalyzes 17αhydroxylation.This is therefore a dual function protein.The lyase activity is important in both the adrenals and

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 441CholesterolHOSCC CH 3C OPregnenolone17α-HYDROXYLASECH 3C O— OHHO17-Hydroxypregnenolone17,20-LYASEOHODehydroepiandrosterone3β-HYDROXYSTEROID DEHYDROGENASE: ∆ 5,4 ISOMERASECH 3COCCH 3— OHOOP450c17P450c17OProgesteroneO17-HydroxyprogesteroneO∆ 4 ANDROSTENE-3,17-DION21-HYDROXYLASECH 2 OHCH 2 OHCOCO— OHO11-DeoxycorticosteroneO11-Deoxycortisol11β-HYDROXYLASECH 2 OHCH 2 OHHOCOHOCO— OHOCorticosteroneOCORTISOL18-HYDROXYLASE18-HYDROXYDEHYDROGENASEHOHOCCH 2 OHCOOALDOSTERONEFigure 42–4. Pathways involved in the synthesis of the three major classes ofadrenal steroids (mineralocorticoids, glucocorticoids, and androgens). Enzymesare shown in the rectangular boxes, and the modifications at each step areshaded. Note that the 17α-hydroxylase and 17,20-lyase activities are both part ofone enzyme, designated P450c17. (Slightly modified and reproduced, with permission,from Harding BW: In: Endocrinology, vol 2. DeGroot LJ [editor]. Grune & Stratton,1979.)

442 / CHAPTER 42the gonads and acts exclusively on 17α-hydroxy-containingmolecules. Adrenal androgen production increasesmarkedly if glucocorticoid biosynthesis is impeded by thelack of one of the hydroxylases (adrenogenital syndrome).DHEA is really a prohormone, since the actionsof 3β-OHSD and ∆ 5,4 -isomerase convert the weak androgenDHEA into the more potent androstenedione.Small amounts of androstenedione are also formed in theadrenal by the action of the lyase on 17α-hydroxyprogesterone.Reduction of androstenedione at the C 17 positionresults in the formation of testosterone, the most potentadrenal androgen. Small amounts of testosterone are producedin the adrenal by this mechanism, but most of thisconversion occurs in the testes.Testicular SteroidogenesisTesticular androgens are synthesized in the interstitialtissue by the Leydig cells. The immediate precursor ofthe gonadal steroids, as for the adrenal steroids, is cholesterol.The rate-limiting step, as in the adrenal, is deliveryof cholesterol to the inner membrane of the mitochondriaby the transport protein StAR. Once in theproper location, cholesterol is acted upon by the sidechain cleavage enzyme P450scc. The conversion of cholesterolto pregnenolone is identical in adrenal, ovary,and testis. In the latter two tissues, however, the reactionis promoted by LH rather than ACTH.The conversion of pregnenolone to testosterone requiresthe action of five enzyme activities contained inthree proteins: (1) 3β-hydroxysteroid dehydrogenase (3β-OHSD) and ∆ 5,4 -isomerase; (2) 17α-hydroxylase and17,20-lyase; and (3) 17β-hydroxysteroid dehydrogenase(17β-OHSD). This sequence, referred to as the progesterone(or 4 ) pathway, is shown on the right side of Figure42–5. Pregnenolone can also be converted to testosteroneby the dehydroepiandrosterone (or 5 ) pathway,which is illustrated on the left side of Figure 42–5. The ∆ 5route appears to be most used in human testes.The five enzyme activities are localized in the microsomalfraction in rat testes, and there is a close functionalassociation between the activities of 3β-OHSDand ∆ 5,4 -isomerase and between those of a 17α-hydroxylaseand 17,20-lyase. These enzyme pairs, both containedin a single protein, are shown in the general reactionsequence in Figure 42–5.Dihydrotestosterone Is Formed FromTestosterone in Peripheral TissuesTestosterone is metabolized by two pathways. One involvesoxidation at the 17 position, and the other involvesreduction of the A ring double bond and the 3-ketone.Metabolism by the first pathway occurs in manytissues, including liver, and produces 17-ketosteroids thatare generally inactive or less active than the parent compound.Metabolism by the second pathway, which is lessefficient, occurs primarily in target tissues and producesthe potent metabolite dihydrotestosterone (DHT).The most significant metabolic product of testosteroneis DHT, since in many tissues, includingprostate, external genitalia, and some areas of the skin,this is the active form of the hormone. The plasma contentof DHT in the adult male is about one-tenth thatof testosterone, and approximately 400 µg of DHT isproduced daily as compared with about 5 mg of testosterone.About 50–100 µg of DHT are secreted by thetestes. The rest is produced peripherally from testosteronein a reaction catalyzed by the NADPH-dependent5-reductase (Figure 42–6). Testosterone canthus be considered a prohormone, since it is convertedinto a much more potent compound (dihydrotestosterone)and since most of this conversion occurs outsidethe testes. Some estradiol is formed from the peripheralaromatization of testosterone, particularly in males.Ovarian SteroidogenesisThe estrogens are a family of hormones synthesized in avariety of tissues. 17β-Estradiol is the primary estrogenof ovarian origin. In some species, estrone, synthesizedin numerous tissues, is more abundant. In pregnancy,relatively more estriol is produced, and this comes fromthe placenta. The general pathway and the subcellularlocalization of the enzymes involved in the early stepsof estradiol synthesis are the same as those involved inandrogen biosynthesis. Features unique to the ovary areillustrated in Figure 42–7.Estrogens are formed by the aromatization of androgensin a complex process that involves three hydroxylationsteps, each of which requires O 2 and NADPH. Thearomatase enzyme complex is thought to include aP450 monooxygenase. Estradiol is formed if the substrateof this enzyme complex is testosterone, whereas estroneresults from the aromatization of androstenedione.The cellular source of the various ovarian steroids hasbeen difficult to unravel, but a transfer of substrates betweentwo cell types is involved. Theca cells are the sourceof androstenedione and testosterone. These are convertedby the aromatase enzyme in granulosa cells to estrone andestradiol, respectively. Progesterone, a precursor for allsteroid hormones, is produced and secreted by the corpusluteum as an end-product hormone because these cells donot contain the enzymes necessary to convert progesteroneto other steroid hormones (Figure 42–8).Significant amounts of estrogens are produced bythe peripheral aromatization of androgens. In humanmales, the peripheral aromatization of testosterone toestradiol (E 2 ) accounts for 80% of the production ofthe latter. In females, adrenal androgens are important

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 443CH 3OCH 3OCCHOPregnenoloneHOProgesterone17α-HYDROXYLASE*17α-HYDROXYLASE*CH 3C OOHHO17α-Hydroxypregnenolone17,20-LYASE*OHODehydroepiandrosterone3β–HYDROXYSTEROID DEHYDROGENASE AND ∆ 5,4 ISOMERASECH 3C OO17α-Hydroxyprogesterone17,20-LYASE*OOAndrostenedioneOH17β-HYDROXYSTEROIDDEHYDROGENASE17β-HYDROXYSTEROIDDEHYDROGENASEFigure 42–5. Pathways of testosteronebiosynthesis. The pathway onthe left side of the figure is called the ∆ 5or dehydroepiandrosterone pathway;the pathway on the right side is calledthe ∆ 4 or progesterone pathway. The asteriskindicates that the 17α-hydroxylaseand 17,20-lyase activities reside in asingle protein, P450c17.HO∆ 5 -AndrostenediolOHOTESTOSTERONEOH

444 / CHAPTER 42OHOH5α-REDUCTASENADPHOOHTestosteroneDIHYDROTESTOSTERONE (DHT)Figure 42–6. Dihydrotestosterone is formed from testosterone through action of theenzyme 5α-reductase.CholesterolPregnenolone17α-HydroxypregnenoloneDehydroepiandrosteroneProgesterone17α-HydroxyprogesteroneAndrostenedioneTestosteroneAROMATASEAROMATASEOOHOthermetabolitesHOHOESTRONE (E 1 )17β-ESTRADIOL (E 2 )16α-HydroxylaseOHOther metabolitesOHHOEstriolFigure 42–7. Biosynthesis of estrogens. (Slightly modified and reproduced, with permission, from GanongWF: Review of Medical Physiology, 20th ed. McGraw-Hill, 2001.)

AcetateCholesterolCH 3C OTHE DIVERSITY OF THE ENDOCRINE SYSTEM / 445most of the precursor for 1,25(OH) 2 -D 3 synthesis isproduced in the malpighian layer of the epidermis from7-dehydrocholesterol in an ultraviolet light-mediated,nonenzymatic photolysis reaction. The extent of thisconversion is related directly to the intensity of the exposureand inversely to the extent of pigmentation inthe skin. There is an age-related loss of 7-dehydrocholesterolin the epidermis that may be related to the negativecalcium balance associated with old age.HOOPregnenoloneProgesteroneCH 3C OFigure 42–8. Biosynthesis of progesterone in thecorpus luteum.substrates, since as much as 50% of the E 2 producedduring pregnancy comes from the aromatization of androgens.Finally, conversion of androstenedione toestrone is the major source of estrogens in postmenopausalwomen. Aromatase activity is present inadipose cells and also in liver, skin, and other tissues.Increased activity of this enzyme may contribute to the“estrogenization” that characterizes such diseases as cirrhosisof the liver, hyperthyroidism, aging, and obesity.1,25(OH) 2 -D 3 (Calcitriol) Is SynthesizedFrom a Cholesterol Derivative1,25(OH) 2 -D 3 is produced by a complex series of enzymaticreactions that involve the plasma transport of precursormolecules to a number of different tissues (Figure42–9). One of these precursors is vitamin D—really nota vitamin, but this common name persists. The activemolecule, 1,25(OH) 2 -D 3 , is transported to other organswhere it activates biologic processes in a manner similarto that employed by the steroid hormones.A. SKINSmall amounts of the precursor for 1,25(OH) 2 -D 3 synthesisare present in food (fish liver oil, egg yolk), butB. LIVERA specific transport protein called the vitamin D-bindingprotein binds vitamin D 3 and its metabolites andmoves vitamin D 3 from the skin or intestine to theliver, where it undergoes 25-hydroxylation, the firstobligatory reaction in the production of 1,25(OH) 2 -D 3 . 25-Hydroxylation occurs in the endoplasmic reticulumin a reaction that requires magnesium, NADPH,molecular oxygen, and an uncharacterized cytoplasmicfactor. Two enzymes are involved: an NADPH-dependentcytochrome P450 reductase and a cytochromeP450. This reaction is not regulated, and it also occurswith low efficiency in kidney and intestine. The25(OH) 2 -D 3 enters the circulation, where it is themajor form of vitamin D found in plasma, and is transportedto the kidney by the vitamin D-binding protein.C. KIDNEY25(OH) 2 -D 3 is a weak agonist and must be modifiedby hydroxylation at position C 1 for full biologic activity.This is accomplished in mitochondria of the renalproximal convoluted tubule by a three-componentmonooxygenase reaction that requires NADPH, Mg 2+ ,molecular oxygen, and at least three enzymes: (1) aflavoprotein, renal ferredoxin reductase; (2) an iron sulfurprotein, renal ferredoxin; and (3) cytochrome P450.This system produces 1,25(OH) 2 -D 3 , which is the mostpotent naturally occurring metabolite of vitamin D.CATECHOLAMINES & THYROIDHORMONES ARE MADE FROM TYROSINECatecholamines Are Synthesized in FinalForm & Stored in Secretion GranulesThree amines—dopamine, norepinephrine, and epinephrine—aresynthesized from tyrosine in the chromaffincells of the adrenal medulla. The major productof the adrenal medulla is epinephrine. This compoundconstitutes about 80% of the catecholamines in themedulla, and it is not made in extramedullary tissue. Incontrast, most of the norepinephrine present in organsinnervated by sympathetic nerves is made in situ (about80% of the total), and most of the rest is made in othernerve endings and reaches the target sites via the circu-

446 / CHAPTER 42Sunlight7-Dehydrocholesterol Previtamin D 3 Vitamin D 3SKINLIVER25-HydroxylaseOthermetabolites25-Hydroxycholecalciferol (25[OH]-D 3 )24-Hydroxylase1 α-Hydroxylase24,25(OH) 2 -D 31,24,25(OH) 3 -D 3KIDNEY1,25(OH) 2 -D 3242725OH26CH 2CH 2HOHOHOOH7-DehydrocholesterolVitamin D 31,25(OH) 2 -D 3Figure 42–9. Formation and hydroxylation of vitamin D 3 . 25-Hydroxylation takes place in the liver, and theother hydroxylations occur in the kidneys. 25,26(OH) 2 -D 3 and 1,25,26(OH) 3 -D 3 are probably formed as well. Theformulas of 7-dehydrocholesterol, vitamin D 3 , and 1,25(OH) 2 -D 3 are also shown. (Modified and reproduced, withpermission, from Ganong WF: Review of Medical Physiology, 20th ed. McGraw-Hill, 2001.)lation. Epinephrine and norepinephrine may be producedand stored in different cells in the adrenalmedulla and other chromaffin tissues.The conversion of tyrosine to epinephrine requiresfour sequential steps: (1) ring hydroxylation; (2) decarboxylation;(3) side chain hydroxylation to form norepinephrine;and (4) N-methylation to form epinephrine.The biosynthetic pathway and the enzymes involved areillustrated in Figure 42–10.A. TYROSINE HYDROXYLASE IS RATE-LIMITINGFOR CATECHOLAMINE BIOSYNTHESISTyrosine is the immediate precursor of catecholamines,and tyrosine hydroxylase is the rate-limiting enzyme incatecholamine biosynthesis. Tyrosine hydroxylase isfound in both soluble and particle-bound forms only intissues that synthesize catecholamines; it functions as anoxidoreductase, with tetrahydropteridine as a cofactor, toconvert L-tyrosine to L-dihydroxyphenylalanine (L-dopa).As the rate-limiting enzyme, tyrosine hydroxylase is regulatedin a variety of ways. The most important mechanisminvolves feedback inhibition by the catecholamines,which compete with the enzyme for the pteridine cofactor.Catecholamines cannot cross the blood-brain barrier;hence, in the brain they must be synthesized locally. Incertain central nervous system diseases (eg, Parkinson’sdisease), there is a local deficiency of dopamine synthesis.L-Dopa, the precursor of dopamine, readily crosses theblood-brain barrier and so is an important agent in thetreatment of Parkinson’s disease.B. DOPA DECARBOXYLASE IS PRESENT IN ALL TISSUESThis soluble enzyme requires pyridoxal phosphate forthe conversion of L-dopa to 3,4-dihydroxyphenylethylamine(dopamine). Compounds that resemble L-dopa,such as α-methyldopa, are competitive inhibitors ofthis reaction. α-Methyldopa is effective in treatingsome kinds of hypertension.

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 447HOHOHOHOHOHOHOTyrosineDopaDOPAMINEHCHOCCHTYROSINEHYDROXYLASEHCHOCCHOHNH 2OHNH 2DOPADECARBOXYLASEHCHHCHDOPAMINEβ-HYDROXYLASEHOHNOREPINEPHRINEHOHOPNMTCHOCHEPINEPHRINEHCHHCHNH 2NH 2CH 3NHFigure 42–10. Biosynthesis of catecholamines.(PNMT, phenylethanolamine-N-methyltransferase.)C. DOPAMINE -HYDROXYLASE (DBH) CATALYZESTHE CONVERSION OF DOPAMINE TO NOREPINEPHRINEDBH is a monooxygenase and uses ascorbate as an electrondonor, copper at the active site, and fumarate asmodulator. DBH is in the particulate fraction of themedullary cells, probably in the secretion granule; thus,the conversion of dopamine to norepinephrine occursin this organelle.D. PHENYLETHANOLAMINE-N-METHYLTRANSFERASE(PNMT) CATALYZES THE PRODUCTIONOF EPINEPHRINEPNMT catalyzes the N-methylation of norepinephrineto form epinephrine in the epinephrine-forming cellsof the adrenal medulla. Since PNMT is soluble, it is assumedthat norepinephrine-to-epinephrine conversionoccurs in the cytoplasm. The synthesis of PNMT is inducedby glucocorticoid hormones that reach themedulla via the intra-adrenal portal system. This specialsystem provides for a 100-fold steroid concentrationgradient over systemic arterial blood, and this highintra-adrenal concentration appears to be necessary forthe induction of PNMT.T 3 & T 4 Illustrate the Diversityin Hormone SynthesisThe formation of triiodothyronine (T 3 ) and tetraiodothyronine(thyroxine; T 4 ) (see Figure 42–2) illustratesmany of the principles of diversity discussed inthis chapter. These hormones require a rare element(iodine) for bioactivity; they are synthesized as part of avery large precursor molecule (thyroglobulin); they arestored in an intracellular reservoir (colloid); and there isperipheral conversion of T 4 to T 3 , which is a muchmore active hormone.The thyroid hormones T 3 and T 4 are unique in thatiodine (as iodide) is an essential component of both. Inmost parts of the world, iodine is a scarce component ofsoil, and for that reason there is little in food. A complexmechanism has evolved to acquire and retain thiscrucial element and to convert it into a form suitablefor incorporation into organic compounds. At the sametime, the thyroid must synthesize thyronine from tyrosine,and this synthesis takes place in thyroglobulin(Figure 42–11).Thyroglobulin is the precursor of T 4 and T 3 . It is alarge iodinated, glycosylated protein with a molecularmass of 660 kDa. Carbohydrate accounts for 8–10% ofthe weight of thyroglobulin and iodide for about0.2–1%, depending upon the iodine content in thediet. Thyroglobulin is composed of two large subunits.It contains 115 tyrosine residues, each of which is a potentialsite of iodination. About 70% of the iodidein thyroglobulin exists in the inactive precursors,monoiodotyrosine (MIT) and diiodotyrosine (DIT),while 30% is in the iodothyronyl residues, T 4 and T 3 .When iodine supplies are sufficient, the T 4 :T 3 ratio isabout 7:1. In iodine deficiency, this ratio decreases, asdoes the DIT:MIT ratio. Thyroglobulin, a large moleculeof about 5000 amino acids, provides the confor-

448 / CHAPTER 42FOLLICULAR SPACE WITH COLLOID–IOxidationPEROXIDASEH 2 O 2O 2MIT MITT 3 MITDITDITDIT DITIodination*TgbDITCoupling*I + +TgbTgb T 4MITMITMITDITDIT DITDIT T 4NADPH NADP +H +TgbPhagocytosisandpinocytosisTHYROID CELLTgbLysosomesTgbSecondarylysosomeTyrosine–I–IDeiodination*DEIODINASEMITDITHydrolysisTransporterConcentration*Na + -K + ATPaseT 3 , T 4Release–IEXTRACELLULAR SPACEFigure 42–11. Model of iodide metabolism in the thyroid follicle. A follicular cell is shown facing the follicularlumen (top) and the extracellular space (at bottom). Iodide enters the thyroid primarily through a transporter(bottom left). Thyroid hormone synthesis occurs in the follicular space through a series of reactions, many ofwhich are peroxidase-mediated. Thyroid hormones, stored in the colloid in the follicular space, are released fromthyroglobulin by hydrolysis inside the thyroid cell. (Tgb, thyroglobulin; MIT, monoiodotyrosine; DIT, diiodotyrosine;T 3 , triiodothyronine; T 4 , tetraiodothyronine.) Asterisks indicate steps or processes that are inherited enzymedeficiencies which cause congenital goiter and often result in hypothyroidism.T 3 , T 4

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 449mation required for tyrosyl coupling and iodide organificationnecessary in the formation of the diaminoacidthyroid hormones. It is synthesized in the basal portionof the cell and moves to the lumen, where it is a storageform of T 3 and T 4 in the colloid; several weeks’ supplyof these hormones exist in the normal thyroid. Withinminutes after stimulation of the thyroid by TSH, colloidreenters the cell and there is a marked increase ofphagolysosome activity. Various acid proteases andpeptidases hydrolyze the thyroglobulin into its constituentamino acids, including T 4 and T 3 , which aredischarged from the basal portion of the cell (see Figure42–11). Thyroglobulin is thus a very large prohormone.Iodide Metabolism InvolvesSeveral Discrete StepsThe thyroid is able to concentrate I − against a strongelectrochemical gradient. This is an energy-dependentprocess and is linked to the Na + -K + ATPase-dependentthyroidal I − transporter. The ratio of iodide in thyroidto iodide in serum (T:S ratio) is a reflection of the activityof this transporter. This activity is primarily controlledby TSH and ranges from 500:1 in animalschronically stimulated with TSH to 5:1 or less in hypophysectomizedanimals (no TSH). The T:S ratio inhumans on a normal iodine diet is about 25:1.The thyroid is the only tissue that can oxidize I − to ahigher valence state, an obligatory step in I − organificationand thyroid hormone biosynthesis. This step involvesa heme-containing peroxidase and occurs at theluminal surface of the follicular cell. Thyroperoxidase, atetrameric protein with a molecular mass of 60 kDa, requireshydrogen peroxide as an oxidizing agent. TheH 2 O 2 is produced by an NADPH-dependent enzymeresembling cytochrome c reductase. A number of compoundsinhibit I − oxidation and therefore its subsequentincorporation into MIT and DIT. The most importantof these are the thiourea drugs. They are used asantithyroid drugs because of their ability to inhibit thyroidhormone biosynthesis at this step. Once iodinationoccurs, the iodine does not readily leave the thyroid.Free tyrosine can be iodinated, but it is not incorporatedinto proteins since no tRNA recognizes iodinatedtyrosine.The coupling of two DIT molecules to form T 4 —orof an MIT and DIT to form T 3 —occurs within thethyroglobulin molecule. A separate coupling enzymehas not been found, and since this is an oxidativeprocess it is assumed that the same thyroperoxidase catalyzesthis reaction by stimulating free radical formationof iodotyrosine. This hypothesis is supported bythe observation that the same drugs which inhibit I − oxidationalso inhibit coupling. The formed thyroid hormonesremain as integral parts of thyroglobulin untilthe latter is degraded, as described above.A deiodinase removes I − from the inactive monoanddiiodothyronine molecules in the thyroid. Thismechanism provides a substantial amount of the I − usedin T 3 and T 4 biosynthesis. A peripheral deiodinase intarget tissues such as pituitary, kidney, and liver selectivelyremoves I − from the 5′ position of T 4 to make T 3(see Figure 42–2), which is a much more active molecule.In this sense, T 4 can be thought of as a prohormone,though it does have some intrinsic activity.SEVERAL HORMONES ARE MADE FROMLARGER PEPTIDE PRECURSORSFormation of the critical disulfide bridges in insulin requiresthat this hormone be first synthesized as part of alarger precursor molecule, proinsulin. This is conceptuallysimilar to the example of the thyroid hormones,which can only be formed in the context of a muchlarger molecule. Several other hormones are synthesizedas parts of large precursor molecules, not because ofsome special structural requirement but rather as amechanism for controlling the available amount of theactive hormone. PTH and angiotensin II are examplesof this type of regulation. Another interesting exampleis the POMC protein, which can be processed intomany different hormones in a tissue-specific manner.These examples are discussed in detail below.Insulin Is Synthesized as a Preprohormone& Modified Within the CellInsulin has an AB heterodimeric structure with one intrachain(A6–A11) and two interchain disulfide bridges(A7–B7 and A20–B19) (Figure 42–12). The A and Bchains could be synthesized in the laboratory, but attemptsat a biochemical synthesis of the mature insulinmolecule yielded very poor results. The reason for thisbecame apparent when it was discovered that insulin issynthesized as a preprohormone (molecular weight approximately11,500), which is the prototype for peptidesthat are processed from larger precursor molecules. Thehydrophobic 23-amino-acid pre-, or leader, sequence directsthe molecule into the cisternae of the endoplasmicreticulum and then is removed. This results in the 9000-MW proinsulin molecule, which provides the conformationnecessary for the proper and efficient formation ofthe disulfide bridges. As shown in Figure 42–12, the sequenceof proinsulin, starting from the amino terminal,is B chain—connecting (C) peptide—A chain. Theproinsulin molecule undergoes a series of site-specificpeptide cleavages that result in the formation of equimolaramounts of mature insulin and C peptide. These enzymaticcleavages are summarized in Figure 42–12.

450 / CHAPTER 4220NH 2 –Ala Leu Pro Gln Leu Ser Gly Ala Gly Pro GlyGly GlyLeuConnecting peptideLeuGluGlu10GlyValSerGlnLeu31GlyGlnValLysGlnArgLeu1 GlyIleVal AsnGlu21GluCysAla1 PheSGlnSTyrGlu 1ValCysAsnA chainCysThrSerGln Leu Glu ArgAsnGlnIle Cys Ser Leu TyrSArgSMisThr1030Leu SLysCysProInsulinSThrGlyTyrSerPheMisB chainPheLeuGlyValGluArg10Glu AlaCys GlyLeu Tyr Leu Val20Figure 42–12. Structure of human proinsulin. Insulin and C-peptide molecules are connected at two sites bydipeptide links. An initial cleavage by a trypsin-like enzyme (open arrows) followed by several cleavages by a carboxypeptidase-likeenzyme (solid arrows) results in the production of the heterodimeric (AB) insulin molecule(light blue) and the C-peptide.–COOHAspParathyroid Hormone (PTH) Is Secreted asan 84-Amino-Acid PeptideThe immediate precursor of PTH is proPTH, whichdiffers from the native 84-amino-acid hormone by havinga highly basic hexapeptide amino terminal extension.The primary gene product and the immediate precursorfor proPTH is the 115-amino-acid preproPTH.This differs from proPTH by having an additional 25-amino-acid amino terminal extension that, in commonwith the other leader or signal sequences characteristicof secreted proteins, is hydrophobic. The completestructure of preproPTH and the sequences of proPTHand PTH are illustrated in Figure 42–13. PTH 1–34 hasfull biologic activity, and the region 25–34 is primarilyresponsible for receptor binding.The biosynthesis of PTH and its subsequent secretionare regulated by the plasma ionized calcium (Ca 2+ )concentration through a complex process. An acute decreaseof Ca 2+ results in a marked increase of PTHmRNA, and this is followed by an increased rate ofPTH synthesis and secretion. However, about 80–90%of the proPTH synthesized cannot be accounted for asintact PTH in cells or in the incubation medium of experimentalsystems. This finding led to the conclusionthat most of the proPTH synthesized is quickly degraded.It was later discovered that this rate of degradationdecreases when Ca 2+ concentrations are low, and itincreases when Ca 2+ concentrations are high. Very specificfragments of PTH are generated during its proteolyticdigestion (Figure 42–13). A number of proteolyticenzymes, including cathepsins B and D, havebeen identified in parathyroid tissue. Cathepsin Bcleaves PTH into two fragments: PTH 1–36 andPTH 37–84 . PTH 37–84 is not further degraded; however,PTH 1–36 is rapidly and progressively cleaved into diandtripeptides. Most of the proteolysis of PTH occurswithin the gland, but a number of studies confirm thatPTH, once secreted, is proteolytically degraded in othertissues, especially the liver, by similar mechanisms.

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 451–31Leader (pre) sequence–6–10–20Ser10 20Glu Ile Gln Phe Met His Asn Leu Gly Lys His Leu Ser Ser Met Glu Arg Val Glu Trp Leu Arg Lys LysLeuProLys Gly Asp Ser Arg Ala Leu Phe Cys Ile Ala Leu Met Val Ile Met Val Lys Val Met Asp Lys Ala Ser Met MetSersequence ValLysLys(2)(1)–1 Arg (3)1 AlaValNH 260ValGluSerAsnValLeuFull biologic activity sequenceGln30AspValHisAsnC-fragment sequencePhe5040ValAsp Glu Lys Lys Arg Pro Arg Gln Ser Ser Gly Asp Arg Tyr Ala Ile Ser Ala Gly Leu AlaHisGlnLys70 80Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asp Val Leu Ile Lys Ala Lys Pro GlnFigure 42–13. Structure of bovine preproparathyroid hormone. Arrows indicate sites cleaved by processingenzymes in the parathyroid gland (1–5) and in the liver after secretion of the hormone (4–5). Thebiologically active region of the molecule is flanked by sequence not required for activity on target receptors.(Slightly modified and reproduced, with permission, from Habener JF: Recent advances in parathyroidhormone research. Clin Biochem 1981;14:223.)COOH(5)(4)Angiotensin II Is Also SynthesizedFrom a Large PrecursorThe renin-angiotensin system is involved in the regulationof blood pressure and electrolyte metabolism(through production of aldosterone). The primary hormoneinvolved in these processes is angiotensin II, anoctapeptide made from angiotensinogen (Figure42–14). Angiotensinogen, a large α 2 -globulin made inliver, is the substrate for renin, an enzyme produced inthe juxtaglomerular cells of the renal afferent arteriole.The position of these cells makes them particularly sensitiveto blood pressure changes, and many of the physiologicregulators of renin release act through renalbaroreceptors. The juxtaglomerular cells are also sensitiveto changes of Na + and Cl − concentration in therenal tubular fluid; therefore, any combination of factorsthat decreases fluid volume (dehydration, decreasedblood pressure, fluid or blood loss) or decreases NaClconcentration stimulates renin release. Renal sympatheticnerves that terminate in the juxtaglomerular cellsmediate the central nervous system and postural effectson renin release independently of the baroreceptor andsalt effects, a mechanism that involves the β-adrenergicreceptor. Renin acts upon the substrate angiotensinogento produce the decapeptide angiotensin I.Angiotensin-converting enzyme, a glycoproteinfound in lung, endothelial cells, and plasma, removestwo carboxyl terminal amino acids from the decapeptideangiotensin I to form angiotensin II in a step thatis not thought to be rate-limiting. Various nonapeptideanalogs of angiotensin I and other compounds act ascompetitive inhibitors of converting enzyme and areused to treat renin-dependent hypertension. These are

452 / CHAPTER 42AngiotensinogenAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu (~400 more amino acids)RENINAngiotensin IAsp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-LeuCONVERTING ENZYMEANGIOTENSIN IIAsp-Arg-Val-Tyr-Ile-His-Pro-PheAMINOPEPTIDASEAngiotensin IIIArg-Val-Tyr-Ile-His-Pro-PheANGIOTENSINASESDegradation productsFigure 42–14. Formation and metabolism of angiotensins. Small arrows indicatecleavage sites.referred to as angiotensin-converting enzyme (ACE)inhibitors. Angiotensin II increases blood pressure bycausing vasoconstriction of the arteriole and is a verypotent vasoactive substance. It inhibits renin releasefrom the juxtaglomerular cells and is a potent stimulatorof aldosterone production. This results in Na + retention,volume expansion, and increased blood pressure.In some species, angiotensin II is converted to theheptapeptide angiotensin III (Figure 42–14), an equallypotent stimulator of aldosterone production. In humans,the plasma level of angiotensin II is four timesgreater than that of angiotensin III, so most effects areexerted by the octapeptide. Angiotensins II and III arerapidly inactivated by angiotensinases.Angiotensin II binds to specific adrenal cortexglomerulosa cell receptors. The hormone-receptor interactiondoes not activate adenylyl cyclase, and cAMPdoes not appear to mediate the action of this hormone.The actions of angiotensin II, which are to stimulatethe conversion of cholesterol to pregnenolone and ofcorticosterone to 18-hydroxycorticosterone and aldosterone,may involve changes in the concentration of intracellularcalcium and of phospholipid metabolites bymechanisms similar to those described in Chapter 43.Complex Processing Generatesthe Pro-opiomelanocortin (POMC)Peptide FamilyThe POMC family consists of peptides that act as hormones(ACTH, LPH, MSH) and others that may serveas neurotransmitters or neuromodulators (endorphins)(see Figure 42–15). POMC is synthesized as a precursormolecule of 285 amino acids and is processed differentlyin various regions of the pituitary.The POMC gene is expressed in the anterior and intermediatelobes of the pituitary. The most conservedsequences between species are within the amino terminalfragment, the ACTH region, and the β-endorphinregion. POMC or related products are found in severalother vertebrate tissues, including the brain, placenta,gastrointestinal tract, reproductive tract, lung, and lymphocytes.The POMC protein is processed differently in the anteriorlobe than in the intermediate lobe. The intermediatelobe of the pituitary is rudimentary in adult humans,but it is active in human fetuses and in pregnant womenduring late gestation and is also active in many animalspecies. Processing of the POMC protein in the peripheraltissues (gut, placenta, male reproductive tract) resem-

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 453POMC (1–134)ACTH (1–39)β-LPH (42–134)α-MSH(1–13)CLIP(18–39)γ-LPH(42–101)β-Endorphin(104–134)β-MSH(84–101)γ-Endorphin(104–118)α-Endorphin(104–117)Figure 42–15. Products of pro-opiomelanocortin (POMC) cleavage.(MSH, melanocyte-stimulating hormone; CLIP, corticotropin-like intermediatelobe peptide; LPH, lipotropin.)bles that in the intermediate lobe. There are three basicpeptide groups: (1) ACTH, which can give rise toα-MSH and corticotropin-like intermediate lobe peptide(CLIP); (2) β-lipotropin (β-LPH), which can yieldγ-LPH, β-MSH, and β-endorphin (and thus α- andγ-endorphins); and (3) a large amino terminal peptide,which generates γ-MSH. The diversity of these productsis due to the many dibasic amino acid clusters that arepotential cleavage sites for trypsin-like enzymes. Each ofthe peptides mentioned is preceded by Lys-Arg, Arg-Lys,Arg-Arg, or Lys-Lys residues. After the prehormone segmentis cleaved, the next cleavage, in both anterior andintermediate lobes, is between ACTH and β-LPH, resultingin an amino terminal peptide with ACTH and aβ-LPH segment (Figure 42–15). ACTH 1–39 is subsequentlycleaved from the amino terminal peptide, and inthe anterior lobe essentially no further cleavages occur. Inthe intermediate lobe, ACTH 1–39 is cleaved into α-MSH(residues 1–13) and CLIP (18–39); β-LPH (42–134) isconverted to γ-LPH (42–101) and β-endorphin (104–134). β-MSH (84–101) is derived from γ-LPH.There are extensive additional tissue-specific modificationsof these peptides that affect activity. Thesemodifications include phosphorylation, acetylation,glycosylation, and amidation.THERE IS VARIATION IN THE STORAGE& SECRETION OF HORMONESAs mentioned above, the steroid hormones and1,25(OH) 2 -D 3 are synthesized in their final activeform. They are also secreted as they are made, and thusthere is no intracellular reservoir of these hormones.The catecholamines, also synthesized in active form, arestored in granules in the chromaffin cells in the adrenalmedulla. In response to appropriate neural stimulation,these granules are released from the cell through exocytosis,and the catecholamines are released into the circulation.A several-hour reserve supply of catecholaminesexists in the chromaffin cells.Parathyroid hormone also exists in storage vesicles.As much as 80–90% of the proPTH synthesized is degradedbefore it enters this final storage compartment,especially when Ca 2+ levels are high in the parathyroidcell (see above). PTH is secreted when Ca 2+ is low inthe parathyroid cells, which contain a several-hour supplyof the hormone.The human pancreas secretes about 40–50 units of insulindaily, which represents about 15–20% of the hormonestored in the B cells. Insulin and the C-peptide (seeFigure 42–12) are normally secreted in equimolaramounts. Stimuli such as glucose, which provokes insulinsecretion, therefore trigger the processing of proinsulin toinsulin as an essential part of the secretory response.A several-week supply of T 3 and T 4 exists in the thyroglobulinthat is stored in colloid in the lumen of thethyroid follicles. These hormones can be released uponstimulation by TSH. This is the most exaggerated exampleof a prohormone, as a molecule containing approximately5000 amino acids must be first synthesized,then degraded, to supply a few molecules of theactive hormones T 4 and T 3 .The diversity in storage and secretion of hormonesis illustrated in Table 42–5.

454 / CHAPTER 42Table 42–5. Diversity in the storage of hormones.HormoneSteroids and 1,25(OH) 2 -D 3Catecholamines and PTHInsulinT 3 and T 4Supply Stored in CellNoneHoursDaysWeeksTable 42–6. Comparison of receptors withtransport proteins.Feature Receptors Transport ProteinsConcentration Very low Very high(thousands/cell) (billions/µL)Binding affinity High (pmol/L to Low (µmol/L range)nmol/L range)Binding specificity Very high LowSaturability Yes NoReversibility Yes YesSignal transduction Yes NoSOME HORMONES HAVE PLASMATRANSPORT PROTEINSThe class I hormones are hydrophobic in chemical natureand thus are not very soluble in plasma. These hormones,principally the steroids and thyroid hormones,have specialized plasma transport proteins that serve severalpurposes. First, these proteins circumvent the solubilityproblem and thereby deliver the hormone to thetarget cell. They also provide a circulating reservoir ofthe hormone that can be substantial, as in the case of thethyroid hormones. Hormones, when bound to the transportproteins, cannot be metabolized, thereby prolongingtheir plasma half-life (t 1/2 ). The binding affinity of agiven hormone to its transporter determines the boundversus free ratio of the hormone. This is important becauseonly the free form of a hormone is biologically active.In general, the concentration of free hormone inplasma is very low, in the range of 10 –15 to 10 –9 mol/L. Itis important to distinguish between plasma transportproteins and hormone receptors. Both bind hormonesbut with very different characteristics (Table 42–6).The hydrophilic hormones—generally class II andof peptide structure—are freely soluble in plasma anddo not require transport proteins. Hormones such asinsulin, growth hormone, ACTH, and TSH circulatein the free, active form and have very short plasma halflives.A notable exception is IGF-I, which is transportedbound to members of a family of binding proteins.Thyroid Hormones Are Transportedby Thyroid-Binding GlobulinMany of the principles discussed above are illustrated ina discussion of thyroid-binding proteins. One-half totwo-thirds of T 4 and T 3 in the body is in an extrathyroidalreservoir. Most of this circulates in bound form,ie, bound to a specific binding protein, thyroxinebindingglobulin (TBG). TBG, a glycoprotein with amolecular mass of 50 kDa, binds T 4 and T 3 and has thecapacity to bind 20 µg/dL of plasma. Under normalcircumstances, TBG binds—noncovalently—nearly allof the T 4 and T 3 in plasma, and it binds T 4 with greateraffinity than T 3 (Table 42–7). The plasma half-life ofT 4 is correspondingly four to five times that of T 3 . Thesmall, unbound (free) fraction is responsible for the biologicactivity. Thus, in spite of the great difference intotal amount, the free fraction of T 3 approximates thatof T 4 , and given that T 3 is intrinsically more active thanT 4 , most biologic activity is attributed to T 3 . TBG doesnot bind any other hormones.Glucocorticoids Are Transportedby Corticosteroid-Binding GlobulinHydrocortisone (cortisol) also circulates in plasma inprotein-bound and free forms. The main plasma bindingprotein is an α-globulin called transcortin, or corticosteroid-bindingglobulin (CBG). CBG is producedin the liver, and its synthesis, like that of TBG, isincreased by estrogens. CBG binds most of the hormonewhen plasma cortisol levels are within the normalrange; much smaller amounts of cortisol are bound toalbumin. The avidity of binding helps determine thebiologic half-lives of various glucocorticoids. Cortisolbinds tightly to CBG and has a t 1/2 of 1.5–2 hours,while corticosterone, which binds less tightly, has a t 1/2of less than 1 hour (Table 42–8). The unbound (free)cortisol constitutes about 8% of the total and representsthe biologically active fraction. Binding to CBG is notrestricted to glucocorticoids. Deoxycorticosterone andTable 42–7. Comparison of T 4 and T 3 in plasma.TotalFree Hormonet 1 ⁄2Hormone Percent in Blood(µg/dL) of Total ng/dL Molarity (days)T 4 8 0.03 ~2.24 3.0 × 10 −11 6.5T 3 0.15 0.3 ~0.4 ~0.6 × 10 −11 1.5

THE DIVERSITY OF THE ENDOCRINE SYSTEM / 455Table 42–8. Approximate affinities of steroids forserum-binding proteins.SHBG 1 CBG 1Dihydrotestosterone 1 > 100Testosterone 2 > 100Estradiol 5 > 10Estrone > 10 > 100Progesterone > 100 ~ 2Cortisol > 100 ~ 3Corticosterone > 100 ~ 51 Affinity expressed as K d (nmol/L).progesterone interact with CBG with sufficient affinityto compete for cortisol binding. Aldosterone, the mostpotent natural mineralocorticoid, does not have a specificplasma transport protein. Gonadal steroids bindvery weakly to CBG (Table 42–8).Gonadal Steroids Are Transportedby Sex Hormone-Binding GlobulinMost mammals, humans included, have a plasma β-globulin that binds testosterone with specificity, relativelyhigh affinity, and limited capacity (Table 42–8).This protein, usually called sex hormone-bindingglobulin (SHBG) or testosterone-estrogen-bindingglobulin (TEBG), is produced in the liver. Its productionis increased by estrogens (women have twice theserum concentration of SHBG as men), certain types ofliver disease, and hyperthyroidism; it is decreased byandrogens, advancing age, and hypothyroidism. Manyof these conditions also affect the production of CBGand TBG. Since SHBG and albumin bind 97–99% ofcirculating testosterone, only a small fraction of thehormone in circulation is in the free (biologically active)form. The primary function of SHBG may be torestrict the free concentration of testosterone in theserum. Testosterone binds to SHBG with higher affinitythan does estradiol (Table 42–8). Therefore, achange in the level of SHBG causes a greater change inthe free testosterone level than in the free estradiol level.Estrogens are bound to SHBG and progestins toCBG. SHBG binds estradiol about five times less avidlythan it binds testosterone or DHT, while progesteroneand cortisol have little affinity for this protein (Table42–8). In contrast, progesterone and cortisol bind withnearly equal affinity to CBG, which in turn has littleavidity for estradiol and even less for testosterone,DHT, or estrone.These binding proteins also provide a circulatingreservoir of hormone, and because of the relatively largebinding capacity they probably buffer against suddenchanges in the plasma level. Because the metabolicclearance rates of these steroids are inversely related tothe affinity of their binding to SHBG, estrone is clearedmore rapidly than estradiol, which in turn is clearedmore rapidly than testosterone or DHT.SUMMARY• The presence of a specific receptor defines the targetcells for a given hormone.• Receptors are proteins that bind specific hormonesand generate an intracellular signal (receptor-effectorcoupling).• Some hormones have intracellular receptors; othersbind to receptors on the plasma membrane.• Hormones are synthesized from a number of precursormolecules, including cholesterol, tyrosine per se,and all the constituent amino acids of peptides andproteins.• A number of modification processes alter the activityof hormones. For example, many hormones are synthesizedfrom larger precursor molecules.• The complement of enzymes in a particular cell typeallows for the production of a specific class of steroidhormone.• Most of the lipid-soluble hormones are bound torather specific plasma transport proteins.REFERENCESBartalina L: Thyroid hormone-binding proteins: update 1994. EndocrRev 1994;13:140.Beato M et al: Steroid hormone receptors: many actors in search ofa plot. Cell 1995;83:851.Dai G, Carrasco L, Carrasco N: Cloning and characterization ofthe thyroid iodide transporter. Nature 1996;379:458.DeLuca HR: The vitamin D story: a collaborative effort of basicscience and clinical medicine. FASEB J 1988;2:224.Douglass J, Civelli O, Herbert E: Polyprotein gene expression:Generation of diversity of neuroendocrine peptides. AnnuRev Biochem 1984;53:665.Miller WL: Molecular biology of steroid hormone biosynthesis.Endocr Rev 1988;9:295.Nagatsu T: Genes for human catecholamine-synthesizing enzymes.Neurosci Res 1991;12:315.Russell DW, Wilson JD: Steroid 5 alpha-reductase: two genes/twoenzymes. Annu Rev Biochem 1994;63:25.Russell J et al: Interaction between calcium and 1,25-dihydroxyvitaminD 3 in the regulation of preproparathyroid hormoneand vitamin D receptor mRNA in avian parathyroids. Endocrinology1993;132:2639.Steiner DF et al: The new enzymology of precursor processing endoproteases.J Biol Chem 1992;267:23435.

Hormone Action &Signal Transduction 43Daryl K. Granner, MDBIOMEDICAL IMPORTANCEThe homeostatic adaptations an organism makes to aconstantly changing environment are in large part accomplishedthrough alterations of the activity andamount of proteins. Hormones provide a major meansof facilitating these changes. A hormone-receptor interactionresults in generation of an intracellular signalthat can either regulate the activity of a select set ofgenes, thereby altering the amount of certain proteinsin the target cell, or affect the activity of specific proteins,including enzymes and transporter or channelproteins. The signal can influence the location of proteinsin the cell and can affect general processes such asprotein synthesis, cell growth, and replication, perhapsthrough effects on gene expression. Other signalingmolecules—including cytokines, interleukins, growthfactors, and metabolites—use some of the same generalmechanisms and signal transduction pathways. Excessive,deficient, or inappropriate production and releaseof hormones and of these other regulatory moleculesare major causes of disease. Many pharmacotherapeuticagents are aimed at correcting or otherwise influencingthe pathways discussed in this chapter.HORMONES TRANSDUCE SIGNALS TOAFFECT HOMEOSTATIC MECHANISMSThe general steps involved in producing a coordinatedresponse to a particular stimulus are illustrated inFigure 43–1. The stimulus can be a challenge or athreat to the organism, to an organ, or to the integrityof a single cell within that organism. Recognition of thestimulus is the first step in the adaptive response. At theorganismic level, this generally involves the nervous systemand the special senses (sight, hearing, pain, smell,touch). At the organismic or cellular level, recognitioninvolves physicochemical factors such as pH, O 2 tension,temperature, nutrient supply, noxious metabolites,and osmolarity. Appropriate recognition results inthe release of one or more hormones that will governgeneration of the necessary adaptive response. For purposesof this discussion, the hormones are categorizedas described in Chapter 42, ie, based on the location oftheir specific cellular receptors and the type of signalsgenerated. Group I hormones interact with an intracellularreceptor and group II hormones with receptorrecognition sites located on the extracellular surface ofthe plasma membrane of target cells. The cytokines, interleukins,and growth factors should also be consideredin this latter category. These molecules, of critical importancein homeostatic adaptation, are hormones inthe sense that they are produced in specific cells, havethe equivalent of autocrine, paracrine, and endocrineactions, bind to cell surface receptors, and activatemany of the same signal transduction pathways employedby the more traditional group II hormones.SIGNAL GENERATIONThe Ligand-Receptor Complex Is theSignal for Group I HormonesThe lipophilic group I hormones diffuse through theplasma membrane of all cells but only encounter theirspecific, high-affinity intracellular receptors in targetcells. These receptors can be located in the cytoplasm orin the nucleus of target cells. The hormone-receptorcomplex first undergoes an activation reaction. Asshown in Figure 43–2, receptor activation occurs by atleast two mechanisms. For example, glucocorticoidsdiffuse across the plasma membrane and encountertheir cognate receptor in the cytoplasm of target cells.Ligand-receptor binding results in the dissociation ofheat shock protein 90 (hsp90) from the receptor. Thisstep appears to be necessary for subsequent nuclear localizationof the glucocorticoid receptor. This receptoralso contains nuclear localization sequences that assistin the translocation from cytoplasm to nucleus. Thenow activated receptor moves into the nucleus (Figure43–2) and binds with high affinity to a specific DNAsequence called the hormone response element(HRE). In the case illustrated, this is a glucocorticoidresponse element, or GRE. Consensus sequences forHREs are shown in Table 43–1. The DNA-bound, ligandedreceptor serves as a high-affinity binding site for456

HORMONE ACTION & SIGNAL TRANSDUCTION / 457STIMULUSRecognitionGroup I hormonesGroup II hormonesHormone releaseHormone•receptor complexMany different signalsSignal generationEffectsGenetranscriptionTransportersChannelsProteintranslocationProteinmodificationCOORDINATED RESPONSE TO STIMULUSFigure 43–1. Hormonal involvement in responses to a stimulus. A challenge to the integrityof the organism elicits a response that includes the release of one or more hormones.These hormones generate signals at or within target cells, and these signals regulate a varietyof biologic processes which provide for a coordinated response to the stimulus or challenge.See Figure 43–8 for a specific example.one or more coactivator proteins, and accelerated genetranscription typically ensues when this occurs. By contrast,certain hormones such as the thyroid hormonesand retinoids diffuse from the extracellular fluid acrossthe plasma membrane and go directly into the nucleus.In this case, the cognate receptor is already bound tothe HRE (the thyroid hormone response element[TRE], in this example). However, this DNA-boundreceptor fails to activate transcription because it is complexedwith a corepressor. Indeed, this receptorcorepressorcomplex serves as an active repressor of genetranscription. The association of ligand with these receptorsresults in dissociation of the corepressor. Theliganded receptor is now capable of binding one ormore coactivators with high affinity, resulting in the activationof gene transcription. The relationship of hormonereceptors to other nuclear receptors and to coregulatorsis discussed in more detail below.By selectively affecting gene transcription and theconsequent production of appropriate target mRNAs,the amounts of specific proteins are changed and metabolicprocesses are influenced. The influence of each ofthese hormones is quite specific; generally, the hormoneaffects less than 1% of the genes, mRNA, or proteinsin a target cell; sometimes only a few are affected.The nuclear actions of steroid, thyroid, and retinoidhormones are quite well defined. Most evidence suggeststhat these hormones exert their dominant effecton modulating gene transcription, but they—and manyof the hormones in the other classes discussed below—can act at any step of the “information pathway” illustratedin Figure 43–3. Direct actions of steroids in thecytoplasm and on various organelles and membraneshave also been described.GROUP II (PEPTIDE &CATECHOLAMINE) HORMONESHAVE MEMBRANE RECEPTORS& USE INTRACELLULAR MESSENGERSMany hormones are water-soluble, have no transportproteins (and therefore have a short plasma half-life),and initiate a response by binding to a receptor locatedin the plasma membrane (see Tables 42–3 and 42–4).The mechanism of action of this group of hormonescan best be discussed in terms of the intracellular signalsthey generate. These signals include cAMP (cyclicAMP; 3′,5′-adenylic acid; see Figure 18–5), a nucleotidederived from ATP through the action ofadenylyl cyclase; cGMP, a nucleotide formed by guanylylcyclase; Ca 2+ ; and phosphatidylinositides. Manyof these second messengers affect gene transcription, asdescribed in the previous paragraph; but they also influ-

458 / CHAPTER 43−Cytoplasm−++TRETRETREGREGREGREhsp++hspNucleusFigure 43–2. Regulation of gene expression by class I hormones.Steroid hormones readily gain access to the cytoplasmic compartmentof target cells. Glucocorticoid hormones (solid triangles) encountertheir cognate receptor in the cytoplasm, where it exists in a complexwith heat shock protein 90 (hsp). Ligand binding causes dissociation ofhsp and a conformational change of the receptor. The receptor•ligandcomplex then traverses the nuclear membrane and binds to DNA withspecificity and high affinity at a glucocorticoid response element (GRE).This event triggers the assembly of a number of transcription coregulators(+ ), and enhanced transcription ensues. By contrast, thyroid hormonesand retinoic acid () directly enter the nucleus, where theircognate receptors are already bound to the appropriate response elementswith an associated transcription repressor complex (− ). Thiscomplex, which consists of molecules such as N-CoR or SMRT (seeTable 43–6) in the absence of ligand, actively inhibits transcription. Ligandbinding results in dissociation of the repressor complex from thereceptor, allowing an activator complex to assemble. The gene is thenactively transcribed.ence a variety of other biologic processes, as shown inFigure 43–1.G Protein-Coupled Receptors (GPCR)Many of the group II hormones bind to receptors thatcouple to effectors through a GTP-binding protein intermediary.These receptors typically have seven hydrophobicplasma membrane-spanning domains. Thisis illustrated by the seven interconnected cylinders extendingthrough the lipid bilayer in Figure 43–4. Receptorsof this class, which signal through guanine nucleotide-boundprotein intermediates, are known asG protein-coupled receptors, or GPCRs. To date,over 130 G protein-linked receptor genes have beencloned from various mammalian species. A wide varietyof responses are mediated by the GPCRs.cAMP Is the Intracellular Signalfor Many ResponsesCyclic AMP was the first intracellular signal identifiedin mammalian cells. Several components comprise asystem for the generation, degradation, and action ofcAMP.A. ADENYLYL CYCLASEDifferent peptide hormones can either stimulate (s) orinhibit (i) the production of cAMP from adenylyl cy-

HORMONE ACTION & SIGNAL TRANSDUCTION / 459Table 43–1. The DNA sequences of severalhormone response elements (HREs). 1Hormone or Effector HRE DNA SequenceGlucocorticoids GREProgestins PRE GGTACA NNN TGTTCTMineralocorticoids MRE ←AndrogensAREEstrogens ERE AGGTCA ––– TGA/TCCT←Thyroid hormone TRERetinoic acid RARE AGGTCA N3,4,5, AGGTCAVitamin DVDRE←←← ←cAMP CRE TGACGTCA1 Letters indicate nucleotide; N means any one of the four can beused in that position. The arrows pointing in opposite directionsillustrate the slightly imperfect inverted palindromes present inmany HREs; in some cases these are called “half binding sites” becauseeach binds one monomer of the receptor. The GRE, PRE,MRE, and ARE consist of the same DNA sequence. Specificity maybe conferred by the intracellular concentration of the ligand orhormone receptor, by flanking DNA sequences not included inthe consensus, or by other accessory elements. A second group ofHREs includes those for thyroid hormones, estrogens, retinoicacid, and vitamin D. These HREs are similar except for the orientationand spacing between the half palindromes. Spacing determinesthe hormone specificity. VDRE (N=3), TRE (N=4), and RARE(N=5) bind to direct repeats rather than to inverted repeats. Anothermember of the steroid receptor superfamily, the retinoid Xreceptor (RXR), forms heterodimers with VDR, TR, and RARE, andthese constitute the trans-acting factors. cAMP affects gene transcriptionthrough the CRE.NUCLEUSCYTOPLASMGenePrimary transcriptmRNAmRNAProteinTRANSCRIPTIONDegradationMODIFICATION/PROCESSINGDegradationTransportTRANSLATIONActivedegradationModificationdegradationinactiveFigure 43–3. The “information pathway.” Informationflows from the gene to the primary transcript tomRNA to protein. Hormones can affect any of the stepsinvolved and can affect the rates of processing, degradation,or modification of the various products.clase, which is encoded by at least nine different genes(Table 43–2). Two parallel systems, a stimulatory (s)one and an inhibitory (i) one, converge upon a singlecatalytic molecule (C). Each consists of a receptor, R s orR i , and a regulatory complex, G s and G i . G s and G i areeach trimers composed of α, β, and γ subunits. Becausethe α subunit in G s differs from that in G i , the proteins,which are distinct gene products, are designatedα s and α i . The α subunits bind guanine nucleotides.The β and γ subunits are always associated (βγ) and appearto function as a heterodimer. The binding of a hormoneto R s or R i results in a receptor-mediated activationof G, which entails the exchange of GDP by GTPon α and the concomitant dissociation of βγ from α.The α s protein has intrinsic GTPase activity. Theactive form, α s •GTP, is inactivated upon hydrolysis ofthe GTP to GDP; the trimeric G s complex (αβγ) isthen re-formed and is ready for another cycle of activation.Cholera and pertussis toxins catalyze the ADPribosylationof α s and α i-2 (see Table 43–3), respectively.In the case of α s , this modification disrupts theintrinsic GTP-ase activity; thus, α s cannot reassociatewith βγ and is therefore irreversibly activated. ADPribosylationof α i-2 prevents the dissociation of α i-2from βγ, and free α i-2 thus cannot be formed. α s activityin such cells is therefore unopposed.There is a large family of G proteins, and these arepart of the superfamily of GTPases. The G proteinfamily is classified according to sequence homologyinto four subfamilies, as illustrated in Table 43–3.There are 21 α, 5 β, and 8 γ subunit genes. Variouscombinations of these subunits provide a large numberof possible αβγ and cyclase complexes.The α subunits and the βγ complex have actions independentof those on adenylyl cyclase (see Figure43–4 and Table 43–3). Some forms of α i stimulate K +channels and inhibit Ca 2+ channels, and some α s moleculeshave the opposite effects. Members of the G q familyactivate the phospholipase C group of enzymes. Theβγ complexes have been associated with K + channelstimulation and phospholipase C activation. G proteinsare involved in many important biologic processes inaddition to hormone action. Notable examples includeolfaction (α OLF ) and vision (α t ). Some examples arelisted in Table 43–3. GPCRs are implicated in a numberof diseases and are major targets for pharmaceuticalagents.

460 / CHAPTER 43NNHEEα sGDPγβCNo hormone: inactive effectorγβGT PCBound hormone (H): active effectorFigure 43–4. Components of the hormone receptor–G protein effector system. Receptors thatcouple to effectors through G proteins (GPCR) typically have seven membrane-spanning domains. Inthe absence of hormone (left), the heterotrimeric G-protein complex (α, β, γ) is in an inactive guanosinediphosphate (GDP)-bound form and is probably not associated with the receptor. This complex isanchored to the plasma membrane through prenylated groups on the βγ subunits (wavy lines) andperhaps by myristoylated groups on α subunits (not shown). On binding of hormone (H ) to the receptor,there is a presumed conformational change of the receptor—as indicated by the tilted membranespanning domains—and activation of the G-protein complex. This results from the exchange ofGDP with guanosine triphosphate (GTP) on the α subunit, after which α and βγ dissociate. The α subunitbinds to and activates the effector (E). E can be adenylyl cyclase, Ca 2+ , Na + , or Cl − channels (α s ), orit could be a K + channel (α i ), phospholipase Cβ (α q ), or cGMP phosphodiesterase (α t ). The βγ subunitcan also have direct actions on E. (Modified and reproduced, with permission, from Granner DK in: Principlesand Practice of Endocrinology and Metabolism, 3rd ed. Becker KL [editor]. Lippincott, 2000.)α sTable 43–2. Subclassification of group II.Ahormones.Hormones That Stimulate Hormones That InhibitAdenylyl CyclaseAdenylyl Cyclase(H s ) (H l )ACTHAcetylcholineADHα 2 -Adrenergicsβ-AdrenergicsAngiotensin IICalcitoninSomatostatinCRHFSHGlucagonhCGLHLPHMSHPTHTSHB. PROTEIN KINASEIn prokaryotic cells, cAMP binds to a specific proteincalled catabolite regulatory protein (CRP) that bindsdirectly to DNA and influences gene expression. In eukaryoticcells, cAMP binds to a protein kinase calledprotein kinase A (PKA) that is a heterotetrameric moleculeconsisting of two regulatory subunits (R) and twocatalytic subunits (C). cAMP binding results in the followingreaction:4 cAMP + R 2C2a R 2⋅(4 cAMP)+ 2CThe R 2 C 2 complex has no enzymatic activity, butthe binding of cAMP by R dissociates R from C,thereby activating the latter (Figure 43–5). The activeC subunit catalyzes the transfer of the γ phosphate ofATP to a serine or threonine residue in a variety of proteins.The consensus phosphorylation sites are -Arg-Arg/Lys-X-Ser/Thr- and -Arg-Lys-X-X-Ser-, where Xcan be any amino acid.Protein kinase activities were originally described asbeing “cAMP-dependent” or “cAMP-independent.” This

Table 43–3. Classes and functions of selected G proteins. 1,2HORMONE ACTION & SIGNAL TRANSDUCTION / 461Class or Type Stimulus Effector EffectG sαsGlucagon, β-adrenergics ↑ Adenylyl cyclase Gluconeogenesis, lipolysis,↑ Cardiac Ca 2+ , Cl − , and Na + channels glycogenolysisα olf Odorant ↑ Adenylyl cyclase OlfactionG iαi-1,2,3 Acetylcholine, ↓ Adenylyl cyclase Slowed heart rateα 2 -adrenergics ↑ Potassium channelsM 2 cholinergics ↓ Calcium channelsα 0 Opioids, endorphins ↑ Potassium channels Neuronal electrical activityG qαqα t Light ↑ cGMP phosphodiesterase VisionM 1 cholinergicsα 1 -Adrenergics ↑ Phospholipase C-β1 ↑ Muscle contractionandα 11 α 1 -Adrenergics ↑ Phospholipase c-β2 ↑ Blood pressureG 12α12? Cl − channel ?1 Modified and reproduced, with permission, from Granner DK in: Principles and Practice of Endocrinology and Metabolism, 3rded. Becker KL (editor). Lippincott, 2000.2 The four major classes or families of mammalian G proteins (G s , G i , G q , and G 12 ) are based on protein sequence homology.Representative members of each are shown, along with known stimuli, effectors, and well-defined biologic effects. Nine isoformsof adenylyl cyclase have been identified (isoforms I–IX). All isoforms are stimulated by α s ; α i isoforms inhibit types Vand VI, and α 0 inhibits types I and V. At least 16 different α subunits have been identified.classification has changed, as protein phosphorylation isnow recognized as being a major regulatory mechanism.Several hundred protein kinases have now beendescribed. The kinases are related in sequence andstructure within the catalytic domain, but each is aunique molecule with considerable variability with respectto subunit composition, molecular weight, autophosphorylation,K m for ATP, and substrate specificity.C. PHOSPHOPROTEINSThe effects of cAMP in eukaryotic cells are all thoughtto be mediated by protein phosphorylation-dephosphorylation,principally on serine and threonine residues.The control of any of the effects of cAMP, includingsuch diverse processes as steroidogenesis, secretion, iontransport, carbohydrate and fat metabolism, enzyme induction,gene regulation, synaptic transmission, andcell growth and replication, could be conferred by aspecific protein kinase, by a specific phosphatase, or byspecific substrates for phosphorylation. These substrateshelp define a target tissue and are involved in definingthe extent of a particular response within a given cell.For example, the effects of cAMP on gene transcriptionare mediated by the protein cyclic AMP response elementbinding protein (CREB). CREB binds to acAMP responsive element (CRE) (see Table 43–1) inits nonphosphorylated state and is a weak activator oftranscription. When phosphorylated by PKA, CREBbinds the coactivator CREB-binding protein CBP/p300 (see below) and as a result is a much more potenttranscription activator.D. PHOSPHODIESTERASESActions caused by hormones that increase cAMP concentrationcan be terminated in a number of ways, includingthe hydrolysis of cAMP to 5′-AMP by phosphodiesterases(see Figure 43–5). The presence of thesehydrolytic enzymes ensures a rapid turnover of the signal(cAMP) and hence a rapid termination of the biologicprocess once the hormonal stimulus is removed.There are at least 11 known members of the phosphodiesterasefamily of enzymes. These are subject to regulationby their substrates, cAMP and cGMP; by hormones;and by intracellular messengers such as calcium,probably acting through calmodulin. Inhibitors ofphosphodiesterase, most notably methylated xanthinederivatives such as caffeine, increase intracellular cAMPand mimic or prolong the actions of hormones throughthis signal.

462 / CHAPTER 43ActiveadenylylcyclaseCellmembraneATP • Mg 2+cAMPPhosphodiesterase5′-AMPR 2 C 2Inactive PKAC 2Active PKA+ R 2Mg 2+ • ATPProtein PhosphoproteinPhosphatasePhysiologiceffectsFigure 43–5. Hormonal regulation of cellular processes throughcAMP-dependent protein kinase (PKA). PKA exists in an inactive form asan R 2 C 2 heterotetramer consisting of two regulatory and two catalyticsubunits. The cAMP generated by the action of adenylyl cyclase (activatedas shown in Figure 43–4) binds to the regulatory (R) subunit ofPKA. This results in dissociation of the regulatory and catalytic subunitsand activation of the latter. The active catalytic subunits phosphorylatea number of target proteins on serine and threonine residues. Phosphatasesremove phosphate from these residues and thus terminatethe physiologic response. A phosphodiesterase can also terminate theresponse by converting cAMP to 5′-AMP.E. PHOSPHOPROTEIN PHOSPHATASESGiven the importance of protein phosphorylation, it isnot surprising that regulation of the protein dephosphorylationreaction is another important controlmechanism (see Figure 43–5). The phosphoproteinphosphatases are themselves subject to regulation byphosphorylation-dephosphorylation reactions and by avariety of other mechanisms, such as protein-proteininteractions. In fact, the substrate specificity of thephosphoserine-phosphothreonine phosphatases may bedictated by distinct regulatory subunits whose bindingis regulated hormonally. The best-studied role of regulationby the dephosphorylation of proteins is that ofglycogen metabolism in muscle. Two major types ofphosphoserine-phosphothreonine phosphatases havebeen described. Type I preferentially dephosphorylatesthe β subunit of phosphorylase kinase, whereas type IIdephosphorylates the α subunit. Type I phosphatase isimplicated in the regulation of glycogen synthase, phosphorylase,and phosphorylase kinase. This phosphataseis itself regulated by phosphorylation of certain of itssubunits, and these reactions are reversed by the actionof one of the type II phosphatases. In addition, twoheat-stable protein inhibitors regulate type I phosphataseactivity. Inhibitor-1 is phosphorylated and activatedby cAMP-dependent protein kinases; and inhibitor-2,which may be a subunit of the inactivephosphatase, is also phosphorylated, possibly by glycogensynthase kinase-3.cGMP Is Also an Intracellular SignalCyclic GMP is made from GTP by the enzyme guanylylcyclase, which exists in soluble and membraneboundforms. Each of these isozymes has unique physiologicproperties. The atriopeptins, a family of peptidesproduced in cardiac atrial tissues, cause natriuresis, diuresis,vasodilation, and inhibition of aldosterone secretion.These peptides (eg, atrial natriuretic factor) bindto and activate the membrane-bound form of guanylylcyclase. This results in an increase of cGMP by as muchas 50-fold in some cases, and this is thought to mediatethe effects mentioned above. Other evidence linkscGMP to vasodilation. A series of compounds, includingnitroprusside, nitroglycerin, nitric oxide, sodiumnitrite, and sodium azide, all cause smooth muscle re-

HORMONE ACTION & SIGNAL TRANSDUCTION / 463laxation and are potent vasodilators. These agents increasecGMP by activating the soluble form of guanylylcyclase, and inhibitors of cGMP phosphodiesterase (thedrug sildenafil [Viagra], for example) enhance and prolongthese responses. The increased cGMP activatescGMP-dependent protein kinase (PKG), which in turnphosphorylates a number of smooth muscle proteins.Presumably, this is involved in relaxation of smoothmuscle and vasodilation.Several Hormones Act ThroughCalcium or PhosphatidylinositolsIonized calcium is an important regulator of a variety ofcellular processes, including muscle contraction, stimulus-secretioncoupling, the blood clotting cascade, enzymeactivity, and membrane excitability. It is also anintracellular messenger of hormone action.A. CALCIUM METABOLISMThe extracellular calcium (Ca 2+ ) concentration is about5 mmol/L and is very rigidly controlled. Although substantialamounts of calcium are associated with intracellularorganelles such as mitochondria and the endoplasmicreticulum, the intracellular concentration of free orionized calcium (Ca 2+ ) is very low: 0.05–10 µmol/L. Inspite of this large concentration gradient and a favorabletransmembrane electrical gradient, Ca 2+ is restrainedfrom entering the cell. A considerable amountof energy is expended to ensure that the intracellularCa 2+ is controlled, as a prolonged elevation of Ca 2+ inthe cell is very toxic. A Na + /Ca 2+ exchange mechanismthat has a high capacity but low affinity pumps Ca 2+out of cells. There also is a Ca 2+ /proton ATPase-dependentpump that extrudes Ca 2+ in exchange for H + . Thishas a high affinity for Ca 2+ but a low capacity and isprobably responsible for fine-tuning cytosolic Ca 2+ .Furthermore, Ca 2+ ATPases pump Ca 2+ from the cytosolto the lumen of the endoplasmic reticulum. Thereare three ways of changing cytosolic Ca 2+ : (1) Certainhormones (class II.C, Table 42–3) by binding to receptorsthat are themselves Ca 2+ channels, enhance membranepermeability to Ca 2+ and thereby increase Ca 2+influx. (2) Hormones also indirectly promote Ca 2+ influxby modulating the membrane potential at theplasma membrane. Membrane depolarization opensvoltage-gated Ca 2+ channels and allows for Ca 2+ influx.(3) Ca 2+ can be mobilized from the endoplasmic reticulum,and possibly from mitochondrial pools.An important observation linking Ca 2+ to hormoneaction involved the definition of the intracellular targetsof Ca 2+ action. The discovery of a Ca 2+ -dependent regulatorof phosphodiesterase activity provided the basisfor a broad understanding of how Ca 2+ and cAMP interactwithin cells.B. CALMODULINThe calcium-dependent regulatory protein is calmodulin,a 17-kDa protein that is homologous to the muscleprotein troponin C in structure and function.Calmodulin has four Ca 2+ binding sites, and full occupancyof these sites leads to a marked conformationalchange, which allows calmodulin to activate enzymesand ion channels. The interaction of Ca 2+ with calmodulin(with the resultant change of activity of the latter)is conceptually similar to the binding of cAMP to PKAand the subsequent activation of this molecule.Calmodulin can be one of numerous subunits of complexproteins and is particularly involved in regulatingvarious kinases and enzymes of cyclic nucleotide generationand degradation. A partial list of the enzymes regulateddirectly or indirectly by Ca 2+ , probably throughcalmodulin, is presented in Table 43–4.In addition to its effects on enzymes and ion transport,Ca 2+ /calmodulin regulates the activity of manystructural elements in cells. These include the actinmyosincomplex of smooth muscle, which is under β-adrenergic control, and various microfilament-mediatedprocesses in noncontractile cells, including cellmotility, cell conformation changes, mitosis, granule release,and endocytosis.C. CALCIUM IS AMEDIATOR OF HORMONE ACTIONA role for Ca 2+ in hormone action is suggested by theobservations that the effect of many hormones is (1)blunted by Ca 2+ -free media or when intracellular calciumis depleted; (2) can be mimicked by agents thatincrease cytosolic Ca 2+ , such as the Ca 2+ ionophoreA23187; and (3) influences cellular calcium flux. Theregulation of glycogen metabolism in liver by vasopressinand α-adrenergic catecholamines provides agood example. This is shown schematically in Figures18–6 and 18–7.Table 43–4. Enzymes and proteins regulated bycalcium or calmodulin.Adenylyl cyclaseCa 2+ -dependent protein kinasesCa 2+ -Mg 2+ ATPaseCa 2+ -phospholipid-dependent protein kinaseCyclic nucleotide phosphodiesteraseSome cytoskeletal proteinsSome ion channels (eg, L-type calcium channels)Nitric oxide synthasePhosphorylase kinasePhosphoprotein phosphatase 2BSome receptors (eg, NMDA-type glutamate receptor)

464 / CHAPTER 43A number of critical metabolic enzymes are regulatedby Ca 2+ , phosphorylation, or both, includingglycogen synthase, pyruvate kinase, pyruvate carboxylase,glycerol-3-phosphate dehydrogenase, and pyruvatedehydrogenase.D. PHOSPHATIDYLINOSITIDE METABOLISM AFFECTSCA 2+ -DEPENDENT HORMONE ACTIONSome signal must provide communication between thehormone receptor on the plasma membrane and the intracellularCa 2+ reservoirs. This is accomplished byproducts of phosphatidylinositol metabolism. Cell surfacereceptors such as those for acetylcholine, antidiuretichormone, and α 1 -type catecholamines are, whenoccupied by their respective ligands, potent activatorsof phospholipase C. Receptor binding and activation ofphospholipase C are coupled by the G q isoforms (Table43–3 and Figure 43–6). Phospholipase C catalyzes thehydrolysis of phosphatidylinositol 4,5-bisphosphate toinositol trisphosphate (IP 3 ) and 1,2-diacylglycerol (Figure43–7). Diacylglycerol is itself capable of activatingprotein kinase C (PKC), the activity of which also dependsupon Ca 2+ . IP 3 , by interacting with a specific intracellularreceptor, is an effective releaser of Ca 2+ fromPIP 2ReceptorG proteinPhospholipase CDiacylglycerolCa 2+MitochondrionEndoplasmic reticulumInositol–P 3(IP 3 )+Protein kinase C(PKC)Ca 2+CalmodulinCa 2+ -Calmodulin+Specificcalmodulin kinase+Multifunctionalcalmodulin kinaseProteinsPhosphoproteinsOtherproteinsPhysiologic responsesFigure 43–6. Certain hormone-receptor interactions result in the activation of phospholipase C. This appearsto involve a specific G protein, which also may activate a calcium channel. Phospholipase C results ingeneration of inositol trisphosphate (IP 3 ), which liberates stored intracellular Ca 2+ , and diacylglycerol (DAG),a potent activator of protein kinase C (PKC). In this scheme, the activated PKC phosphorylates specific substrates,which then alter physiologic processes. Likewise, the Ca 2+ -calmodulin complex can activate specifickinases, two of which are shown here. These actions result in phosphorylation of substrates, and this leads toaltered physiologic responses. This figure also shows that Ca 2+ can enter cells through voltage- or ligandgatedCa 2+ channels. The intracellular Ca 2+ is also regulated through storage and release by the mitochondriaand endoplasmic reticulum. (Courtesy of JH Exton.)

HORMONE ACTION & SIGNAL TRANSDUCTION / 465R 1R 2PFigure 43–7. Phospholipase C cleaves PIP 2into diacylglycerol and inositol trisphosphate. R 1generally is stearate, and R 2 is usually arachidonate.IP 3 can be dephosphorylated (to the inactiveI-1,4-P 2 ) or phosphorylated (to the potentiallyactive I-1,3,4,5-P 4 ).OHOHOHPhosphatidylinositol 4,5-bisphosphate(PIP 2 )PPPhospholipase COHR 1POHR 2OHPPOH1,2-Diacylglycerol(DAG)Inositol 1,4,5-trisphosphate(IP 3 )intracellular storage sites in the endoplasmic reticulum.Thus, the hydrolysis of phosphatidylinositol 4,5-bisphosphateleads to activation of PKC and promotes anincrease of cytoplasmic Ca 2+ . As shown in Figure 43–4,the activation of G proteins can also have a direct actionon Ca 2+ channels. The resulting elevations of cytosolicCa 2+ activate Ca 2+ –calmodulin-dependent kinasesand many other Ca 2+ –calmodulin-dependent enzymes.Steroidogenic agents—including ACTH and cAMPin the adrenal cortex; angiotensin II, K + , serotonin,ACTH, and cAMP in the zona glomerulosa of theadrenal; LH in the ovary; and LH and cAMP in theLeydig cells of the testes—have been associated with increasedamounts of phosphatidic acid, phosphatidylinositol,and polyphosphoinositides (see Chapter 14) inthe respective target tissues. Several other examplescould be cited.The roles that Ca 2+ and polyphosphoinositide breakdownproducts might play in hormone action are presentedin Figure 43–6. In this scheme the activated proteinkinase C can phosphorylate specific substrates,which then alter physiologic processes. Likewise, theCa 2+ -calmodulin complex can activate specific kinases.These then modify substrates and thereby alter physiologicresponses.Some Hormones Act Througha Protein Kinase CascadeSingle protein kinases such as PKA, PKC, and Ca 2+ -calmodulin (CaM)-kinases, which result in the phosphorylationof serine and threonine residues in targetproteins, play a very important role in hormone action.The discovery that the EGF receptor contains an intrinsictyrosine kinase activity that is activated by the bindingof the ligand EGF was an important breakthrough.The insulin and IGF-I receptors also contain intrinsicligand-activated tyrosine kinase activity. Several receptors—generallythose involved in binding ligands involvedin growth control, differentiation, and the inflammatoryresponse—either have intrinsic tyrosinekinase activity or are associated with proteins that aretyrosine kinases. Another distinguishing feature of thisclass of hormone action is that these kinases preferentiallyphosphorylate tyrosine residues, and tyrosinephosphorylation is infrequent (< 0.03% of total aminoacid phosphorylation) in mammalian cells. A third distinguishingfeature is that the ligand-receptor interactionthat results in a tyrosine phosphorylation event initiatesa cascade that may involve several protein kinases,phosphatases, and other regulatory proteins.A. INSULIN TRANSMITS SIGNALSBY SEVERAL KINASE CASCADESThe insulin, epidermal growth factor (EGF), and IGF-Ireceptors have intrinsic protein tyrosine kinase activitieslocated in their cytoplasmic domains. These activitiesare stimulated when the receptor binds ligand. The receptorsare then autophosphorylated on tyrosineresidues, and this initiates a complex series of events(summarized in simplified fashion in Figure 43–8). Thephosphorylated insulin receptor next phosphorylatesinsulin receptor substrates (there are at least four ofthese molecules, called IRS 1–4) on tyrosine residues.Phosphorylated IRS binds to the Src homology 2(SH2) domains of a variety of proteins that are directlyinvolved in mediating different effects of insulin. Oneof these proteins, PI-3 kinase, links insulin receptor activationto insulin action through activation of a numberof molecules, including the kinase PDK1 (phosphoinositide-dependentkinase-1). This enzyme propagatesthe signal through several other kinases, including PKB(akt), SKG, and aPKC (see legend to Figure 43–8 fordefinitions and expanded abbreviations). An alternative

466 / CHAPTER 43RECOGNITION(HYPERGLYCEMIA)INSULINP-Y Y-P Y YSIGNALGENERATIONYPTENIRS 1-4Y+P-YPI3 -kinaseIRS 1-4Y-PGRB2/mSOS+PKBSGKaPKC?p70S6K+mTORp21RasRaf-1MEKMAPkinaseEFFECTSProtein translocation Enzyme activity Gene transcriptionGlucose transporterInsulin receptorIGF-II receptorInsulin receptorProtein phosphatasesPhosphodiesterases*OthersPEPCKGlucagonIGFBP1HKIIGlucokinase> 100 othersCell growthDNA synthesisEarly responsegenesFigure 43–8. Insulin signaling pathways. The insulin signaling pathways provide an excellent example of the“recognition → hormone release → signal generation → effects” paradigm outlined in Figure 43–1. Insulin is releasedin response to hyperglycemia. Binding of insulin to a target cell-specific plasma membrane receptor results ina cascade of intracellular events. Stimulation of the intrinsic tyrosine kinase activity of the insulin receptor marks theinitial event, resulting in increased tyrosine (Y) phosphorylation (Y → Y-P) of the receptor and then one or more ofthe insulin receptor substrate molecules (IRS 1–4). This increase in phosphotyrosine stimulates the activity of manyintracellular molecules such as GTPases, protein kinases, and lipid kinases, all of which play a role in certain metabolicactions of insulin. The two best-described pathways are shown. First, phosphorylation of an IRS molecule(probably IRS-2) results in docking and activation of the lipid kinase, PI-3 kinase, which generates novel inositol lipidsthat may act as “second messenger” molecules. These, in turn, activate PDK1 and then a variety of downstream signalingmolecules, including protein kinase B (PKB or akt), SGK, and aPKC. An alternative pathway involves the activationof p70S6K and perhaps other as yet unidentified kinases. Second, phosphorylation of IRS (probably IRS-1) resultsin docking of GRB2/mSOS and activation of the small GTPase, p21RAS, which initiates a protein kinase cascadethat activates Raf-1, MEK, and the p42/p44 MAP kinase isoforms. These protein kinases are important in the regulationof proliferation and differentiation of several cell types. The mTOR pathway provides an alternative way of activatingp70S6K and appears to be involved in nutrient signaling as well as insulin action. Each of these cascades mayinfluence different physiologic processes, as shown. Each of the phosphorylation events is reversible through theaction of specific phosphatases. For example, the lipid phosphatase PTEN dephosphorylates the product of the PI-3kinase reaction, thereby antagonizing the pathway and terminating the signal. Representative effects of major actionsof insulin are shown in each of the boxes. The asterisk after phosphodiesterase indicates that insulin indirectlyaffects the activity of many enzymes by activating phosphodiesterases and reducing intracellular cAMP levels.(IGFBP, insulin-like growth factor binding protein; IRS 1–4, insulin receptor substrate isoforms 1–4); PI-3 kinase, phosphatidylinositol3-kinase; PTEN, phosphatase and tensin homolog deleted on chromosome 10; PKD1, phosphoinositide-dependentkinase; PKB, protein kinase B; SGK, serum and glucocorticoid-regulated kinase; aPKC, atypical proteinkinase C; p70S6K, p70 ribosomal protein S6 kinase; mTOR, mammalian target of rapamycin; GRB2, growth factorreceptor binding protein 2; mSOS, mammalian son of sevenless; MEK, MAP kinase kinase and ERK kinase; MAPkinase, mitogen-activated protein kinase.)

HORMONE ACTION & SIGNAL TRANSDUCTION / 467pathway downstream from PKD1 involves p70S6K andperhaps other as yet unidentified kinases. A second majorpathway involves mTOR. This enzyme is directly regulatedby amino acids and insulin and is essential forp70S6K activity. This pathway provides a distinctionbetween the PKB and p70S6K branches downstreamfrom PKD1. These pathways are involved in proteintranslocation, enzyme activity, and the regulation, byinsulin, of genes involved in metabolism (Figure 43–8).Another SH2 domain-containing protein is GRB2,which binds to IRS-1 and links tyrosine phosphorylationto several proteins, the result of which is activationof a cascade of threonine and serine kinases. A pathwayshowing how this insulin-receptor interaction activatesthe mitogen-activated protein (MAP) kinase pathwayand the anabolic effects of insulin is illustrated in Figure43–8. The exact roles of many of these dockingproteins, kinases, and phosphatases remain to be established.B. THE JAK/STAT PATHWAY IS USEDBY HORMONES AND CYTOKINESTyrosine kinase activation can also initiate a phosphorylationand dephosphorylation cascade that involves theaction of several other protein kinases and the counterbalancingactions of phosphatases. Two mechanismsare employed to initiate this cascade. Some hormones,such as growth hormone, prolactin, erythropoietin, andthe cytokines, initiate their action by activating a tyrosinekinase, but this activity is not an integral part ofthe hormone receptor. The hormone-receptor interactionpromotes binding and activation of cytoplasmicprotein tyrosine kinases, such as Tyk-2, Jak1, orJak2. These kinases phosphorylate one or more cytoplasmicproteins, which then associate with other dockingproteins through binding to SH2 domains. Onesuch interaction results in the activation of a family ofcytosolic proteins called signal transducers and activatorsof transcription (STATs). The phosphorylatedSTAT protein dimerizes and translocates into the nucleus,binds to a specific DNA element such as the interferonresponse element, and activates transcription.This is illustrated in Figure 43–9. Other SH2 dockingevents may result in the activation of PI 3-kinase, theMAP kinase pathway (through SHC or GRB2), or Gprotein-mediated activation of phospholipase C (PLCγ)with the attendant production of diacylglycerol and activationof protein kinase C. It is apparent that there isa potential for “cross-talk” when different hormones activatethese various signal transduction pathways.LigandRRRRRRJAKJAKP JAK JAK P P JAK JAK PPPPPPXSH2PPPPSTAT Px = SHCGRB2PLCγPI-3KGAPDimerizationandnucleartranslocationFigure 43–9. Initiation of signal transduction by receptors linked to Jak kinases.The receptors (R) that bind prolactin, growth hormone, interferons, and cytokineslack endogenous tyrosine kinase. Upon ligand binding, these receptorsdimerize and an associated protein (Jak1, Jak2, or TYK) is phosphorylated. Jak-P,an active kinase, phosphorylates the receptor on tyrosine residues. The STAT proteinsassociate with the phosphorylated receptor and then are themselves phosphorylatedby Jak-P. STATP dimerizes, translocates to the nucleus, binds to specificDNA elements, and regulates transcription. The phosphotyrosine residues ofthe receptor also bind to several SH2 domain-containing proteins. This results inactivation of the MAP kinase pathway (through SHC or GRB2), PLCγ, or PI-3 kinase.

468 / CHAPTER 43C. THE NF-B PATHWAY ISREGULATED BY GLUCOCORTICOIDSThe transcription factor NF-κB is a heterodimericcomplex typically composed of two subunits termedp50 and p65 (Figure 43–10). Normally, NF-κB is keptsequestered in the cytoplasm in a transcriptionally inactiveform by members of the inhibitor of NF-κB (IκB)family. Extracellular stimuli such as proinflammatorycytokines, reactive oxygen species, and mitogens lead toactivation of the IκB kinase complex, IKK, which is aheterohexameric structure consisting of α, β, and γ subunits.IKK phosphorylates IκB on two serine residues,and this targets IκB for ubiquitination and subsequentdegradation by the proteasome. Following IκB degradation,free NF-κB can now translocate to the nucleus,where it binds to a number of gene promoters and activatestranscription, particularly of genes involved in theinflammatory response. Transcriptional regulation byNF-κB is mediated by a variety of coactivators such asCREB binding protein (CBP), as described below (Figure43–13).Glucocorticoid hormones are therapeutically usefulagents for the treatment of a variety of inflammatory andimmune diseases. Their anti-inflammatory and immunomodulatoryactions are explained in part by the inhibitionof NF-κB and its subsequent actions. Evidencefor three mechanisms for the inhibition of NF-κB byglucocorticoids has been presented: (1) Glucocorticoidsincrease IκB mRNA, which leads to an increase of IκBprotein and more efficient sequestration of NF-κB in thecytoplasm. (2) The glucocorticoid receptor competeswith NF-κB for binding to coactivators. (3) The glucocorticoidreceptor directly binds to the p65 subunit ofNF-κB and inhibits its activation (Figure 43–10).HORMONES CAN INFLUENCESPECIFIC BIOLOGIC EFFECTS BYMODULATING TRANSCRIPTIONThe signals generated as described above have to betranslated into an action that allows the cell to effectivelyadapt to a challenge (Figure 43–1). Much of thisNF-κB ActivatorsProinflammatory cytokinesBacterial and viral infectionReactive oxygen speciesMitogensIKKcomplexγ γ1αβ βαPPIκBProteasomeUbiquitinp50IκBp65p50p652CytoplasmNucleusCoactivators3p50p65Target geneFigure 43–10. Regulation of the NF-κB pathway. NF-κB consists of two subunits,p50 and p65, which regulate transcription of many genes when in thenucleus. NF-κB is restricted from entering the nucleus by IκB, an inhibitor ofNF-κB. IκB binds to—and masks—the nuclear localization signal of NF-κB.This cytoplasmic protein is phosphorylated by an IKK complex which is activatedby cytokines, reactive oxygen species, and mitogens. PhosphorylatedIκB can be ubiquitinylated and degraded, thus releasing its hold on NF-κB. Glucocorticoidsaffect many steps in this process, as described in the text.

HORMONE ACTION & SIGNAL TRANSDUCTION / 469Figure 43–11. The hormone response transcriptionunit. The hormone response transcription unitis an assembly of DNA elements and bound proteinsthat interact, through protein-protein interactions,with a number of coactivator or corepressormolecules. An essential component is the hormoneresponse element which binds the ligand ()-bound receptor (R). Also important are the accessoryfactor elements (AFEs) with bound transcriptionfactors. More than two dozen of theseaccessory factors (AFs), which are often members ofthe nuclear receptor superfamily, have been linkedto hormone effects on transcription. The AFs can interactwith each other, with the liganded nuclearreceptors, or with coregulators. These componentscommunicate with the basal transcription complexthrough a coregulator complex that can consist ofone or more members of the p160, corepressor,mediator-related, or CBP/p300 families (seeTable 43–6).AFEHREAFRAFEAFRp160p300BTCGenecoding regionadaptation is accomplished through alterations in therates of transcription of specific genes. Many differentobservations have led to the current view of how hormonesaffect transcription. Some of these are as follows:(1) Actively transcribed genes are in regions of “open”chromatin (defined by a susceptibility to the enzymeDNase I), which allows for the access of transcriptionfactors to DNA. (2) Genes have regulatory regions, andtranscription factors bind to these to modulate the frequencyof transcription initiation. (3) The hormonereceptorcomplex can be one of these transcription factors.The DNA sequence to which this binds is called ahormone response element (HRE; see Table 43–1 forexamples). (4) Alternatively, other hormone-generatedsignals can modify the location, amount, or activity oftranscription factors and thereby influence binding tothe regulatory or response element. (5) Members of alarge superfamily of nuclear receptors act with—or in amanner analogous to—hormone receptors. (6) Thesenuclear receptors interact with another large group ofcoregulatory molecules to effect changes in the transcriptionof specific genes.Several Hormone Response Elements(HREs) Have Been DefinedHormone response elements resemble enhancer elementsin that they are not strictly dependent on positionand location. They generally are found within afew hundred nucleotides upstream (5′) of the transcriptioninitiation site, but they may be located within thecoding region of the gene, in introns. HREs were definedby the strategy illustrated in Figure 39–11. Theconsensus sequences illustrated in Table 43–1 were arrivedat through analysis of several genes regulated by agiven hormone using simple, heterologous reporter systems(see Figure 39–10). Although these simple HREsbind the hormone-receptor complex more avidly thansurrounding DNA—or DNA from an unrelatedsource—and confer hormone responsiveness to a reportergene, it soon became apparent that the regulatorycircuitry of natural genes must be much morecomplicated. Glucocorticoids, progestins, mineralocorticoids,and androgens have vastly different physiologicactions. How could the specificity required for these effectsbe achieved through regulation of gene expressionby the same HRE (Table 43–1)? Questions like thishave led to experiments which have allowed for elaborationof a very complex model of transcription regulation.For example, the HRE must associate with otherDNA elements (and associated binding proteins) tofunction optimally. The extensive sequence similaritynoted between steroid hormone receptors, particularlyin their DNA-binding domains, led to discovery of thenuclear receptor superfamily of proteins. These—anda large number of coregulator proteins—allow for awide variety of DNA-protein and protein-protein interactionsand the specificity necessary for highly regulatedphysiologic control. A schematic of such an assembly isillustrated in Figure 43–11.

470 / CHAPTER 43A/BCDEFNAF-1DBDHingeLBDAF-2C××GR, MR, PRAR, ERTR, RAR, VDRPPARα, β, γFXR, CAR, LXR,PXR/SXRCOUP-TF, TR2, GEN8HNF-4, TLXReceptors:Binding:Ligand:DNA element:Steroid classHomodimersSteroidsInvertedrepeatRXR partneredHeterodimers9-Cis RA + (x)Direct repeatsOrphansHomodimers?Direct repeatsFigure 43–12. The nuclear receptor superfamily. Members of this family aredivided into six structural domains (A–F). Domain A/B is also called AF-1, or themodulator region, because it is involved in activating transcription. The C domainconsists of the DNA-binding domain (DBD). The D region contains thehinge, which provides flexibility between the DBD and the ligand-binding domain(LBD, region E). The amino (N) terminal part of region E contains AF-2, anotherdomain important for transactivation. The F region is poorly defined. Thefunctions of these domains are discussed in more detail in the text. Receptorswith known ligands, such as the steroid hormones, bind as homodimers on invertedrepeat half-sites. Other receptors form heterodimers with the partnerRXR on direct repeat elements. There can be nucleotide spacers of one to fivebases between these direct repeats (DR1–5). Another class of receptors forwhich ligands have not been determined (orphan receptors) bind as homodimersto direct repeats and occasionally as monomers to a single half-site.There Is a Large Family of NuclearReceptor ProteinsThe nuclear receptor superfamily consists of a diverse setof transcription factors that were discovered because of asequence similarity in their DNA-binding domains. Thisfamily, now with more than 50 members, includes thenuclear hormone receptors discussed above, a number ofother receptors whose ligands were discovered after thereceptors were identified, and many putative or orphanreceptors for which a ligand has yet to be discovered.These nuclear receptors have several common structuralfeatures (Figure 43–12). All have a centrally locatedDNA-binding domain (DBD) that allows thereceptor to bind with high affinity to a response element.The DBD contains two zinc finger binding motifs(see Figure 39–14) that direct binding either as homodimers,as heterodimers (usually with a retinoid Xreceptor [RXR] partner), or as monomers. The targetresponse element consists of one or two half-site consensussequences arranged as an inverted or direct repeat.The spacing between the latter helps determinebinding specificity. Thus, a direct repeat with three,four, or five nucleotide spacer regions specifies thebinding of the vitamin D, thyroid, and retinoic acid receptors,respectively, to the same consensus responseelement (Table 43–1). A multifunctional ligandbindingdomain (LBD) is located in the carboxyl terminalhalf of the receptor. The LBD binds hormonesor metabolites with selectivity and thus specifies a particularbiologic response. The LBD also contains domainsthat mediate the binding of heat shock proteins,dimerization, nuclear localization, and transactivation.The latter function is facilitated by the carboxyl terminaltranscription activation function (AF-2 domain),which forms a surface required for the interaction with

HORMONE ACTION & SIGNAL TRANSDUCTION / 471Insulin,EGF, etcGPCRGroup IHormonesGH, Prl,Cytokines, etcTNF, etcRAS IRSMEKMAPKcAMPPKARetinoic acid,estrogen,vitamin D,glucocorticoids,etcJakSTATsNFκB•IκBNFκBPlasmamembraneAP-1CREBNuclearreceptorsSTATsNFκBNuclearmembraneCBPp300Figure 43–13. Several signal transduction pathways converge onCBP/p300. Ligands that associate with membrane or nuclear receptors eventuallyconverge on CBP/p300. Several different signal transduction pathwaysare employed. EGF, epidermal growth factor; GH, growth hormone; Prl, prolactin;TNF, tumor necrosis factor; other abbreviations are expanded in thetext.coactivators. A highly variable hinge region separatesthe DBD from the LBD. This region provides flexibilityto the receptor, so it can assume different DNAbindingconformations. Finally, there is a highly variableamino terminal region that contains another transactivationdomain referred to as AF-1. Less well defined,the AF-1 domain may provide for distinctphysiologic functions through the binding of differentcoregulator proteins. This region of the receptor,through the use of different promoters, alternativesplice sites, and multiple translation initiation sites,provides for receptor isoforms that share DBD andLBD identity but exert different physiologic responsesbecause of the association of various coregulators withthis variable amino terminal AF-1 domain.It is possible to sort this large number of receptorsinto groups in a variety of ways. Here they are discussedaccording to the way they bind to their respective DNAelements (Figure 43–12). Classic hormone receptors forglucocorticoids (GR), mineralocorticoids (MR), estrogens(ER), androgens (AR), and progestins (PR) bindas homodimers to inverted repeat sequences. Otherhormone receptors such as thyroid (TR), retinoic acid(RAR), and vitamin D (VDR) and receptors that bindvarious metabolite ligands such as PPAR α β, and γ,FXR, LXR, PXR/SXR, and CAR bind as heterodimers,with retinoid X receptor (RXR) as a partner, to directrepeat sequences (see Figure 43–12 and Table 43–5).Another group of orphan receptors that as yet have noknown ligand bind as homodimers or monomers to directrepeat sequences.As illustrated in Table 43–5, the discovery of the nuclearreceptor superfamily has led to an important understandingof how a variety of metabolites and xenobioticsregulate gene expression and thus the metabolism,detoxification, and elimination of normal body productsand exogenous agents such as pharmaceuticals.Not surprisingly, this area is a fertile field for investigationof new therapeutic interventions.A Large Number of Nuclear ReceptorCoregulators Also Participatein Regulating TranscriptionChromatin remodeling, transcription factor modificationby various enzyme activities, and the communicationbetween the nuclear receptors and the basal transcriptionapparatus are accomplished by protein-protein interactionswith one or more of a class of coregulatormolecules. The number of these coregulator moleculesnow exceeds 100, not counting species variations andsplice variants. The first of these to be described was theCREB-binding protein, CBP. CBP, through anamino terminal domain, binds to phosphorylated serine137 of CREB and mediates transactivation in responseto cAMP. It thus is described as a coactivator. CBP and

472 / CHAPTER 43Table 43–5. Nuclear receptors with special ligands. 1Receptor Partner Ligand Process AffectedPeroxisome PPARα RXR (DR1) Fatty acids Peroxisome proliferationProliferator- PPARβ Fatty acidsactivated PPARγ Fatty acids Lipid and carbohydrate metabolismEicosanoids,thiazolidinedionesFarnesoid X FXR RXR (DR4) Farnesol, bile acids Bile acid metabolismLiver X LXR RXR (DR4) Oxysterols Cholesterol metabolismXenobiotic X CAR RXR (DR5) AndrostanesPhenobarbital Protection against certain drugs, toxicXenobioticsmetabolites, and xenobioticsPXR RXR (DR3) PregnanesXenobiotics1 Many members of the nuclear receptor superfamily were discovered by cloning, and the correspondingligands were then identified. These ligands are not hormones in the classic sense, but they do have asimilar function in that they activate specific members of the nuclear receptor superfamily. The receptorsdescribed here form heterodimers with RXR and have variable nucleotide sequences separating thedirect repeat binding elements (DR1–5). These receptors regulate a variety of genes encoding cytochromep450s (CYP), cytosolic binding proteins, and ATP-binding cassette (ABC) transporters to influencemetabolism and protect cells against drugs and noxious agents.its close relative, p300, interact directly or indirectlywith a number of signaling molecules, including activatorprotein-1 (AP-1), signal transducers and activatorsof transcription (STATs), nuclear receptors, and CREB(Figure 39–11). CBP/p300 also binds to the p160family of coactivators described below and to a numberof other proteins, including viral transcription factorEla, the p90 rsk protein kinase, and RNA helicase A. It isimportant to note that CBP/p300 also has intrinsichistone acetyltransferase (HAT) activity. The importanceof this is described below. Some of the many actionsof CBP/p300, which appear to depend on intrinsicenzyme activities and its ability to serve as a scaffoldfor the binding of other proteins, are illustrated in Figure43–11. Other coregulators may serve similar functions.Several other families of coactivator molecules havebeen described. Members of the p160 family of coactivators,all of about 160 kDa, include (1) SRC-1 andNCoA-1; (2) GRIP 1, TIF2, and NCoA-2; and (3)p/CIP, ACTR, AIB1, RAC3, and TRAM-1 (Table43–6). The different names for members within a subfamilyoften represent species variations or minor splicevariants. There is about 35% amino acid identity betweenmembers of the different subfamilies. The p160coactivators share several properties. They (1) bindnuclear receptors in an agonist and AF-2 transactivationdomain-dependent manner; (2) have a conservedamino terminal basic helix-loop-helix (bHLH) motif(see Chapter 39); (3) have a weak carboxyl terminaltransactivation domain and a stronger amino terminalTable 43–6. Some mammalian coregulatorproteins.I. 300-kDa family of coactivatorsCBP CREB-binding proteinp300 Protein of 300 kDaII. 160-kDa family of coactivatorsA. SRC-1 Steroid receptor coactivator 1NCoA-1 Nuclear receptor coactivator 1B. TIF2 Transcriptional intermediary factor 2GRIP1 Glucocorticoid receptor-interacting proteinNCoA-2 Nuclear receptor coactivator 2C. p/CIP p300/CBP cointegrator-associated protein 1ACTR Activator of the thyroid and retinoic acidreceptorsAIB Amplified in breast cancerRAC3 Receptor-associated coactivator 3TRAM-1 TR activator molecule 1III. CorepressorsNCoR Nuclear receptor corepressorSMRT Silencing mediator for RXR and TRIV. Mediator-related proteinsTRAPs Thyroid hormone receptor-associatedproteinsDRIPs Vitamin D receptor-interacting proteinsARC Activator-recruited cofactor

HORMONE ACTION & SIGNAL TRANSDUCTION / 473transactivation domain in a region that is required forthe CBP/p16O interaction; (4) contain at least three ofthe LXXLL motifs required for protein-protein interactionwith other coactivators; and (5) often have HATactivity. The role of HAT is particularly interesting, asmutations of the HAT domain disable many of thesetranscription factors. Current thinking holds that theseHAT activities acetylate histones and result in remodelingof chromatin into a transcription-efficient environment;however, other protein substrates for HATmediatedacetylation have been reported. Histoneacetylation/deacetylation is proposed to play a criticalrole in gene expression.A small number of proteins, including NCoR andSMRT, comprise the corepressor family. They function,at least in part, as described in Figure 43–2. Anotherfamily includes the TRAPs, DRIPs, and ARC(Table 43–6). These so-called mediator-related proteinsrange in size from 80 kDa to 240 kDa and arethought to be involved in linking the nuclear receptorcoactivatorcomplex to RNA polymerase II and theother components of the basal transcription apparatus.The exact role of these coactivators is presentlyunder intensive investigation. Many of these proteinshave intrinsic enzymatic activities. This is particularlyinteresting in view of the fact that acetylation, phosphorylation,methylation, and ubiquitination—as wellas proteolysis and cellular translocation—have beenproposed to alter the activity of some of these coregulatorsand their targets.It appears that certain combinations of coregulators—andthus different combinations of activators andinhibitors—are responsible for specific ligand-inducedactions through various receptors.SUMMARY• Hormones, cytokines, interleukins, and growth factorsuse a variety of signaling mechanisms to facilitatecellular adaptive responses.• The ligand-receptor complex serves as the initial signalfor members of the nuclear receptor family.• Class II hormones, which bind to cell surface receptors,generate a variety of intracellular signals. Theseinclude cAMP, cGMP, Ca 2+ , phosphatidylinositides,and protein kinase cascades.• Many hormone responses are accomplished throughalterations in the rate of transcription of specificgenes.• The nuclear receptor superfamily of proteins plays acentral role in the regulation of gene transcription.• These receptors, which may have hormones, metabolites,or drugs as ligands, bind to specific DNA elementsas homodimers or as heterodimers with RXR.Some—orphan receptors—have no known ligandbut bind DNA and influence transcription.• Another large family of coregulator proteins remodelchromatin, modify other transcription factors, andbridge the nuclear receptors to the basal transcriptionapparatus.REFERENCESArvanitakis L et al: Constitutively signaling G-protein-coupled receptorsand human disease. Trends Endocrinol Metab1998;9:27.Berridge M: Inositol triphosphate and calcium signalling. Nature1993;361:315.Chawla A et al: Nuclear receptors and lipid physiology: opening theX files. Science 2001;294:1866.Darnell JE Jr, Kerr IM, Stark GR: Jak-STAT pathways and transcriptionalactivation in response to IFNs and other extracellularsignaling proteins. Science 1994;264:1415.Fantl WJ, Johnson DE, Williams LT: Signalling by receptor tyrosinekinases. Annu Rev Biochem 1993;62:453.Giguère V: Orphan nuclear receptors: from gene to function. EndocrRev 1999;20:689.Grunstein M: Histone acetylation in chromatin structure and transcription.Nature 1997;389:349.Hanoune J, Defer N: Regulation and role of adenylyl cyclase isoforms.Annu Rev Pharmacol Toxicol 2001;41:145.Hermanson O, Glass CK, Rosenfeld MG: Nuclear receptor coregulators:multiple modes of receptor modification. Trends EndocrinolMetab 2002;13:55.Jaken S: Protein kinase C isozymes and substrates. Curr Opin CellBiol 1996;8:168.Lucas P, Granner D: Hormone response domains in gene transcription.Annu Rev Biochem 1992;61:1131.Montminy M: Transcriptional regulation by cyclic AMP. AnnuRev Biochem 1997;66:807Morris AJ, Malbon CC: Physiological regulation of G proteinlinkedsignaling. Physiol Rev 1999;79:1373.Walton KM, Dixon JE: Protein tyrosine phosphatases. Annu RevBiochem 1993;62:101.

SECTION VISpecial TopicsNutrition, Digestion, & Absorption 44David A. Bender, PhD, & Peter A. Mayes, PhD, DScBIOMEDICAL IMPORTANCEBesides water, the diet must provide metabolic fuels(mainly carbohydrates and lipids), protein (for growthand turnover of tissue proteins), fiber (for roughage),minerals (elements with specific metabolic functions),and vitamins and essential fatty acids (organic compoundsneeded in small amounts for essential metabolicand physiologic functions). The polysaccharides, triacylglycerols,and proteins that make up the bulk of thediet must be hydrolyzed to their constituent monosaccharides,fatty acids, and amino acids, respectively, beforeabsorption and utilization. Minerals and vitaminsmust be released from the complex matrix of food beforethey can be absorbed and utilized.Globally, undernutrition is widespread, leading toimpaired growth, defective immune systems, and reducedwork capacity. By contrast, in developed countries,there is often excessive food consumption (especiallyof fat), leading to obesity and to the developmentof cardiovascular disease and some forms of cancer. Deficienciesof vitamin A, iron, and iodine pose majorhealth concerns in many countries, and deficiencies ofother vitamins and minerals are a major cause of illhealth. In developed countries, nutrient deficiency israre, though there are vulnerable sections of the populationat risk. Intakes of minerals and vitamins that areadequate to prevent deficiency may be inadequate topromote optimum health and longevity.Excessive secretion of gastric acid, associated withHelicobacter pylori infection, can result in the developmentof gastric and duodenal ulcers; small changes inthe composition of bile can result in crystallization ofcholesterol as gallstones; failure of exocrine pancreaticsecretion (as in cystic fibrosis) leads to undernutritionand steatorrhea. Lactose intolerance is due to lactasedeficiency leading to diarrhea and intestinal discomfort.Absorption of intact peptides that stimulate antibodyresponses causes allergic reactions, and celiac diseaseis an allergic reaction to wheat gluten.DIGESTION & ABSORPTIONOF CARBOHYDRATESThe digestion of complex carbohydrates is by hydrolysisto liberate oligosaccharides, then free mono- and disaccharides.The increase in blood glucose after a testdose of a carbohydrate compared with that after anequivalent amount of glucose is known as the glycemicindex. Glucose and galactose have an index of 1, as dolactose, maltose, isomaltose, and trehalose, which giverise to these monosaccharides on hydrolysis. Fructoseand the sugar alcohols are absorbed less rapidly andhave a lower glycemic index, as does sucrose. Theglycemic index of starch varies between near 1 to nearzero due to variable rates of hydrolysis, and that of nonstarchpolysaccharides is zero. Foods that have a lowglycemic index are considered to be more beneficialsince they cause less fluctuation in insulin secretion.Amylases Catalyzethe Hydrolysis of StarchThe hydrolysis of starch by salivary and pancreaticamylases catalyze random hydrolysis of α(1→4) glycosidebonds, yielding dextrins, then a mixture of glucose,maltose, and isomaltose (from the branch points inamylopectin).474

NUTRITION, DIGESTION, & ABSORPTION / 475Disaccharidases Are BrushBorder EnzymesThe disaccharidases—maltase, sucrase-isomaltase (abifunctional enzyme catalyzing hydrolysis of sucrose andisomaltose), lactase, and trehalase—are located on thebrush border of the intestinal mucosal cells where the resultantmonosaccharides and others arising from the dietare absorbed. In most people, apart from those of northernEuropean genetic origin, lactase is gradually lostthrough adolescence, leading to lactose intolerance.Lactose remains in the intestinal lumen, where it is asubstrate for bacterial fermentation to lactate, resultingin discomfort and diarrhea.There Are Two Separate Mechanismsfor the Absorption of Monosaccharidesin the Small IntestineGlucose and galactose are absorbed by a sodium-dependentprocess. They are carried by the same transportprotein (SGLT 1) and compete with each other for intestinalabsorption (Figure 44–1). Other monosaccharidesare absorbed by carrier-mediated diffusion. Becausethey are not actively transported, fructose andsugar alcohols are only absorbed down their concentrationgradient, and after a moderately high intake somemay remain in the intestinal lumen, acting as a substratefor bacterial fermentation.DIGESTION & ABSORPTION OF LIPIDSThe major lipids in the diet are triacylglycerols and, to alesser extent, phospholipids. These are hydrophobicmolecules and must be hydrolyzed and emulsified tovery small droplets (micelles) before they can be absorbed.The fat-soluble vitamins—A, D, E, and K—and a variety of other lipids (including cholesterol) areabsorbed dissolved in the lipid micelles. Absorption ofthe fat-soluble vitamins is impaired on a very low fatdiet.Hydrolysis of triacylglycerols is initiated by lingualand gastric lipases that attack the sn-3 ester bond, forming1,2-diacylglycerols and free fatty acids, aiding emulsification.Pancreatic lipase is secreted into the smallintestine and requires a further pancreatic protein, colipase,for activity. It is specific for the primary esterlinks—ie, positions 1 and 3 in triacylglycerols—resultingin 2-monoacylglycerols and free fatty acids as themajor end-products of luminal triacylglycerol digestion.Monoacylglycerols are hydrolyzed with difficulty toglycerol and free fatty acids, so that less than 25% of ingestedtriacylglycerol is completely hydrolyzed to glyceroland fatty acids (Figure 44–2). Bile salts, formed inthe liver and secreted in the bile, enable emulsificationGLUT 5GlucoseGalactoseFructoseGlucoseGalactoseGlucoseFructoseGalactoseTo capillariesGlucoseNa +Na + -K +pumpNa + ATP3Na +2K +2K +ADP+ P iGLUT 2SGLT 1transporterproteinBrushborderIntestinalepitheliumFigure 44–1. Transport of glucose, fructose, andgalactose across the intestinal epithelium. The SGLT 1transporter is coupled to the Na + -K + pump, allowingglucose and galactose to be transported against theirconcentration gradients. The GLUT 5 Na + -independentfacilitative transporter allows fructose as well as glucoseand galactose to be transported with their concentrationgradients. Exit from the cell for all the sugarsis via the GLUT 2 facilitative transporter.of the products of lipid digestion into micelles and liposomestogether with phospholipids and cholesterolfrom the bile. Because the micelles are soluble, theyallow the products of digestion, including the fatsolublevitamins, to be transported through the aqueousenvironment of the intestinal lumen and permit closecontact with the brush border of the mucosal cells, allowinguptake into the epithelium, mainly of the jejunum.The bile salts pass on to the ileum, wheremost are absorbed into the enterohepatic circulation(Chapter 26). Within the intestinal epithelium,1-monoacylglycerols are hydrolyzed to fatty acids andglycerol and 2-monoacylglycerols are re-acylated to triacylglycerolsvia the monoacylglycerol pathway. Glycerolreleased in the intestinal lumen is not reutilized butpasses into the portal vein; glycerol released within the

INTESTINALLUMEN1AcylPANCREATICAcylLIPASE2AcylAcyl3AcylFAOHTriacylglycerol, 100% 1,2-DiacylglycerolPANCREATICLIPASEFA72%OHAcylOHINTESTINALEPITHELIUMMonoacylglycerol pathwayAcylAcylAcylLYMPHATICVESSELS(LACTEALS)Absorptionfrombile saltmicelleOHAcylOH2-MonoacylglycerolTriacylglycerolAcylAcylAcylChylomicrons476FAISOMERASEPANCREATICLIPASEFAAcylOHOH1-MonoacylglycerolOHOHACYL-CoASYNTHETASEATP, CoA6%ACYL-CoASYNTHETASEAcylOHOHAcyl-CoAATPCoAFAATPINTESTINALLIPASEPhosphatidic acid pathwayOHOHOHGlycerolATPGLYCEROLKINASEOHOHPGlycerol3-phosphateAcylAcylAcylOHGlycerolGlycolysisPORTAL VEIN22%GlycerolFigure 44–2. Digestion and absorption of triacylglycerols. The values given for percentage uptake may varywidely but indicate the relative importance of the three routes shown.

NUTRITION, DIGESTION, & ABSORPTION / 477epithelium is reutilized for triacylglycerol synthesis viathe normal phosphatidic acid pathway (Chapter 24).All long-chain fatty acids absorbed are converted to triacylglycerolin the mucosal cells and, together with theother products of lipid digestion, secreted as chylomicronsinto the lymphatics, entering the blood stream viathe thoracic duct (Chapter 25).DIGESTION & ABSORPTION OF PROTEINSFew peptide bonds that are hydrolyzed by proteolyticenzymes are accessible without prior denaturation of dietaryproteins (by heat in cooking and by the action ofgastric acid).Several Groups of Enzymes Catalyzethe Digestion of ProteinsThere are two main classes of proteolytic digestive enzymes(proteases), with different specificities for theamino acids forming the peptide bond to be hydrolyzed.Endopeptidases hydrolyze peptide bonds between specificamino acids throughout the molecule. They are thefirst enzymes to act, yielding a larger number of smallerfragments, eg, pepsin in the gastric juice and trypsin,chymotrypsin, and elastase secreted into the small intestineby the pancreas. Exopeptidases catalyze the hydrolysisof peptide bonds, one at a time, from the endsof polypeptides. Carboxypeptidases, secreted in thepancreatic juice, release amino acids from the free carboxylterminal, and aminopeptidases, secreted by theintestinal mucosal cells, release amino acids from theamino terminal. Dipeptides, which are not substrates forexopeptidases, are hydrolyzed in the brush border of intestinalmucosal cells by dipeptidases.The proteases are secreted as inactive zymogens; theactive site of the enzyme is masked by a small region ofits peptide chain, which is removed by hydrolysis of aspecific peptide bond. Pepsinogen is activated to pepsinby gastric acid and by activated pepsin (autocatalysis). Inthe small intestine, trypsinogen, the precursor oftrypsin, is activated by enteropeptidase, which is secretedby the duodenal epithelial cells; trypsin can thenactivate chymotrypsinogen to chymotrypsin, proelastaseto elastase, procarboxypeptidase to carboxypeptidase,and proaminopeptidase to aminopeptidase.Free Amino Acids & Small Peptides AreAbsorbed by Different MechanismsThe end product of the action of endopeptidases andexopeptidases is a mixture of free amino acids, di- andtripeptides, and oligopeptides, all of which are absorbed.Free amino acids are absorbed across the intestinal mucosaby sodium-dependent active transport. There areseveral different amino acid transporters, with specificityfor the nature of the amino acid side chain (large orsmall; neutral, acidic, or basic). The various amino acidscarried by any one transporter compete with each otherfor absorption and tissue uptake. Dipeptides and tripeptidesenter the brush border of the intestinal mucosalcells, where they are hydrolyzed to free amino acids,which are then transported into the hepatic portal vein.Relatively large peptides may be absorbed intact, eitherby uptake into mucosal epithelial cells (transcellular) orby passing between epithelial cells (paracellular). Manysuch peptides are large enough to stimulate antibody formation—thisis the basis of allergic reactions to foods.DIGESTION & ABSORPTIONOF VITAMINS & MINERALSVitamins and minerals are released from food duringdigestion—though this is not complete—and the availabilityof vitamins and minerals depends on the type offood and, especially for minerals, the presence of chelatingcompounds. The fat-soluble vitamins are absorbedin the lipid micelles that result from fat digestion;water-soluble vitamins and most mineral salts areabsorbed from the small intestine either by active transportor by carrier-mediated diffusion followed by bindingto intracellular binding proteins to achieve concentrationupon uptake. Vitamin B 12 absorption requires aspecific transport protein, intrinsic factor; calcium absorptionis dependent on vitamin D; zinc absorptionprobably requires a zinc-binding ligand secreted by theexocrine pancreas; and the absorption of iron is limited.Calcium Absorption Is Dependenton Vitamin DIn addition to its role in regulating calcium homeostasis,vitamin D is required for the intestinal absorptionof calcium. Synthesis of the intracellular calciumbindingprotein, calbindin, required for calcium absorption,is induced by vitamin D, which also affectsthe permeability of the mucosal cells to calcium, an effectthat is rapid and independent of protein synthesis.Phytic acid (inositol hexaphosphate) in cereals bindscalcium in the intestinal lumen, preventing its absorption.Other minerals, including zinc, are also chelatedby phytate. This is mainly a problem among peoplewho consume large amounts of unleavened wholewheat products; yeast contains an enzyme, phytase,which dephosphorylates phytate, so rendering it inactive.High concentrations of fatty acids in the intestinallumen—as a result of impaired fat absorption—canalso reduce calcium absorption by forming insolublecalcium salts; a high intake of oxalate can sometimescause deficiency, since calcium oxalate is insoluble.

478 / CHAPTER 44Iron Absorption Is Limited& Strictly Controlled but IsEnhanced by Vitamin C & EthanolAlthough iron deficiency is a common problem, about10% of the population are genetically at risk of ironoverload (hemochromatosis), and elemental iron canlead to nonenzymic generation of free radicals. Absorptionof iron is strictly regulated. Inorganic iron is accumulatedin intestinal mucosal cells bound to an intracellularprotein, ferritin. Once the ferritin in the cell issaturated with iron, no more can enter. Iron can onlyleave the mucosal cell if there is transferrin in plasmato bind to. Once transferrin is saturated with iron, anythat has accumulated in the mucosal cells will be lostwhen the cells are shed. As a result of this mucosal barrier,only about 10% of dietary iron is normally absorbedand only 1–5% from many plant foods.Inorganic iron is absorbed only in the Fe 2+ (reduced)state, and for that reason the presence of reducing agentswill enhance absorption. The most effective compoundis vitamin C, and while intakes of 40–60 mg of vitaminC per day are more than adequate to meet requirements,an intake of 25–50 mg per meal will enhance iron absorption,especially when iron salts are used to treat irondeficiency anemia. Ethanol and fructose also enhanceiron absorption. Heme iron from meat is absorbed separatelyand is considerably more available than inorganiciron. However, the absorption of both inorganic andheme iron is impaired by calcium—a glass of milk witha meal significantly reduces availability.ENERGY BALANCE:OVER- & UNDERNUTRITIONAfter the provision of water, the body’s first requirementis for metabolic fuels—fats, carbohydrates, and aminoacids from proteins (and ethanol) (Table 27–1). Food intakein excess of energy expenditure leads to obesity,while intake less than expenditure leads to emaciationand wasting, as in marasmus and kwashiorkor. Bothobesity and severe undernutrition are associated with increasedmortality. The body mass index, defined asweight in kilograms divided by height in meters squared,is commonly used as a way of expressing relative obesityto height. A desirable range is between 20 and 25.Energy Requirements Are Estimated byMeasurement of Energy ExpenditureEnergy expenditure can be determined directly by measuringheat output from the body but is normally estimatedindirectly from the consumption of oxygen.There is an energy expenditure of 20 kJ/L of oxygenconsumed regardless of whether the fuel being metabolizedis carbohydrate, fat, or protein. Measurement ofthe ratio of the volume of carbon dioxide produced tovolume of oxygen consumed (respiratory quotient; RQ)is an indication of the mixture of metabolic fuels beingoxidized (Table 27–1). A more recent technique permitsestimation of total energy expenditure over a periodof 1–2 weeks using dual isotopically labeled water,2 H 2 18 O. 2 H is lost from the body only in water, while18 O is lost in both water and carbon dioxide; the differencein the rate of loss of the two labels permits estimationof total carbon dioxide production and thus oxygenconsumption and energy expenditure.Basal metabolic rate (BMR) is the energy expenditureby the body when at rest—but not asleep—undercontrolled conditions of thermal neutrality, measuredat about 12 hours after the last meal, and depends onweight, age, and gender. Total energy expenditure dependson the basal metabolic rate, the energy requiredfor physical activity, and the energy cost of synthesizingreserves in the fed state. It is therefore possible to calculatean individual’s energy requirement from bodyweight, age, gender, and level of physical activity. Bodyweight affects BMR because there is a greater amountof active tissue in a larger body. The decrease in BMRwith increasing age, even when body weight remainsconstant, is due to muscle tissue replacement by adiposetissue, which is metabolically much less active.Similarly, women have a significantly lower BMR thando men of the same body weight because women’s bodieshave proportionately more adipose tissue than men.Energy Requirements IncreaseWith ActivityThe most useful way of expressing the energy cost ofphysical activities is as a multiple of BMR. Sedentaryactivities use only about 1.1–1.2 × BMR. By contrast,vigorous exertion, such as climbing stairs, cross-countryskiing, walking uphill, etc, may use 6–8 × BMR.Ten Percent of the Energy Yield of a MealMay Be Expended in Forming ReservesThere is a considerable increase in metabolic rate after ameal, a phenomenon known as diet-induced thermogenesis.A small part of this is the energy cost of secretingdigestive enzymes and of active transport of theproducts of digestion; the major part is due to synthesizingreserves of glycogen, triacylglycerol, and protein.There Are Two Extreme Formsof UndernutritionMarasmus can occur in both adults and children andoccurs in vulnerable groups of all populations. Kwash-

NUTRITION, DIGESTION, & ABSORPTION / 479iorkor only affects children and has only been reportedin developing countries. The distinguishing feature ofkwashiorkor is that there is fluid retention, leading toedema. Marasmus is a state of extreme emaciation; it isthe outcome of prolonged negative energy balance. Notonly have the body’s fat reserves been exhausted, butthere is wastage of muscle as well, and as the conditionprogresses there is loss of protein from the heart, liver,and kidneys. The amino acids released by the catabolismof tissue proteins are used as a source of metabolicfuel and as substrates for gluconeogenesis to maintain asupply of glucose for the brain and red blood cells. As aresult of the reduced synthesis of proteins, there is impairedimmune response and more risk from infections.Impairment of cell proliferation in the intestinal mucosaoccurs, resulting in reduction in surface area of theintestinal mucosa and reduction in absorption of suchnutrients as are available.Patients With Advanced Cancer& AIDS Are MalnourishedPatients with advanced cancer, HIV infection andAIDS, and a number of other chronic diseases are frequentlyundernourished—the condition is calledcachexia. Physically, they show all the signs of marasmus,but there is considerably more loss of body proteinthan occurs in starvation. The secretion of cytokinesin response to infection and cancer increases thecatabolism of tissue protein. This differs from marasmus,in which protein synthesis is reduced but catabolismin unaffected. Patients are hypermetabolic, ie,there is a considerable increase in basal metabolic rate.Many tumors metabolize glucose anaerobically to releaselactate. This is used for gluconeogenesis in theliver, which is energy-consuming with a net cost of sixATP for each mole of glucose cycled (Chapter 19).There is increased stimulation of uncoupling proteinsby cytokines, leading to thermogenesis and increasedoxidation of metabolic fuels. Futile cycling of lipids occursbecause hormone-sensitive lipase is activated by aproteoglycan secreted by tumors, resulting in liberationof fatty acids from adipose tissue and ATP-expensivereesterification in the liver to triacylglycerols, which areexported in VLDL.Kwashiorkor AffectsUndernourished ChildrenIn addition to the wasting of muscle tissue, loss of intestinalmucosa, and impaired immune responses seenin marasmus, children with kwashiorkor show a numberof characteristic features. The defining characteristicis edema, associated with a decreased concentration ofplasma proteins. In addition, there is enlargement ofthe liver due to accumulation of fat. It was formerly believedthat the cause of kwashiorkor was a lack of protein,with a more or less adequate energy intake; however,analysis of the diets of affected children shows thatthis is not so. Children with kwashiorkor are lessstunted than those with marasmus, and the edema beginsto improve early in treatment, when the child isstill receiving a low-protein diet. Very commonly, aninfection precipitates kwashiorkor. Superimposed ongeneral food deficiency, there is probably a deficiencyof the antioxidant nutrients such as zinc, copper,carotene, and vitamins C and E. The respiratory burstin response to infection leads to the production of oxygenand halogen free radicals as part of the cytotoxicaction of stimulated macrophages. This added oxidantstress may well trigger the development of kwashiorkor.PROTEIN & AMINO ACID REQUIREMENTSProtein Requirements Can Be Determinedby Measuring Nitrogen BalanceThe state of protein nutrition can be determined bymeasuring the dietary intake and output of nitrogenouscompounds from the body. Although nucleic acids alsocontain nitrogen, protein is the major dietary source ofnitrogen and measurement of total nitrogen intakegives a good estimate of protein intake (mg N × 6.25 =mg protein, as nitrogen is 16% of most proteins). Theoutput of nitrogen from the body is mainly in urea andsmaller quantities of other compounds in urine andundigested protein in feces, and significant amountsmay also be lost in sweat and shed skin.The difference between intake and output of nitrogenouscompounds is known as nitrogen balance. Threestates can be defined: In a healthy adult, nitrogen balanceis in equilibrium when intake equals output, andthere is no change in the total body content of protein.In a growing child, a pregnant woman, or in recoveryfrom protein loss, the excretion of nitrogenous compoundsis less than the dietary intake and there is net retentionof nitrogen in the body as protein, ie, positivenitrogen balance. In response to trauma or infection—or if the intake of protein is inadequate to meet requirements—thereis net loss of protein nitrogen from thebody, ie, negative nitrogen balance. The continual catabolismof tissue proteins creates the requirement fordietary protein even in an adult who is not growing,though some of the amino acids released can be reutilized.Nitrogen balance studies show that the averagedaily requirement is 0.6 g of protein per kilogram ofbody weight (the factor 0.75 should be used to allow forindividual variation), or approximately 50 g/d. Averageintakes of protein in developed countries are about80–100 g/d, ie, 14–15% of energy intake. Because

480 / CHAPTER 44growing children are increasing the protein in the body,they have a proportionately greater requirement thanadults and should be in positive nitrogen balance. Evenso, the need is relatively small compared with the requirementfor protein turnover. In some countries, proteinintake may be inadequate to meet these requirements,resulting in stunting of growth.There Is a Loss of Body Protein inResponse to Trauma & InfectionOne of the metabolic reactions to major trauma, suchas a burn, a broken limb, or surgery, is an increase inthe net catabolism of tissue proteins. As much as 6–7%of the total body protein may be lost over 10 days. Prolongedbed rest results in considerable loss of proteinbecause of atrophy of muscles. Protein is catabolized asnormal, but without the stimulus of exercise it is notcompletely replaced. Lost protein is replaced duringconvalescence, when there is positive nitrogen balance.A normal diet is adequate to permit this replacement.The Requirement Is Not for Protein Itselfbut for Specific Amino AcidsNot all proteins are nutritionally equivalent. More ofsome than of others is needed to maintain nitrogenbalance because different proteins contain differentamounts of the various amino acids. The body’s requirementis for specific amino acids in the correctproportions to replace the body proteins. The aminoacids can be divided into two groups: essential andnonessential. There are nine essential or indispensableamino acids, which cannot be synthesized in the body:histidine, isoleucine, leucine, lysine, methionine, phenylalanine,threonine, tryptophan, and valine. If one ofthese is lacking or inadequate, then—regardless of thetotal intake of protein—it will not be possible to maintainnitrogen balance since there will not be enough ofthat amino acid for protein synthesis.Two amino acids—cysteine and tyrosine—can besynthesized in the body, but only from essential aminoacid precursors (cysteine from methionine and tyrosinefrom phenylalanine). The dietary intakes of cysteineand tyrosine thus affect the requirements for methionineand phenylalanine. The remaining 11 amino acidsin proteins are considered to be nonessential or dispensable,since they can be synthesized as long as there isenough total protein in the diet—ie, if one of theseamino acids is omitted from the diet, nitrogen balancecan still be maintained. However, only three aminoacids—alanine, aspartate, and glutamate—can be consideredto be truly dispensable; they are synthesizedfrom common metabolic intermediates (pyruvate, oxaloacetate,and α-ketoglutarate, respectively). The remainingamino acids are considered as nonessential, butunder some circumstances the requirement for themmay outstrip the organism’s capacity for synthesis.SUMMARY• Digestion involves hydrolyzing food molecules intosmaller molecules for absorption through thegastrointestinal epithelium. Polysaccharides areabsorbed as monosaccharides; triacylglycerols as2-monoacylglycerols, fatty acids, and glycerol; andproteins as amino acids.• Digestive disorders arise as a result of (1) enzyme deficiency,eg, lactase and sucrase; (2) malabsorption,eg, of glucose and galactose due to defects in theNa + -glucose cotransporter (SGLT 1); (3) absorptionof unhydrolyzed polypeptides, leading to immunologicresponses, eg, as in celiac disease; and (4) precipitationof cholesterol from bile as gallstones.• Besides water, the diet must provide metabolic fuels(carbohydrate and fat) for bodily growth and activity;protein for synthesis of tissue proteins; fiber forroughage; minerals for specific metabolic functions;certain polyunsaturated fatty acids of the n-3 and n-6families for eicosanoid synthesis and other functions;and vitamins, organic compounds needed in smallamounts for many varied essential functions.• Twenty different amino acids are required for proteinsynthesis, of which nine are essential in thehuman diet. The quantity of protein required is affectedby protein quality, energy intake, and physicalactivity.• Undernutrition occurs in two extreme forms: marasmusin adults and children and kwashiorkor in children.Overnutrition from excess energy intake is associatedwith diseases such as obesity, type 2 diabetesmellitus, atherosclerosis, cancer, and hypertension.REFERENCESBender DA, Bender AE: Nutrition: A Reference Handbook. OxfordUniv Press, 1997.Büller HA, Grand RJ: Lactose intolerance. Annu Rev Med1990;41:141.Fuller MF, Garlick PJ: Human amino acid requirements. AnnuRev Nutr 1994;14:217.Garrow JS, James WPT, Ralph A: Human Nutrition and Dietetics,10th ed. Churchill-Livingstone, 2000.National Academy of Sciences report on diet and health. Nutr Rev1989;47:142.Nielsen FH: Nutritional significance of the ultratrace elements.Nutr Rev 1988;46:337.

Vitamins & Minerals 45David A. Bender, PhD, & Peter A. Mayes, PhD, DScBIOMEDICAL IMPORTANCE481Vitamins are a group of organic nutrients required insmall quantities for a variety of biochemical functionsand which, generally, cannot be synthesized by thebody and must therefore be supplied in the diet.The lipid-soluble vitamins are apolar hydrophobiccompounds that can only be absorbed efficiently whenthere is normal fat absorption. They are transported inthe blood, like any other apolar lipid, in lipoproteins orattached to specific binding proteins. They have diversefunctions, eg, vitamin A, vision; vitamin D, calciumand phosphate metabolism; vitamin E, antioxidant; vitaminK, blood clotting. As well as dietary inadequacy,conditions affecting the digestion and absorption of thelipid-soluble vitamins—such as steatorrhea and disordersof the biliary system—can all lead to deficiencysyndromes, including: night blindness and xerophthalmia(vitamin A); rickets in young children and osteomalaciain adults (vitamin D); neurologic disordersand anemia of the newborn (vitamin E); and hemorrhageof the newborn (vitamin K). Toxicity can resultfrom excessive intake of vitamins A and D. Vitamin Aand β-carotene (provitamin A), as well as vitamin E, areantioxidants and have possible roles in atherosclerosisand cancer prevention.The water-soluble vitamins comprise the B complexand vitamin C and function as enzyme cofactors. Folicacid acts as a carrier of one-carbon units. Deficiency ofa single vitamin of the B complex is rare, since poordiets are most often associated with multiple deficiencystates. Nevertheless, specific syndromes are characteristicof deficiencies of individual vitamins, eg, beriberi(thiamin); cheilosis, glossitis, seborrhea (riboflavin);pellagra (niacin); peripheral neuritis (pyridoxine);megaloblastic anemia, methylmalonic aciduria, andpernicious anemia (vitamin B 12 ); and megaloblasticanemia (folic acid). Vitamin C deficiency leads toscurvy.Inorganic mineral elements that have a function inthe body must be provided in the diet. When the intakeis insufficient, deficiency symptoms may arise, eg, anemia(iron), cretinism and goiter (iodine). If present inexcess as with selenium, toxicity symptoms may occur.THE DETERMINATION OF NUTRIENTREQUIREMENTS DEPENDS ON THECRITERIA OF ADEQUACY CHOSENFor any nutrient, particularly minerals and vitamins,there is a range of intakes between that which is clearlyinadequate, leading to clinical deficiency disease, andthat which is so much in excess of the body’s metaboliccapacity that there may be signs of toxicity. Betweenthese two extremes is a level of intake that is adequatefor normal health and the maintenance of metabolic integrity.Individuals do not all have the same requirementfor nutrients even when calculated on the basis ofbody size or energy expenditure. There is a range of individualrequirements of up to 25% around the mean.Therefore, in order to assess the adequacy of diets, it isnecessary to set a reference level of intake high enoughto ensure that no one will either suffer from deficiencyor be at risk of toxicity. If it is assumed that individualrequirements are distributed in a statistically normalfashion around the observed mean requirement, then arange of +/− 2 × the standard deviation (SD) aroundthe mean will include the requirements of 95% of thepopulation.THE VITAMINS ARE A DISPARATE GROUPOF COMPOUNDS WITH A VARIETYOF METABOLIC FUNCTIONSA vitamin is defined as an organic compound that is requiredin the diet in small amounts for the maintenanceof normal metabolic integrity. Deficiency causes a specificdisease, which is cured or prevented only by restoringthe vitamin to the diet (Table 45–1). However, vitaminD, which can be made in the skin after exposureto sunlight, and niacin, which can be formed from theessential amino acid tryptophan, do not strictly conformto this definition.

482 / CHAPTER 45Table 45–1. The vitamins.Vitamin Functions Deficiency DiseaseA Retinol, β-carotene Visual pigments in the retina; regulation of Night blindness, xerophthalmia;gene expression and cell differentiation;keratinization of skinβ-carotene is an antioxidantD Calciferol Maintenance of calcium balance; enhances Rickets = poor mineralization of bone;intestinal absorption of Ca 2+ and mobilizes osteomalacia = bone demineralizationbone mineralE Tocopherols, tocotrienols Antioxidant, especially in cell membranes Extremely rare—serious neurologicdysfunctionK Phylloquinone, Coenzyme in formation of γ -carboxyglutamate Impaired blood clotting, hemormenaquinonesin enzymes of blood clotting and bone matrix rhagic diseaseB 1 Thiamin Coenzyme in pyruvate and α−ketoglutarate, Peripheral nerve damage (beriberi) ordehydrogenases, and transketolase; poorly central nervous system lesionsdefined function in nerve conduction(Wernicke-Korsakoff syndrome)B 2 Riboflavin Coenzyme in oxidation and reduction reactions; Lesions of corner of mouth, lips, andprosthetic group of flavoproteinstongue; seborrheic dermatitisNiacin Nicotinic acid, Coenzyme in oxidation and reduction reactions, Pellagra—photosensitive dermatitis,nicotinamide functional part of NAD and NADP depressive psychosisB 6 Pyridoxine, pyridoxal, Coenzyme in transamination and decarboxy- Disorders of amino acid metabolism,pyridoxamine lation of amino acids and glycogen convulsionsphosphorylase; role in steroid hormone actionFolic acid Coenzyme in transfer of one-carbon fragments Megaloblastic anemiaB 12 Cobalamin Coenzyme in transfer of one-carbon fragments Pernicious anemia = megaloblasticand metabolism of folic acidanemia with degeneration of thespinal cordPantothenic acidFunctional part of CoA and acyl carrier protein:fatty acid synthesis and metabolismH Biotin Coenzyme in carboxylation reactions in gluco- Impaired fat and carbohydrate metabneogenesisand fatty acid synthesisolism, dermatitisC Ascorbic acid Coenzyme in hydroxylation of proline and Scurvy—impaired wound healing,lysine in collagen synthesis; antioxidant;loss of dental cement, subcutaneousenhances absorption of ironhemorrhageLIPID-SOLUBLE VITAMINSRETINOIDS & CAROTENOIDSHAVE VITAMIN A ACTIVITY(Figure 45–1)Retinoids comprise retinol, retinaldehyde, andretinoic acid (preformed vitamin A, found only infoods of animal origin); carotenoids, found in plants,comprise carotenes and related compounds, known asprovitamin A, as they can be cleaved to yield retinaldehydeand thence retinol and retinoic acid. The α-, β-,and γ-carotenes and cryptoxanthin are quantitativelythe most important provitamin A carotenoids. Althoughit would appear that one molecule of β-carotene should yield two of retinol, this is not so inpractice; 6 µg of β-carotene is equivalent to 1 µg ofpreformed retinol. The total amount of vitamin A infoods is therefore expressed as micrograms of retinolequivalents. Beta-carotene and other provitamin Acarotenoids are cleaved in the intestinal mucosa bycarotene dioxygenase, yielding retinaldehyde, which isreduced to retinol, esterified, and secreted in chylomi-

VITAMINS & MINERALS / 483H 3 CCH 3CH 3 CH 3H 3 CCH 3HCH CH 3 C CH 33 3β-CaroteneH 3 CCH 3CH 3 CH 3CH 2 OHH 3 CCH 3CH 3 CH 3CHOCH 3RetinolCH 3RetinaldehydeFigure 45–1. β-Carotene and the major vitaminA vitamers. * Shows the site of cleavage ofβ-carotene into two molecules of retinaldehydeby carotene dioxygenase.H 3 CCHCH 3 CH 3 CH3H CHCOOH 3 C33CH 3All-trans-retinoic acidCH 3H 3 C9-cis-retinoic acidCOOHcrons together with esters formed from dietary retinol.The intestinal activity of carotene dioxygenase is low,so that a relatively large proportion of ingested β-carotene may appear in the circulation unchanged.While the principal site of carotene dioxygenase attackis the central bond of β-carotene, asymmetric cleavagemay also occur, leading to the formation of 8′-, 10′-,and 12′-apo-carotenals, which are oxidized to retinoicacid but cannot be used as sources of retinol or retinaldehyde.Vitamin A Has a Function in VisionIn the retina, retinaldehyde functions as the prostheticgroup of the light-sensitive opsin proteins, formingrhodopsin (in rods) and iodopsin (in cones). Any onecone cell contains only one type of opsin and is sensitiveto only one color. In the pigment epithelium of theretina, all-trans-retinol is isomerized to 11-cis-retinoland oxidized to 11-cis-retinaldehyde. This reacts with alysine residue in opsin, forming the holoproteinrhodopsin. As shown in Figure 45–2, the absorption oflight by rhodopsin causes isomerization of the retinaldehydefrom 11-cis to all-trans, and a conformationalchange in opsin. This results in the release of retinaldehydefrom the protein and the initiation of a nerve impulse.The formation of the initial excited form ofrhodopsin, bathorhodopsin, occurs within picosecondsof illumination. There is then a series of conformationalchanges leading to the formation of metarhodopsin II,which initiates a guanine nucleotide amplification cascadeand then a nerve impulse. The final step is hydrolysisto release all-trans-retinaldehyde and opsin. The keyto initiation of the visual cycle is the availability of11-cis-retinaldehyde, and hence vitamin A. In deficiency,both the time taken to adapt to darkness and theability to see in poor light are impaired.Retinoic Acid Has a Rolein the Regulation of GeneExpression & Tissue DifferentiationA most important function of vitamin A is in the controlof cell differentiation and turnover. All-transretinoicacid and 9-cis-retinoic acid (Figure 45–1) regulategrowth, development, and tissue differentiation;they have different actions in different tissues. Like thesteroid hormones and vitamin D, retinoic acid binds tonuclear receptors that bind to response elements ofDNA and regulate the transcription of specific genes.There are two families of nuclear retinoid receptors: theretinoic acid receptors (RARs) bind all-trans-retinoicacid or 9-cis-retinoic acid, and the retinoid X receptors(RXRs) bind 9-cis-retinoic acid.Vitamin A Deficiency Is a Major PublicHealth Problem WorldwideVitamin A deficiency is the most important preventablecause of blindness. The earliest sign of deficiency is aloss of sensitivity to green light, followed by impairmentof adaptation to dim light, followed by nightblindness. More prolonged deficiency leads to xerophthalmia:keratinization of the cornea and skin andblindness. Vitamin A also has an important role in differentiationof immune system cells, and mild deficiencyleads to increased susceptibility to infectious diseases.Furthermore, the synthesis of retinol-bindingprotein in response to infection is reduced (it is a negativeacute phase protein), decreasing the circulating vi-

484 / CHAPTER 45H 3 CH 3 CH 3 CH 3 CCH 3CH 3CH 3CH 3 CH 3CH 3CH 3 H 3 CCH 3CH 3CH 3 H 3 CCH 3CH 3CHH 3 C CH 3 CH 33CH 3PhotorhodopsinCH 2 OHCH 3 H 3 CHC=NRhodopsin (visual purple)Conformational changes in proteinLIGHTH 3 CCH 3CH 310 -15 sec45 psecBathorhodopsin30 nsecLumirhodopsin75 µsecMetarhodopsin I10 msecMetarhodopsin IIminutesMetarhodopsin IIIFigure 45–2.GDPGTPCH 3 CH 3CH 2 OHHC=OC=OH 2 NLysine residuein opsin NHC=NAll-trans-retinol11-cis-RetinolcGMPNa+channel openHC=O11-cis-RetinaldehydeInactiveC=Otamin, and therefore there is further impairment of immuneresponses.Vitamin A Is Toxic in ExcessThere is only a limited capacity to metabolize vitaminA, and excessive intakes lead to accumulation beyondthe capacity of binding proteins, so that unbound vitaminA causes tissue damage. Symptoms of toxicity affectthe central nervous system (headache, nausea,NHAll-trans-retinaldehyde + opsinC=ONHTransducin-GTPTransducin-GDP5'GMP+Na channel closedActivephosphodiesteraseThe role of retinaldehyde in vision.P iataxia, and anorexia, all associated with increased cerebrospinalfluid pressure), the liver (hepatomegaly withhistologic changes and hyperlipidemia), calcium homeostasis(thickening of the long bones, hypercalcemiaand calcification of soft tissues), and the skin (excessivedryness, desquamation, and alopecia).VITAMIN D IS REALLY A HORMONEVitamin D is not strictly a vitamin since it can be synthesizedin the skin, and under most conditions that isits major source. Only when sunlight is inadequate is adietary source required. The main function of vitaminD is in the regulation of calcium absorption and homeostasis;most of its actions are mediated by wayof nuclear receptors that regulate gene expression.Deficiency—leading to rickets in children and osteomalaciain adults—continues to be a problem in northernlatitudes, where sunlight exposure is poor.Vitamin D Is Synthesized in the Skin7-Dehydrocholesterol (an intermediate in the synthesisof cholesterol that accumulates in the skin), undergoesa nonenzymic reaction on exposure to ultraviolet light,yielding previtamin D (Figure 45–3). This undergoes afurther reaction over a period of hours to form the vitaminitself, cholecalciferol, which is absorbed into thebloodstream. In temperate climates, the plasma concentrationof vitamin D is highest at the end of summerand lowest at the end of winter. Beyond about 40 degreesnorth or south in winter, there is very little ultravioletradiation of appropriate wavelength.Vitamin D Is Metabolized to the ActiveMetabolite, Calcitriol, in Liver & KidneyIn the liver, cholecalciferol, which has been synthesizedin the skin or derived from food, is hydroxylated toform the 25-hydroxy derivative calcidiol (Figure 45–4).This is released into the circulation bound to a vitaminD-binding globulin which is the main storage form ofthe vitamin. In the kidney, calcidiol undergoes either1-hydroxylation to yield the active metabolite 1,25-dihydroxyvitaminD (calcitriol) or 24-hydroxylation to yieldan inactive metabolite, 24,25-dihydroxyvitamin D (24-hydroxycalcidiol). Ergocalciferol from fortified foodsundergoes similar hydroxylations to yield ercalcitriol.Vitamin D Metabolism Both Regulates& Is Regulated by Calcium HomeostasisThe main function of vitamin D is in the control of calciumhomeostasis, and in turn vitamin D metabolism is

VITAMINS & MINERALS / 485OHLIGHTThermal isomerizationHOCH 37-DehydrocholesterolPrevitamin DHOFigure 45–3. Synthesis of vitamin D in the skin.Cholecalciferol(calciol;vitamin D 3 )CH 2regulated by factors that respond to plasma concentrationsof calcium and phosphate. Calcitriol acts to reduceits own synthesis by inducing the 24-hydroxylase andrepressing the 1-hydroxylase in the kidney. Its principalfunction is to maintain the plasma calcium concentration.Calcitriol achieves this in three ways: it increasesintestinal absorption of calcium, reduces excretion ofcalcium (by stimulating resorption in the distal renaltubules), and mobilizes bone mineral. In addition, calcitriolis involved in insulin secretion, synthesis and secretionof parathyroid and thyroid hormones, inhibitionof production of interleukin by activated T lymphocytesand of immunoglobulin by activated B lymphocytes,differentiation of monocyte precursor cells, and modulationof cell proliferation. In its actions, it behaves likea steroid hormone, binding to a nuclear receptorprotein.Vitamin D Deficiency AffectsChildren & AdultsIn the vitamin D deficiency disease rickets, the bonesof children are undermineralized as a result of poor absorptionof calcium. Similar problems occur in adolescentswho are deficient during their growth spurt. Osteomalaciain adults results from demineralization ofbone in women who have little exposure to sunlight,often after several pregnancies. Although vitamin D isessential for prevention and treatment of osteomalaciain the elderly, there is little evidence that it is beneficialin treating osteoporosis.Vitamin D Is Toxic in ExcessSome infants are sensitive to intakes of vitamin D aslow as 50 µg/d, resulting in an elevated plasma concen-OHOHCalciol-25-hydroxylaseCalcidiol-1-hydroxylaseHOCH 2Cholecalciferol(calciol;vitamin D 3 )HOCH 2 CH 2Calcidiol(25-hydroxycholecalciferol)HOCalcitriol(1,25-hydroxycholecalciferol)OHCalcidiol-24-hydroxylaseOHCalcidiol-24-hydroxylaseOHOHOHCalcidiol-1-hydroxylaseFigure 45–4. Metabolism of vitamin D.CH 224-hydroxycalcidiolHOHOCH 2CalcitetrolOH

486 / CHAPTER 45tration of calcium. This can lead to contraction ofblood vessels, high blood pressure, and calcinosis—thecalcification of soft tissues. Although excess dietary vitaminD is toxic, excessive exposure to sunlight does notlead to vitamin D poisoning because there is a limitedcapacity to form the precursor 7-dehydrocholesteroland to take up cholecalciferol from the skin.VITAMIN E DOES NOT HAVE A PRECISELYDEFINED METABOLIC FUNCTIONNo unequivocal unique function for vitamin E hasbeen defined. However, it does act as a lipid-soluble antioxidantin cell membranes, where many of its functionscan be provided by synthetic antioxidants. VitaminE is the generic descriptor for two families ofcompounds, the tocopherols and the tocotrienols (Figure45–5). The different vitamers (compounds havingsimilar vitamin activity) have different biologic potencies;the most active is D-α-tocopherol, and it is usualto express vitamin E intake in milligrams of D-α-tocopherolequivalents. Synthetic DL-α-tocopherol does nothave the same biologic potency as the naturally occurringcompound.Vitamin E Is the Major Lipid-SolubleAntioxidant in Cell Membranes& Plasma LipoproteinsThe main function of vitamin E is as a chain-breaking,free radical trapping antioxidant in cell membranesand plasma lipoproteins. It reacts with the lipidperoxide radicals formed by peroxidation of polyunsaturatedfatty acids before they can establish a chainreaction. The tocopheroxyl free radical product is relativelyunreactive and ultimately forms nonradicalcompounds. Commonly, the tocopheroxyl radical isHOR 2HOR 2R 1OR 3CH 3R 1OR 3CH 3TocopherolTocotrienolFigure 45–5. The vitamin E vitamers. In α-tocopheroland tocotrienol R 1 , R 2 , and R 3 are all ⎯CH 3 groups.In the β-vitamers R 2 is H; in the γ-vitamers R 1 is H, and inthe δ-vitamers R 1 and R 2 are both H.reduced back to tocopherol by reaction with vitaminC from plasma (Figure 45–6). The resultant monodehydroascorbatefree radical then undergoes enzymic ornonenzymic reaction to yield ascorbate and dehydroascorbate,neither of which is a free radical. Thestability of the tocopheroxyl free radical means that itcan penetrate farther into cells and, potentially, propagatea chain reaction. Therefore, vitamin E may, likeother antioxidants, also have pro-oxidant actions, especiallyat high concentrations. This may explain why,although studies have shown an association betweenhigh blood concentrations of vitamin E and a lowerincidence of atherosclerosis, the effect of high doses ofvitamin E have been disappointing.Dietary Vitamin E Deficiencyin Humans Is UnknownIn experimental animals, vitamin E deficiency results inresorption of fetuses and testicular atrophy. Dietary deficiencyof vitamin E in humans is unknown, thoughpatients with severe fat malabsorption, cystic fibrosis,and some forms of chronic liver disease suffer deficiencybecause they are unable to absorb the vitamin ortransport it, exhibiting nerve and muscle membranedamage. Premature infants are born with inadequate reservesof the vitamin. Their erythrocyte membranes areabnormally fragile as a result of peroxidation, whichleads to hemolytic anemia.VITAMIN K IS REQUIRED FOR SYNTHESISOF BLOOD-CLOTTING PROTEINSVitamin K was discovered as a result of investigationsinto the cause of a bleeding disorder—hemorrhagic(sweet clover) disease—of cattle, and of chickens fed ona fat-free diet. The missing factor in the diet of thechickens was vitamin K, while the cattle feed containeddicumarol, an antagonist of the vitamin. Antagonistsof vitamin K are used to reduce blood coagulation inpatients at risk of thrombosis—the most widely usedagent is warfarin.Three compounds have the biologic activity of vitaminK (Figure 45–7): phylloquinone, the normal dietarysource, found in green vegetables; menaquinones,synthesized by intestinal bacteria, with differinglengths of side-chain; menadione, menadiol, andmenadiol diacetate, synthetic compounds that can bemetabolized to phylloquinone. Menaquinones are absorbedto some extent but it is not clear to what extentthey are biologically active as it is possible to inducesigns of vitamin K deficiency simply by feeding a phylloquinonedeficient diet, without inhibiting intestinalbacterial action.

VITAMINS & MINERALS / 487Free radicalchain reactionPUFAOO PUFA OOHROR2TocOHTocOPHOSPHOLIPASEA 2MEMBRANESCYTOSOLPUFA H(in phospholipid)Vitamin C ox ,GS SGVitamin C red ,GSHPUFA OOH,H 2 O 2GSHSUPEROXIDEDISMUTASECATALASESeGLUTATHIONEPEROXIDASE–O 2SuperoxideH 2 O,PUFA OHFigure 45–6. Interaction and synergism between antioxidant systems operating in the lipidphase (membranes) of the cell and the aqueous phase (cytosol). (R•, free radical; PUFA-OO•, peroxylfree radical of polyunsaturated fatty acid in membrane phospholipid; PUFA-OOH, hydroperoxypolyunsaturated fatty acid in membrane phospholipid released as hydroperoxy free fatty acid intocytosol by the action of phospholipase A 2 ; PUFA-OH, hydroxy polyunsaturated fatty acid; TocOH,vitamin E (α-tocopherol); TocO•, free radical of α-tocopherol; Se, selenium; GSH, reduced glutathione;GS-SG, oxidized glutathione, which is returned to the reduced state after reaction withNADPH catalyzed by glutathione reductase; PUFA-H, polyunsaturated fatty acid.)GSSGVitamin K Is the Coenzymefor Carboxylation of Glutamatein the Postsynthetic Modificationof Calcium-Binding ProteinsVitamin K is the cofactor for the carboxylation of glutamateresidues in the post-synthetic modification of proteinsto form the unusual amino acid γ-carboxyglutamate(Gla), which chelates the calcium ion. Initially,vitamin K hydroquinone is oxidized to the epoxide(Figure 45–8), which activates a glutamate residue inthe protein substrate to a carbanion, that reacts nonenzymicallywith carbon dioxide to form γ-carboxyglutamate.Vitamin K epoxide is reduced to the quinone bya warfarin-sensitive reductase, and the quinone is reducedto the active hydroquinone by either the samewarfarin-sensitive reductase or a warfarin-insensitivequinone reductase. In the presence of warfarin, vitaminK epoxide cannot be reduced but accumulates, and isexcreted. If enough vitamin K (a quinone) is providedin the diet, it can be reduced to the active hydroquinoneby the warfarin-insensitive enzyme, and carboxylationcan continue, with stoichiometric utilizationof vitamin K and excretion of the epoxide. A high doseof vitamin K is the antidote to an overdose of warfarin.Prothrombin and several other proteins of the bloodclotting system (Factors VII, IX and X, and proteins Cand S) each contain between four and six γ-carboxyglutamateresidues which chelate calcium ions and so permitthe binding of the blood clotting proteins to membranes.In vitamin K deficiency or in the presence of warfarin, anabnormal precursor of prothrombin (preprothrombin)containing little or no γ-carboxyglutamate, and incapableof chelating calcium, is released into the circulation.

488 / CHAPTER 45OCH 3HO 3PhylloquinoneOOMenaquinoneOHOHMenadiolCH 3CH 3HnCH 3CH 3CH 3Menadiolo diacetate(acetomenaphthone)Figure 45–7. The vitamin K vitamers. Menadiol (ormenadione) and menadiol diacetate are synthetic compoundsthat are converted to menaquinone in the liverand have vitamin K activity.COOCOOVitamin K Is Also Importantin the Synthesis of BoneCalcium-Binding ProteinsTreatment of pregnant women with warfarin can lead tofetal bone abnormalities (fetal warfarin syndrome). Twoproteins are present in bone that contain γ-carboxyglutamate,osteocalcin and bone matrix Gla protein. Osteocalcinalso contains hydroxyproline, so its synthesis is dependenton both vitamins K and C; in addition, its synthesisis induced by vitamin D. The release into the circulationof osteocalcin provides an index of vitamin D status.WATER-SOLUBLE VITAMINSVITAMIN B 1 (THIAMIN) HAS A KEY ROLEIN CARBOHYDRATE METABOLISMThiamin has a central role in energy-yielding metabolism,and especially the metabolism of carbohydrate(Figure 45–9). Thiamin diphosphate is the coenzymefor three multi-enzyme complexes that catalyze oxidativedecarboxylation reactions: pyruvate dehydrogenasein carbohydrate metabolism; α-ketoglutarate dehydro-OOCCHCH 2COOHNCHCOCarboxyglutamate residueCH 2CH 2HN CH CGlutamate residueOHOHCOOCH 3O 2VITAMIN KEPOXIDASEVitamin K hydroquinoneDisulfideNADP+VITAMIN K QUINONEQUINONEREDUCTASE OREDUCTASESulfhydrylNADPHORCH 3nonenzymicCO 2CH COOCH 2HN CH CGlutamate carbanionOOCH 3ORVitamin K epoxideOSulfhydrylVITAMIN K EPOXIDEREDUCTASEDisulfideROVitamin K quinoneFigure 45–8. The role of vitamin K in thebiosynthesis of γ-carboxyglutamate.

VITAMINS & MINERALS / 489H 3 CNNH 3 C NH 2CH 2NSCH 2CH 2 OHO OCH 2 CH 2 O P O P OO OH 3 CNCH 2S+HThiamin Thiamin diphosphate CarbanionFigure 45–9.Thiamin, thiamin diphosphate, and the carbanion form.genase in the citric acid cycle; and the branched-chainketo-acid dehydrogenase involved in the metabolism ofleucine, isoleucine, and valine. It is also the coenzymefor transketolase, in the pentose phosphate pathway. Ineach case, the thiamin diphosphate provides a reactivecarbon on the thiazole moiety that forms a carbanion,which then adds to the carbonyl group of, for instance,pyruvate. The addition compound then decarboxylates,eliminating CO 2 . Electrical stimulation of nerve leadsto a fall in membrane thiamin triphosphate and releaseof free thiamin. It is likely that thiamin triphosphateacts as a phosphate donor for phosphorylation of thenerve membrane sodium transport channel.Thiamin Deficiency Affectsthe Nervous System & HeartThiamin deficiency can result in three distinct syndromes:a chronic peripheral neuritis, beriberi, whichmay or may not be associated with heart failure andedema; acute pernicious (fulminating) beriberi (shoshinberiberi), in which heart failure and metabolic abnormalitiespredominate, without peripheral neuritis; andWernicke’s encephalopathy with Korsakoff’s psychosis,which is associated especially with alcohol anddrug abuse. The central role of thiamin diphosphate inpyruvate dehydrogenase means that in deficiency there isimpaired conversion of pyruvate to acetyl CoA. In subjectson a relatively high carbohydrate diet, this results inincreased plasma concentrations of lactate and pyruvate,which may cause life-threatening lactic acidosis.Thiamin Nutritional Status CanBe Assessed by ErythrocyteTransketolase ActivationThe activation of apo-transketolase(the enzyme protein)in erythrocyte lysate by thiamin diphosphateadded in vitro has become the accepted index of thiaminnutritional status.VITAMIN B 2 (RIBOFLAVIN) HASA CENTRAL ROLE IN ENERGY-YIELDING METABOLISMRiboflavin fulfills its role in metabolism as the coenzymesflavin mononucleotide (FMN) and flavin adeninedinucleotide (FAD) (Figure 45–10). FMN is formed byATP-dependent phosphorylation of riboflavin, whereasFAD is synthesized by further reaction of FMN withATP in which its AMP moiety is transferred to theOHOHOHOHOHOHOCH 2CHCHCHCH 2 OHCH 2CHCHCHCH 2OPOH 3 C N NOH 3 C N NH 3 C NNH 3 C NNOORiboflavinFMNH 3 C N NOH OH OHCH 2 CH CH CH CH 2OH 3 C NNOFADO OOOH OHNNCH 2NH 2O P O P OO N NO OFigure 45–10. Riboflavin and the coenzymes flavin mononucleotide (FMN) and flavinadenine dinucleotide (FAD).O

490 / CHAPTER 45FMN. The main dietary sources of riboflavin are milkand dairy products. In addition, because of its intenseyellow color, riboflavin is widely used as a food additive.Flavin Coenzymes Are Electron Carriersin Oxidoreduction ReactionsThese include the mitochondrial respiratory chain, keyenzymes in fatty acid and amino acid oxidation, andthe citric acid cycle. Reoxidation of the reduced flavinin oxygenases and mixed-function oxidases proceeds byway of formation of the flavin radical and flavin hydroperoxide,with the intermediate generation of superoxideand perhydroxyl radicals and hydrogen peroxide.Because of this, flavin oxidases make a significant contributionto the total oxidant stress of the body.Riboflavin Deficiency IsWidespread But Not FatalAlthough riboflavin is fundamentally involved in metabolism,and deficiencies are found in most countries,it is not fatal as there is very efficient conservation oftissue riboflavin. Riboflavin deficiency is characterizedby cheilosis, lingual desquamation and a seborrheic dermatitis.Riboflavin nutritional status is assessed by measurementof the activation of erythrocyte glutathionereductase by FAD added in vitro.NIACIN IS NOT STRICTLY A VITAMINNiacin was discovered as a nutrient during studies of pellagra.It is not strictly a vitamin since it can be synthesizedin the body from the essential amino acidtryptophan. Two compounds, nicotinic acid and nicotinamide,have the biologic activity of niacin; its metabolicfunction is as the nicotinamide ring of the coenzymesNAD and NADP in oxidation-reduction reactions(Figure 45–11). About 60 mg of tryptophan is equivalentto 1 mg of dietary niacin. The niacin content of foods isexpressed as mg niacin equivalents = mg preformed niacin+ 1/60 × mg tryptophan. Because most of the niacin incereals is biologically unavailable, this is discounted.NAD Is the Source of ADP-RiboseIn addition to its coenzyme role, NAD is the source ofADP-ribose for the ADP-ribosylation of proteins andpolyADP-ribosylation of nucleoproteins involved in theDNA repair mechanism.Pellagra Is Caused by Deficiencyof Tryptophan & NiacinPellagra is characterized by a photosensitive dermatitis.As the condition progresses, there is dementia, possibly+NCONH 2OH OHOCH 2NCOONicotinic acidO OO P O P OO ONADOH OHdiarrhea, and, if untreated, death. Although the nutritionaletiology of pellagra is well established and tryptophanor niacin will prevent or cure the disease, additionalfactors, including deficiency of riboflavin orvitamin B 6 , both of which are required for synthesis ofnicotinamide from tryptophan, may be important. Inmost outbreaks of pellagra twice as many women asmen are affected, probably the result of inhibition oftryptophan metabolism by estrogen metabolites.Pellagra Can Occur as a Resultof Disease Despite an AdequateIntake of Tryptophan & NiacinA number of genetic diseases that result in defects oftryptophan metabolism are associated with the developmentof pellagra despite an apparently adequate intakeof both tryptophan and niacin. Hartnup disease is arare genetic condition in which there is a defect of themembrane transport mechanism for tryptophan, resultingin large losses due to intestinal malabsorption andfailure of the renal resorption mechanism. In carcinoidsyndrome there is metastasis of a primary liver tumorof enterochromaffin cells which synthesize 5-hydroxytryptamine.Overproduction of 5-hydroxytryptaminemay account for as much as 60% of the body’s tryptophanmetabolism, causing pellagra because of the diversionaway from NAD synthesis.Niacin Is Toxic in ExcessNicotinic acid has been used to treat hyperlipidemiawhen of the order of 1–6 g/d are required, causing dilationof blood vessels and flushing, with skin irritation.Intakes of both nicotinic acid and nicotinamide in excessof 500 mg/d can cause liver damage.NNicotinamideNNNNCH 2NH 2OCONH 2Figure 45–11. Niacin (nicotinic acid and nicotinamide)and nicotinamide adenine dinucleotide (NAD).* Shows the site of phosphorylation in NADP.

VITAMINS & MINERALS / 491VITAMIN B 6 IS IMPORTANT IN AMINOACID & GLYCOGEN METABOLISM& IN STEROID HORMONE ACTIONSix compounds have vitamin B 6 activity (Figure 45–12):pyridoxine, pyridoxal, pyridoxamine, and their 5′-phosphates. The active coenzyme is pyridoxal 5′-phosphate.Approximately 80% of the body’s total vitamin B 6is present as pyridoxal phosphate in muscle, mostly associatedwith glycogen phosphorylase. This is not availablein B 6 deficiency but is released in starvation, when glycogenreserves become depleted, and is then available, especiallyin liver and kidney, to meet increased requirementfor gluconeogenesis from amino acids.Vitamin B 6 Has Several Rolesin MetabolismPyridoxal phosphate is a coenzyme for many enzymesinvolved in amino acid metabolism, especially intransamination and decarboxylation. It is also the cofactorof glycogen phosphorylase, where the phosphategroup is catalytically important. In addition, vitamin B 6is important in steroid hormone action where it removesthe hormone-receptor complex from DNAbinding, terminating the action of the hormones. In vitaminB 6 deficiency, this results in increased sensitivityto the actions of low concentrations of estrogens, androgens,cortisol, and vitamin D.HOCH 2HOCH 2HOCH 2CH 2 OHNOHCH 3PyridoxineHC=ONPyridoxalOHCH 3CH 2 NH 2OHNCH 3PyridoxamineKINASEPHOSPHATASEKINASEPHOSPHATASEOOOPOCH 2CH 2 OHOHOCH 3Pyridoxine phosphateOPOCH 2AMINOTRANSFERASESKINASEPHOSPHATASEONHC=OOHOCH 3Pyridoxal phosphateOPOCH 2NOXIDASECH 2 NH 2OHOCH 3Pyridoxamine phosphateFigure 45–12. Interconversion of the vitamin B 6vitamers.NOXIDASEVitamin B 6 Deficiency Is RareAlthough clinical deficiency disease is rare, there is evidencethat a significant proportion of the populationhave marginal vitamin B 6 status. Moderate deficiencyresults in abnormalities of tryptophan and methioninemetabolism. Increased sensitivity to steroid hormone actionmay be important in the development of hormonedependentcancer of the breast, uterus, and prostate,and vitamin B 6 status may affect the prognosis.Vitamin B 6 Status Is Assessed by AssayingErythrocyte AminotransferasesThe most widely used method of assessing vitamin B 6status is by the activation of erythrocyte aminotransferasesby pyridoxal phosphate added in vitro, expressedas the activation coefficient.In Excess, Vitamin B 6 CausesSensory NeuropathyThe development of sensory neuropathy has been reportedin patients taking 2–7 g of pyridoxine per day fora variety of reasons (there is some slight evidence that itis effective in treating premenstrual syndrome). Therewas some residual damage after withdrawal of these highdoses; other reports suggest that intakes in excess of 200mg/d are associated with neurologic damage.VITAMIN B 12 IS FOUND ONLYIN FOODS OF ANIMAL ORIGINThe term “vitamin B 12 ” is used as a generic descriptorfor the cobalamins—those corrinoids (cobalt containingcompounds possessing the corrin ring) havingthe biologic activity of the vitamin (Figure 45–13).Some corrinoids that are growth factors for microorganismsnot only have no vitamin B 12 activity but mayalso be antimetabolites of the vitamin. Although it issynthesized exclusively by microorganisms, for practicalpurposes vitamin B 12 is found only in foods of animalorigin, there being no plant sources of this vitamin.This means that strict vegetarians (vegans) are atrisk of developing B 12 deficiency. The small amountsof the vitamin formed by bacteria on the surface offruits may be adequate to meet requirements, butpreparations of vitamin B 12 made by bacterial fermentationare available.Vitamin B 12 Absorption Requires TwoBinding ProteinsVitamin B 12 is absorbed bound to intrinsic factor, asmall glycoprotein secreted by the parietal cells of the

492 / CHAPTER 45H 3 CH 2 NCOCH 2 CH 2 NCH 3H 2 NCOCH R2 NCHH 33 C Co NH 3 C N CH 2 CH 2 CONH 2CHH 32 NCOCH 2 CH 3CH 2H 3 CCH 2C ONHCH 2 OCHH 3 COCH 2 CONH 2CH 2 CH 2 CONH 2HOCH 2O HOgastric mucosa. Gastric acid and pepsin release the vitaminfrom protein binding in food and make it availableto bind to cobalophilin, a binding protein secreted inthe saliva. In the duodenum, cobalophilin is hydrolyzed,releasing the vitamin for binding to intrinsicfactor. Pancreatic insufficiency can therefore be a factorin the development of vitamin B 12 deficiency, resultingin the excretion of cobalophilin-bound vitaminB 12 . Intrinsic factor binds the various vitamin B 12 vitamers,but not other corrinoids. Vitamin B 12 is absorbedfrom the distal third of the ileum via receptors thatbind the intrinsic factor-vitamin B 12 complex but notfree intrinsic factor or free vitamin.POONNCH 3CH 3Figure 45–13. Vitamin B 12 (cobalamin). R may bevaried to give the various forms of the vitamin, eg,R = CN – in cyanocobalamin; R = OH – in hydroxocobalamin;R = 5′-deoxyadenosyl in 5′-deoxyadenosylcobalamin;R = H 2 O in aquocobalamin; and R = CH 3 inmethylcobalamin.There Are Three VitaminB 12 -Dependent EnzymesMethylmalonyl CoA mutase, leucine aminomutase,and methionine synthase (Figure 45–14) are vitaminB 12 -dependent enzymes. Methylmalonyl CoA is formedas an intermediate in the catabolism of valine and bythe carboxylation of propionyl CoA arising in the catabolismof isoleucine, cholesterol, and, rarely, fattyacids with an odd number of carbon atoms—or directlyfrom propionate, a major product of microbial fermentationin ruminants. It undergoes vitamin B 12 -dependent rearrangement to succinyl-CoA, catalyzedby methylmalonyl-CoA isomerase (Figure 19–2). Theactivity of this enzyme is greatly reduced in vitamin B 12deficiency, leading to an accumulation of methylmalonyl-CoAand urinary excretion of methylmalonicacid, which provides a means of assessing vitamin B 12nutritional status.Vitamin B 12 Deficiency CausesPernicious AnemiaPernicious anemia arises when vitamin B 12 deficiencyblocks the metabolism of folic acid, leading to functionalfolate deficiency. This impairs erythropoiesis,causing immature precursors of erythrocytes to be releasedinto the circulation (megaloblastic anemia). Thecommonest cause of pernicious anemia is failure of theabsorption of vitamin B 12 rather than dietary deficiency.This can be due to failure of intrinsic factor secretioncaused by autoimmune disease of parietal cellsor to generation of anti-intrinsic factor antibodies.THERE ARE MULTIPLE FORMSOF FOLATE IN THE DIETThe active form of folic acid (pteroyl glutamate) istetrahydrofolate (Figure 45–15). The folates in foodsmay have up to seven additional glutamate residueslinked by γ-peptide bonds. In addition, all of the onecarbonsubstituted folates in Figure 45–15 may also bepresent in foods.The extent to which the different forms of folate canbe absorbed varies, and this must be allowed for in calculatingfolate intakes.SH(CH 2 ) 2H C NH +3COO –HomocysteineMethylMETHIONINESYNTHASEMethylcobalaminH B 124 folateH 3 CS(CH 2 ) 2H C NH + 3COO –MethionineH 4 folateFigure 45–14. Homocysteinuria and the folate trap.Vitamin B 12 deficiency leads to inhibition of methioninesynthase activity causing homocysteinuria and thetrapping of folate as methyltetrahydrofolate.

VITAMINS & MINERALS / 493H 2 NOHHNN5N NHHCH 2 N10OCNHTetrahydrofolate (THF)COOCHCH 2CH 2CO(Glu) nH 2 NHCOHHNNN NHOCH 2HN5-Formyl THFH 2 NOHNNHNNHHC OCH 2 N10-Formyl THFH 2 NHCOHHNNN NHNHHCH 2 N5-Formimino THFH 2 NOHNNN NHCH 2CH 2 N5,10-Methylene THFFigure 45–15. Tetrahydrofolic acid and theone-carbon substituted folates.H 2 NCH 3OHH HN CH NN2N N5-Methyl THFHH 2 NOHNN+N NHCHCH 2N5,10-Methenyl THFTetrahydrofolate Is a Carrierof One-Carbon UnitsTetrahydrofolate can carry one-carbon fragments attachedto N-5 (formyl, formimino, or methyl groups),N-10 (formyl group), or bridging N-5 to N-10 (methyleneor methenyl groups). 5-Formyl-tetrahydrofolate ismore stable than folate and is therefore used pharmaceuticallyin the agent known as folinic acid and in thesynthetic (racemic) compound leucovorin.The major point of entry for one-carbon fragmentsinto substituted folates is methylene tetrahydrofolate(Figure 45–16), which is formed by the reaction ofglycine, serine, and choline with tetrahydrofolate. Serineis the most important source of substituted folatesfor biosynthetic reactions, and the activity of serine hy-Sources of one-carbon unitsSynthesis using one-carbon unitsSerineGlycineMethylene-THFSerineMethyl-THFMethionineCholineTMP + dihydrofolateHistidineFormimino-THFMethenyl-THFDNAFormyl-methionineFormateFormyl-THFPurinesCO 2Figure 45–16.Sources and utilization of one-carbon substituted folates.

494 / CHAPTER 45droxymethyltransferase is regulated by the state of folatesubstitution and the availability of folate. The reaction isreversible, and in liver it can form serine from glycine asa substrate for gluconeogenesis. Methylene, methenyl,and 10-formyl tetrahydrofolates are interconvertible.When one-carbon folates are not required, the oxidationof formyl tetrahydrofolate to yield carbon dioxide providesa means of maintaining a pool of free folate.Inhibitors of Folate MetabolismProvide Cancer Chemotherapy &Antibacterial & Antimalarial DrugsThe methylation of deoxyuridine monophosphate(dUMP) to thymidine monophosphate (TMP), catalyzedby thymidylate synthase, is essential for the synthesisof DNA. The one-carbon fragment of methylene-tetrahydrofolateis reduced to a methyl group withrelease of dihydrofolate, which is then reduced back totetrahydrofolate by dihydrofolate reductase. Thymidylatesynthase and dihydrofolate reductase are especiallyactive in tissues with a high rate of cell division.Methotrexate, an analog of 10-methyl-tetrahydrofolate,inhibits dihydrofolate reductase and has been exploitedas an anticancer drug. The dihydrofolate reductasesof some bacteria and parasites differ from thehuman enzyme; inhibitors of these enzymes can be usedas antibacterial drugs, eg, trimethoprim, and antimalarialdrugs, eg, pyrimethamine.Vitamin B 12 Deficiency Causes FunctionalFolate Deficiency—the Folate TrapWhen acting as a methyl donor, S-adenosylmethionineforms homocysteine, which may be remethylated bymethyltetrahydrofolate catalyzed by methionine synthase,a vitamin B 12 -dependent enzyme (Figure 45–14).The reduction of methylene-tetrahydrofolate to methyltetrahydrofolateis irreversible, and since the major sourceof tetrahydrofolate for tissues is methyl-tetrahydrofolate,the role of methionine synthase is vital and provides a linkbetween the functions of folate and vitamin B 12 . Impairmentof methionine synthase in B 12 deficiency results inthe accumulation of methyl-tetrahydrofolate—the “folatetrap.” There is therefore functional deficiency of folatesecondary to the deficiency of vitamin B 12 .Folate Deficiency CausesMegaloblastic AnemiaDeficiency of folic acid itself—or deficiency of vitaminB 12 , which leads to functional folic acid deficiency—affectscells that are dividing rapidly because they have alarge requirement for thymidine for DNA synthesis.Clinically, this affects the bone marrow, leading tomegaloblastic anemia.Folic Acid Supplements Reducethe Risk of Neural Tube Defects& HyperhomocysteinemiaSupplements of 400 µg/d of folate begun before conceptionresult in a significant reduction in the incidenceof neural tube defects as found in spina bifida. Elevatedblood homocysteine is an associated risk factorfor atherosclerosis, thrombosis, and hypertension.The condition is due to impaired ability to formmethyl-tetrahydrofolate by methylene-tetrahydrofolatereductase, causing functional folate deficiency and resultingin failure to remethylate homocysteine to methionine.People with the causative abnormal variant ofmethylene-tetrahydrofolate reductase do not develophyperhomocysteinemia if they have a relatively high intakeof folate, but it is not yet known whether this affectsthe incidence of cardiovascular disease.Folate Enrichment of FoodsMay Put Some People at RiskFolate supplements will rectify the megaloblastic anemiaof vitamin B 12 deficiency but may hasten the developmentof the (irreversible) nerve damage found in B 12 deficiency.There is also antagonism between folic acid andthe anticonvulsants used in the treatment of epilepsy.DIETARY BIOTIN DEFICIENCYIS UNKNOWNThe structures of biotin, biocytin, and carboxy-biotin(the active metabolic intermediate) are shown in Figure45–17. Biotin is widely distributed in many foods asbiocytin (ε-amino-biotinyl lysine), which is released onproteolysis. It is synthesized by intestinal flora in excessof requirements. Deficiency is unknown except amongpeople maintained for many months on parenteral nutritionand a very small number who eat abnormallylarge amounts of uncooked egg white, which containsavidin, a protein that binds biotin and renders it unavailablefor absorption.Biotin Is a Coenzymeof Carboxylase EnzymesBiotin functions to transfer carbon dioxide in a smallnumber of carboxylation reactions. A holocarboxylasesynthetase acts on a lysine residue of the apoenzymes ofacetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoAcarboxylase, or methylcrotonyl-CoA carboxylaseto react with free biotin to form the biocytin residueof the holoenzyme. The reactive intermediate is 1-Ncarboxybiocytin,formed from bicarbonate in an ATPdependentreaction. The carboxyl group is then transferredto the substrate for carboxylation (Figure 21–1).

VITAMINS & MINERALS / 495OOCHNFigure 45–17.OSNHOHN NHSON NHSBiotinCOOBiotinyl-lysine (biocytin)COCHNCarboxy-biocytinOHNCCHNHCCHNHBiotin, biocytin, and carboxy-biocytin.Biotin also has a role in regulation of the cell cycle,acting to biotinylate key nuclear proteins.AS PART OF CoA AND ACP,PANTOTHENIC ACID ACTS ASA CARRIER OF ACYL RADICALSPantothenic acid has a central role in acyl group metabolismwhen acting as the pantetheine functional moietyof coenzyme A or acyl carrier protein (ACP) (Figure45–18). The pantetheine moiety is formed after combinationof pantothenate with cysteine, which providesOOthe ⎯SH prosthetic group of CoA and ACP. CoAtakes part in reactions of the citric acid cycle, fatty acidsynthesis and oxidation, acetylations, and cholesterolsynthesis. ACP participates in fatty acid synthesis. Thevitamin is widely distributed in all foodstuffs, and deficiencyhas not been unequivocally reported in humanbeings except in specific depletion studies.ASCORBIC ACID IS A VITAMINFOR ONLY SOME SPECIESVitamin C (Figure 45–19) is a vitamin for human beingsand other primates, the guinea pig, bats, passerine birds,and most fishes and invertebrates; other animals synthesizeit as an intermediate in the uronic acid pathway ofglucose metabolism (Chapter 20). In those species forwhich it is a vitamin, there is a block in that pathway dueto absence of gulonolactone oxidase. Both ascorbic acidand dehydroascorbic acid have vitamin activity.Vitamin C Is the Coenzymefor Two Groups of HydroxylasesAscorbic acid has specific roles in the copper-containinghydroxylases and the α-ketoglutarate-linked iron-containinghydroxylases. It also increases the activity of anumber of other enzymes in vitro, though this is a nonspecificreducing action. In addition, it has a number ofnonenzymic effects due to its action as a reducing agentand oxygen radical quencher.Dopamine -hydroxylase is a copper-containingenzyme involved in the synthesis of the catecholaminesnorepinephrine and epinephrine from tyrosine in theadrenal medulla and central nervous system. During hydroxylation,the Cu + is oxidized to Cu 2+ ; reduction backOCCH 2OHOCNHCH 2CH 2CH 2SHCH 2CH 2NHNHCOCOH 3 CCHOHCCH 3CH 2 OHH 3 CCHOHCCH 3CH 2 OOPOOOPOONN NHNCH 2NH 2OFigure 45–18. Pantothenic acid andcoenzyme A. * Shows the site of acylationby fatty acids.Pantothenic acidCoenzyme A (CoASH)OO OHP OO

496 / CHAPTER 45CH 2 OHCH 2 OHCH 2 OHHOCH 2OOHOCH 2OOHOCH 2OOOHOHAscorbate.OOHMonodehydroascorbate(semidehydroascorbate)OODehydroascorbateFigure 45–19. Vitamin C.to Cu + specifically requires ascorbate, which is oxidizedto monodehydroascorbate. Similar actions of ascorbateoccur in tyrosine degradation at the p-hydroxyphenylpyruvatehydroxylase step and at the homogentisatedioxygenase step, which needs Fe 2+ (Figure 30–12).A number of peptide hormones have a carboxyl terminalamide which is derived from a glycine terminalresidue. This glycine is hydroxylated on the α-carbonby a copper-containing enzyme, peptidylglycine hydroxylase,which, again, requires ascorbate for reductionof Cu 2+ .A number of iron-containing, ascorbate-requiringhydroxylases share a common reaction mechanism inwhich hydroxylation of the substrate is linked to decarboxylationof α-ketoglutarate (Figure 28–11). Many ofthese enzymes are involved in the modification of precursorproteins. Proline and lysine hydroxylases arerequired for the postsynthetic modification of procollagento collagen, and proline hydroxylase is also requiredin formation of osteocalcin and the C1q componentof complement. Aspartate β-hydroxylase isrequired for the postsynthetic modification of the precursorof protein C, the vitamin K-dependent proteasewhich hydrolyzes activated factor V in the blood clottingcascade. Trimethyllysine and γ-butyrobetaine hydroxylasesare required for the synthesis of carnitine.Vitamin C Deficiency Causes ScurvySigns of vitamin C deficiency in scurvy include skinchanges, fragility of blood capillaries, gum decay, toothloss, and bone fracture, many of which can be attributedto deficient collagen synthesis.There May Be Benefits From HigherIntakes of Vitamin CAt intakes above approximately 100 mg/d, the body’scapacity to metabolize vitamin C is saturated, and anyfurther intake is excreted in the urine. However, in additionto its other roles, vitamin C enhances the absorptionof iron, and this depends on the presence of the vitaminin the gut. Therefore, increased intakes may bebeneficial. Evidence is unconvincing that high doses ofvitamin C prevent the common cold or reduce the durationof its symptoms.MINERALS ARE REQUIREDFOR BOTH PHYSIOLOGIC &BIOCHEMICAL FUNCTIONSMany of the essential minerals (Table 45–2) are widelydistributed in foods, and most people eating a normalmixed diet are likely to receive adequate intakes. TheTable 45–2. Classification of essential mineralsaccording to their function.FunctionMineralStructural function Calcium, magnesium, phosphateInvolved in membrane Sodium, potassiumfunction: principalcations of extracellularandintracellular fluids,respectivelyFunction as prosthetic Cobalt, copper, iron, molybdegroupsin enzymes num, selenium, zincRegulatory role or role Calcium, chromium, iodine,in hormone action magnesium, manganese, sodium,potassiumKnown to be essential, Silicon, vanadium, nickel, tinbut function unknownHave effects in the Fluoride, lithiumbody, but essentiality isnot establishedWithout known nutritional Aluminum, arsenic, antimony,function but toxic in boron, bromine, cadmium, ceexcesssium, germanium, lead, mercury,silver, strontium

VITAMINS & MINERALS / 497amounts required vary from the order of grams per dayfor sodium and calcium, through milligrams per day(eg, iron) to micrograms per day for the trace elements.In general, mineral deficiencies are encountered whenfoods come from one region, where the soil may be deficientin some minerals, eg, iodine deficiency. Wherethe diet comes from a variety of different regions, mineraldeficiencies are unlikely. However, iron deficiencyis a general problem because if iron losses from thebody are relatively high (eg, from heavy menstrualblood loss), it is difficult to achieve an adequate intaketo replace the losses. Foods from soils containing highlevels of selenium cause toxicity, and increased intakesof common salt (sodium chloride) cause hypertensionin susceptible individuals.SUMMARY• Vitamins are organic nutrients with essential metabolicfunctions, generally required in small amountsin the diet because they cannot be synthesized by thebody. The lipid-soluble vitamins (A, D, E, and K)are hydrophobic molecules requiring normal fat absorptionfor their efficient absorption and the avoidanceof deficiency symptoms.• Vitamin A (retinol), present in carnivorous diets, andthe provitamin (β-carotene), found in plants, formretinaldehyde, utilized in vision, and retinoic acid,which acts in the control of gene expression. VitaminD is a steroid prohormone yielding the active hormonederivative calcitriol, which regulates calciumand phosphate metabolism. Vitamin D deficiencyleads to rickets and osteomalacia.• Vitamin E (tocopherol) is the most important antioxidantin the body, acting in the lipid phase ofmembranes and protecting against the effects of freeradicals. Vitamin K functions as cofactor to a carboxylasethat acts on glutamate residues of clottingfactor precursor proteins to enable them to chelatecalcium.• The water-soluble vitamins of the B complex act asenzyme cofactors. Thiamin is a cofactor in oxidativedecarboxylation of α-keto acids and of transketolasein the pentose phosphate pathway. Riboflavin andniacin are important cofactors in oxidoreduction reactions,respectively present in flavoprotein enzymesand in NAD and NADP.• Pantothenic acid is present in coenzyme A and acylcarrier protein, which act as carriers for acyl groupsin metabolic reactions. Pyridoxine, as pyridoxalphosphate, is the coenzyme for several enzymes ofamino acid metabolism, including the aminotransferases,and of glycogen phosphorylase. Biotin is thecoenzyme for several carboxylase enzymes.• Besides other functions, vitamin B 12 and folic acidtake part in providing one-carbon residues for DNAsynthesis, deficiency resulting in megaloblastic anemia.Vitamin C is a water-soluble antioxidant thatmaintains vitamin E and many metal cofactors in thereduced state.• Inorganic mineral elements that have a function inthe body must be provided in the diet. When insufficient,deficiency symptoms may arise, and if presentin excess they may be toxic.REFERENCESBender DA, Bender AE: Nutrition: A Reference Handbook. OxfordUniv Press, 1997.Bender DA: Nutritional Biochemistry of the Vitamins. 2nd ed. CambridgeUniv Press, 2003.Garrow JS, James WPT, Ralph A: Human Nutrition and Dietetics,10th ed. Churchill-Livingstone, 2000.Halliwell B, Chirico S: Lipid peroxidation: its mechanism, measurement,and significance. Am J Clin Nutr 1993;57(5Suppl):715S.Krinsky NI: Actions of carotenoids in biological systems. Annu RevNutr 1993;13:561.Padh H: Vitamin C: newer insights into its biochemical functions.Nutr Rev 1991;49:65.Shane B: Folylpolyglutamate synthesis and role in the regulation ofone-carbon metabolism. Vitam Horm 1989;45:263.Wiseman H, Halliwell B: Damage to DNA by reactive oxygen andnitrogen species: role in inflammatory disease and progressionto cancer. Biochem J 1996;313:17.

Intracellular Traffic & Sortingof Proteins 46Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEProteins must travel from polyribosomes to many differentsites in the cell to perform their particular functions.Some are destined to be components of specificorganelles, others for the cytosol or for export, and yetothers will be located in the various cellular membranes.Thus, there is considerable intracellular trafficof proteins. Many studies have shown that the Golgiapparatus plays a major role in the sorting of proteinsfor their correct destinations. A major insight was therecognition that for proteins to attain their proper locations,they generally contain information (a signal orcoding sequence) that targets them appropriately. Oncea number of the signals were defined, it became apparentthat certain diseases result from mutations that affectthese signals. In this chapter we discuss the intracellulartraffic of proteins and their sorting and brieflyconsider some of the disorders that result when abnormalitiesoccur.MANY PROTEINS ARE TARGETEDBY SIGNAL SEQUENCES TO THEIRCORRECT DESTINATIONSThe protein biosynthetic pathways in cells can be consideredto be one large sorting system. Many proteinscarry signals (usually but not always specific sequencesof amino acids) that direct them to their destination,thus ensuring that they will end up in the appropriatemembrane or cell compartment; these signals are a fundamentalcomponent of the sorting system. Usually thesignal sequences are recognized and interact with complementaryareas of proteins that serve as receptors forthe proteins that contain them.A major sorting decision is made early in proteinbiosynthesis, when specific proteins are synthesized eitheron free or on membrane-bound polyribosomes.This results in two sorting branches called the cytosolicbranch and the rough endoplasmic reticulum (RER)branch (Figure 46–1). This sorting occurs because proteinssynthesized on membrane-bound polyribosomescontain a signal peptide that mediates their attachmentto the membrane of the ER. Further details on498the signal peptide are given below. Proteins synthesizedon free polyribosomes lack this particular signal peptideand are delivered into the cytosol. There they aredirected to mitochondria, nuclei, and peroxisomes byspecific signals—or remain in the cytosol if they lack asignal. Any protein that contains a targeting sequencethat is subsequently removed is designated as a preprotein.In some cases a second peptide is also removed,and in that event the original protein is known as a preproprotein(eg, preproalbumin; Chapter 50).Proteins synthesized and sorted in the rough ERbranch (Figure 46–2) include many destined for variousmembranes (eg, of the ER, Golgi apparatus, lysosomes,and plasma membrane) and for secretion. Lysosomalenzymes are also included. Thus, such proteinsmay reside in the membranes or lumens of the ER orfollow the major transport route of intracellular proteinsto the Golgi apparatus. Further signal-mediatedsorting of certain proteins occurs in the Golgi apparatus,resulting in delivery to lysosomes, membranes ofthe Golgi apparatus, and other sites. Proteins destinedfor the plasma membrane or for secretion pass throughthe Golgi apparatus but generally are not thought tocarry specific sorting signals; they are believed to reachtheir destinations by default.The entire pathway of ER → Golgi apparatus →plasma membrane is often called the secretory or exocytoticpathway. Events along this route will be givenspecial attention. Most of the proteins reaching theGolgi apparatus or the plasma membrane are carried intransport vesicles; a brief description of the formationof these important particles will be given subsequently.Other proteins destined for secretion are carried in secretoryvesicles (Figure 46–2). These are prominent inthe pancreas and certain other glands. Their mobilizationand discharge are regulated and often referred to as“regulated secretion,” whereas the secretory pathwayinvolving transport vesicles is called “constitutive.”Experimental approaches that have afforded majorinsights to the processes described in this chapter include(1) use of yeast mutants; (2) application of recombinantDNA techniques (eg, mutating or eliminatingparticular sequences in proteins, or fusing newsequences onto them; and (3) development of in vitro

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 499Polyribosomes(1) Cytosolic(2) Rough ERProteinsMitochondrialNuclearPeroxisomalCytosolicER membraneGA membranePlasma membraneSecretoryLysosomal enzymesFigure 46–1. Diagrammatic representation of thetwo branches of protein sorting occurring by synthesison (1) cytosolic and (2) membrane-bound polyribosomes.The mitochondrial proteins listed are encodedby nuclear genes. Some of the signals used in furthersorting of these proteins are listed in Table 46–4. (ER,endoplasmic reticulum; GA, Golgi apparatus.)systems (eg, to study translocation in the ER and mechanismsof vesicle formation).The sorting of proteins belonging to the cytosolicbranch referred to above is described next, starting withmitochondrial proteins.THE MITOCHONDRION BOTH IMPORTS& SYNTHESIZES PROTEINSMitochondria contain many proteins. Thirteen proteins(mostly membrane components of the electrontransport chain) are encoded by the mitochondrialgenome and synthesized in that organelle using its ownprotein-synthesizing system. However, the majority (atleast several hundred) are encoded by nuclear genes,are synthesized outside the mitochondria on cytosolicpolyribosomes, and must be imported. Yeast cells haveproved to be a particularly useful system for analyzingthe mechanisms of import of mitochondrial proteins,partly because it has proved possible to generate a varietyof mutants that have illuminated the fundamentalprocesses involved. Most progress has been made in thestudy of proteins present in the mitochondrial matrix,such as the F 1 ATPase subunits. Only the pathway ofimport of matrix proteins will be discussed in any detailhere.Matrix proteins must pass from cytosolic polyribosomesthrough the outer and inner mitochondrialmembranes to reach their destination. Passage throughthe two membranes is called translocation. They havean amino terminal leader sequence (presequence),about 20–80 amino acids in length, which is not highlyconserved but contains many positively charged aminoacids (eg, Lys or Arg). The presequence is equivalent toa signal peptide mediating attachment of polyribosomesto membranes of the ER (see below), but in this instancetargeting proteins to the matrix; if the leader sequenceis cleaved off, potential matrix proteins will notreach their destination.Translocation is believed to occur posttranslationally,after the matrix proteins are released from the cytosolicpolyribosomes. Interactions with a number ofcytosolic proteins that act as chaperones (see below)and as targeting factors occur prior to translocation.Two distinct translocation complexes are situatedin the outer and inner mitochondrial membranes, referredto (respectively) as TOM (translocase-of-theoutermembrane) and TIM (translocase-of-the-innermembrane). Each complex has been analyzed andfound to be composed of a number of proteins, some ofwhich act as receptors for the incoming proteins andothers as components of the transmembrane poresthrough which these proteins must pass. Proteins mustbe in the unfolded state to pass through the complexes,and this is made possible by ATP-dependentbinding to several chaperone proteins. The roles ofchaperone proteins in protein folding are discussed laterin this chapter. In mitochondria, they are involved intranslocation, sorting, folding, assembly, and degradationof imported proteins. A proton-motive forceacross the inner membrane is required for import; it ismade up of the electric potential across the membrane(inside negative) and the pH gradient (see Chapter12). The positively charged leader sequence may behelped through the membrane by the negative chargein the matrix. The presequence is split off in the matrixby a matrix-processing peptidase (MPP). Contactwith other chaperones present in the matrix is essentialto complete the overall process of import. Interactionwith mt-Hsp70 (Hsp = heat shock protein) ensuresproper import into the matrix and prevents misfoldingor aggregation, while interaction with the mt-Hsp60-Hsp10 system ensures proper folding. The latter proteinsresemble the bacterial GroEL chaperonins, a subclassof chaperones that form complex cage-likeassemblies made up of heptameric ring structures. Theinteractions of imported proteins with the above chaperonesrequire hydrolysis of ATP to drive them.The details of how preproteins are translocated havenot been fully elucidated. It is possible that the electricpotential associated with the inner mitochondrial membranecauses a conformational change in the unfoldedpreprotein being translocated and that this helps to pullit across. Furthermore, the fact that the matrix is morenegative than the intermembrane space may “attract”the positively charged amino terminal of the preprotein

Plasma membraneCytosolEarlyendosomePrelysosome(or late endosome)Constitutive(excretory)transportvesicleSecretorystoragegranuleLysosomeTGNGolgiapparatustransmedialcisCGNEndoplasmicreticulumNuclearenvelopeFigure 46–2. Diagrammatic representation of the rough endoplasmic reticulumbranch of protein sorting. Newly synthesized proteins are inserted into theER membrane or lumen from membrane-bound polyribosomes (small black circlesstudding the cytosolic face of the ER). Those proteins that are transportedout of the ER (indicated by solid black arrows) do so from ribosome-free transitionalelements. Such proteins may then pass through the various subcompartmentsof the Golgi until they reach the TGN, the exit side of the Golgi. In the TGN,proteins are segregated and sorted. Secretory proteins accumulate in secretorystorage granules from which they may be expelled as shown in the upper righthandside of the figure. Proteins destined for the plasma membrane or those thatare secreted in a constitutive manner are carried out to the cell surface in transportvesicles, as indicated in the upper middle area of the figure. Some proteinsmay reach the cell surface via late and early endosomes. Other proteins enterprelysosomes (late endosomes) and are selectively transferred to lysosomes. Theendocytic pathway illustrated in the upper left-hand area of the figure is consideredelsewhere in this chapter. Retrieval from the Golgi apparatus to the ER is notconsidered in this scheme. (CGN, cis-Golgi network; TGN, trans-Golgi network.)(Courtesy of E Degen.)500

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 501to enter the matrix. Close contact between the membranesites in the outer and inner membranes involvedin translocation is necessary.The above describes the major pathway of proteinsdestined for the mitochondrial matrix. However, certainproteins insert into the outer mitochondrialmembrane facilitated by the TOM complex. Othersstop in the intermembrane space, and some insert intothe inner membrane. Yet others proceed into the matrixand then return to the inner membrane or intermembranespace. A number of proteins contain twosignaling sequences—one to enter the mitochondrialmatrix and the other to mediate subsequent relocation(eg, into the inner membrane). Certain mitochondrialproteins do not contain presequences (eg, cytochromec, which locates in the inter membrane space), and otherscontain internal presequences. Overall, proteinsemploy a variety of mechanisms and routes to attaintheir final destinations in mitochondria.General features that apply to the import of proteinsinto organelles, including mitochondria and some ofthe other organelles to be discussed below, are summarizedin Table 46–1.IMPORTINS & EXPORTINS AREINVOLVED IN TRANSPORTOF MACROMOLECULES IN& OUT OF THE NUCLEUSIt has been estimated that more than a million macromoleculesper minute are transported between the nucleusand the cytoplasm in an active eukaryotic cell.Table 46–1. Some general features of proteinimport to organelles. 1• Import of a protein into an organelle usually occurs in threestages: recognition, translocation, and maturation.• Targeting sequences on the protein are recognized in thecytoplasm or on the surface of the organelle.• The protein is unfolded for translocation, a state maintainedin the cytoplasm by chaperones.• Threading of the protein through a membrane requires energyand organellar chaperones on the trans side of themembrane.• Cycles of binding and release of the protein to the chaperoneresult in pulling of its polypeptide chain through themembrane.• Other proteins within the organelle catalyze folding of theprotein, often attaching cofactors or oligosaccharides andassembling them into active monomers or oligomers.1 Data from McNew JA, Goodman JM: The targeting and assemblyof peroxisomal proteins: some old rules do not apply. TrendsBiochem Sci 1998;21:54.These macromolecules include histones, ribosomal proteinsand ribosomal subunits, transcription factors, andmRNA molecules. The transport is bidirectional andoccurs through the nuclear pore complexes (NPCs).These are complex structures with a mass approximately30 times that of a ribosome and are composedof about 100 different proteins. The diameter of anNPC is approximately 9 nm but can increase up to approximately28 nm. Molecules smaller than about 40kDa can pass through the channel of the NPC by diffusion,but special translocation mechanisms exist forlarger molecules. These mechanisms are under intensiveinvestigation, but some important features have alreadyemerged.Here we shall mainly describe nuclear import ofcertain macromolecules. The general picture that hasemerged is that proteins to be imported (cargo molecules)carry a nuclear localization signal (NLS). Oneexample of an NLS is the amino acid sequence (Pro) 2 -(Lys) 4 -Ala-Lys-Val, which is markedly rich in basic lysineresidues. Depending on which NLS it contains, acargo molecule interacts with one of a family of solubleproteins called importins, and the complex docks atthe NPC. Another family of proteins called Ran plays acritical regulatory role in the interaction of the complexwith the NPC and in its translocation through theNPC. Ran proteins are small monomeric nuclear GTPasesand, like other GTPases, exist in either GTPboundor GDP-bound states. They are themselves regulatedby guanine nucleotide exchange factors(GEFs; eg, the protein RCC1 in eukaryotes), which arelocated in the nucleus, and Ran guanine-activatingproteins (GAPs), which are predominantly cytoplasmic.The GTP-bound state of Ran is favored in the nucleusand the GDP-bound state in the cytoplasm. Theconformations and activities of Ran molecules vary dependingon whether GTP or GDP is bound to them(the GTP-bound state is active; see discussion of G proteinsin Chapter 43). The asymmetry between nucleusand cytoplasm—with respect to which of these two nucleotidesis bound to Ran molecules—is thought to becrucial in understanding the roles of Ran in transferringcomplexes unidirectionally across the NPC. Whencargo molecules are released inside the nucleus, the importinsrecirculate to the cytoplasm to be used again.Figure 46–3 summarizes some of the principal featuresin the above process.Other small monomeric GTPases (eg, ARF, Rab,Ras, and Rho) are important in various cellular processessuch as vesicle formation and transport (ARF andRab; see below), certain growth and differentiationprocesses (Ras), and formation of the actin cytoskeleton.A process involving GTP and GDP is also crucialin the transport of proteins across the membrane of theER (see below).

502 / CHAPTER 4612GTP3GDPTargetingαβDockingαβRanGDPRanGEF ?CytoplasmNucleusRanGTPCytoplasmNucleusRanGTPGDPOFFGTP ONRanGAPRan+ P i Ran exchangeGEF+RanGDPRanGDPRanBP1RanBP14α βRanGTPTranslocationα βTermination6RanGTP5PiRanGAPα+RanGTPβRanGDP7?α+βRanGTP+8Recycle factorsFigure 46–3. Schematic representation of the proposed role of Ran in the import of cargocarrying an NLS signal. (1) The targeting complex forms when the NLS receptor (α, an importin)binds NLS cargo and the docking factor (β). (2) Docking occurs at filamentous sites that protrudefrom the NPC. Ran-GDP docks independently. (3) Transfer to the translocation channel istriggered when a RanGEF converts Ran-GDP to Ran-GTP. (4) The NPC catalyzes translocation ofthe targeting complex. (5) Ran-GTP is recycled to Ran-GDP by docked RanGAP. (6) Ran-GTP disruptsthe targeting complex by binding to a site on β that overlaps with a binding site. (7) NLScargo dissociates from α, and Ran-GTP may dissociate from β. (8) α and β factors are recycled tothe cytoplasm. Inset: The Ran translocation switch is off in the cytoplasm and on in the nucleus.Ran-GTP promotes NLS- and NES-directed translocation. However, cytoplasmic Ran is enrichedin Ran-GDP (OFF) by an active RanGAP, and nuclear pools are enriched in Ran-GTP (ON) by anactive GEF. RanBP1 promotes the contrary activities of these two factors. Direct linkage of nuclearand cytoplasmic pools of Ran occurs through the NPC by an unknown shuttling mechanism.P i , inorganic phosphate; NLS, nuclear localization signal; NPC, nuclear pore complex; GEF,guanine nucleotide exchange factor; GAP, guanine-activating protein; NES, nuclear export signal;BP, binding protein. (Reprinted, with permission, from Goldfarb DS: Whose finger is on theswitch? Science 1997;276:1814.)

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 503Proteins similar to importins, referred to as exportins,are involved in export of many macromoleculesfrom the nucleus. Cargo molecules for exportcarry nuclear export signals (NESs). Ran proteins areinvolved in this process also, and it is now establishedthat the processes of import and export share a numberof common features.MOST CASES OF ZELLWEGER SYNDROMEARE DUE TO MUTATIONS IN GENESINVOLVED IN THE BIOGENESISOF PEROXISOMESThe peroxisome is an important organelle involved inaspects of the metabolism of many molecules, includingfatty acids and other lipids (eg, plasmalogens, cholesterol,bile acids), purines, amino acids, and hydrogenperoxide. The peroxisome is bounded by a single membraneand contains more than 50 enzymes; catalase andurate oxidase are marker enzymes for this organelle. Itsproteins are synthesized on cytosolic polyribosomes andfold prior to import. The pathways of import of a numberof its proteins and enzymes have been studied, somebeing matrix components and others membrane components.At least two peroxisomal-matrix targetingsequences (PTSs) have been discovered. One, PTS1, isa tripeptide (ie, Ser-Lys-Leu [SKL], but variations ofthis sequence have been detected) located at the carboxylterminal of a number of matrix proteins, includingcatalase. Another, PTS2, consisting of about 26–36amino acids, has been found in at least four matrix proteins(eg, thiolase) and, unlike PTS1, is cleaved afterentry into the matrix. Proteins containing PTS1 sequencesform complexes with a soluble receptor protein(PTS1R) and proteins containing PTS2 sequencescomplex with another, PTS2R. The resulting complexesthen interact with a membrane receptor, Pex14p.Proteins involved in further transport of proteins intothe matrix are also present. Most peroxisomal membraneproteins have been found to contain neither ofthe above two targeting sequences, but apparently containothers. The import system can handle intactoligomers (eg, tetrameric catalase). Import of matrixproteins requires ATP, whereas import of membraneproteins does not.Interest in import of proteins into peroxisomes hasbeen stimulated by studies on Zellweger syndrome.This condition is apparent at birth and is characterizedby profound neurologic impairment, victims oftendying within a year. The number of peroxisomes canvary from being almost normal to being virtually absentin some patients. Biochemical findings include an accumulationof very long chain fatty acids, abnormalities ofthe synthesis of bile acids, and a marked reduction ofplasmalogens. The condition is believed to be due tomutations in genes encoding certain proteins—socalled peroxins—involved in various steps of peroxisomebiogenesis (such as the import of proteins describedabove), or in genes encoding certain peroxisomalenzymes themselves. Two closely related conditionsare neonatal adrenoleukodystrophy and infantileRefsum disease. Zellweger syndrome and these twoconditions represent a spectrum of overlapping features,with Zellweger syndrome being the most severe(many proteins affected) and infantile Refsum diseasethe least severe (only one or a few proteins affected).Table 46–2 lists some features of these and related conditions.THE SIGNAL HYPOTHESIS EXPLAINSHOW POLYRIBOSOMES BIND TO THEENDOPLASMIC RETICULUMAs indicated above, the rough ER branch is the secondof the two branches involved in the synthesis and sortingof proteins. In this branch, proteins are synthesizedon membrane-bound polyribosomes and translocatedinto the lumen of the rough ER prior to further sorting(Figure 46–2).The signal hypothesis was proposed by Blobel andSabatini partly to explain the distinction between freeand membrane-bound polyribosomes. They found thatproteins synthesized on membrane-bound polyribosomescontained a peptide extension (signal peptide)Table 46–2. Disorders due to peroxisomalabnormalities. 1 MIM Number 2Zellweger syndrome 214100Neonatal adrenoleukodystrophy 202370Infantile Refsum disease 266510Hyperpipecolic acidemia 239400Rhizomelic chondrodysplasia punctata 215100Adrenoleukodystrophy 300100Pseudo-neonatal adrenoleukodystrophy 264470Pseudo-Zellweger syndrome 261510Hyperoxaluria type 1 259900Acatalasemia 115500Glutaryl-CoA oxidase deficiency 2316901 Reproduced, with permission, from Seashore MR, Wappner RS:Genetics in Primary Care & Clinical Medicine. Appleton & Lange,1996.2 MIM = Mendelian Inheritance in Man. Each number specifies a referencein which information regarding each of the above conditionscan be found.

504 / CHAPTER 46at their amino terminals which mediated their attachmentto the membranes of the ER. As noted above,proteins whose entire synthesis occurs on free polyribosomeslack this signal peptide. An important aspect ofthe signal hypothesis was that it suggested—as turnsout to be the case—that all ribosomes have the samestructure and that the distinction between membraneboundand free ribosomes depends solely on the former’scarrying proteins that have signal peptides. Muchevidence has confirmed the original hypothesis. Becausemany membrane proteins are synthesized on membrane-boundpolyribosomes, the signal hypothesis playsan important role in concepts of membrane assembly.Some characteristics of signal peptides are summarizedin Table 46–3.Figure 46–4 illustrates the principal features in relationto the passage of a secreted protein through themembrane of the ER. It incorporates features from theoriginal signal hypothesis and from subsequent work.The mRNA for such a protein encodes an amino terminalsignal peptide (also variously called a leader sequence,a transient insertion signal, a signal sequence,or a presequence). The signal hypothesis proposed thatthe protein is inserted into the ER membrane at thesame time as its mRNA is being translated on polyribosomes,so-called cotranslational insertion. As the signalpeptide emerges from the large subunit of the ribosome,it is recognized by a signal recognition particle(SRP) that blocks further translation after about 70amino acids have been polymerized (40 buried in thelarge ribosomal subunit and 30 exposed). The block isreferred to as elongation arrest. The SRP contains sixproteins and has a 7S RNA associated with it that isclosely related to the Alu family of highly repeatedDNA sequences (Chapter 36). The SRP-imposed blockis not released until the SRP-signal peptide-polyribosomecomplex has bound to the so-called docking protein(SRP-R, a receptor for the SRP) on the ER membrane;the SRP thus guides the signal peptide to theSRP-R and prevents premature folding and expulsionof the protein being synthesized into the cytosol.The SRP-R is an integral membrane protein composedof α and β subunits. The α subunit binds GDPTable 46–3. Some properties of signal peptides.• Usually, but not always, located at the amino terminal• Contain approximately 12–35 amino acids• Methionine is usually the amino terminal amino acid• Contain a central cluster of hydrophobic amino acids• Contain at least one positively charged amino acid neartheir amino terminal• Usually cleaved off at the carboxyl terminal end of an Alaresidue by signal peptidaseand the β subunit spans the membrane. When the SRPsignalpeptide complex interacts with the receptor, theexchange of GDP for GTP is stimulated. This form ofthe receptor (with GTP bound) has a high affinity forthe SRP and thus releases the signal peptide, which bindsto the translocation machinery (translocon) also presentin the ER membrane. The α subunit then hydrolyzes itsbound GTP, restoring GDP and completing a GTP-GDP cycle. The unidirectionality of this cycle helps drivethe interaction of the polyribosome and its signal peptidewith the ER membrane in the forward direction.The translocon consists of a number of membraneproteins that form a protein-conducting channel in theER membrane through which the newly synthesizedprotein may pass. The channel appears to be open onlywhen a signal peptide is present, preserving conductanceacross the ER membrane when it closes. The conductanceof the channel has been measured experimentally.Specific functions of a number of components of thetranslocon have been identified or suggested. TRAM(translocating chain-associated membrane) protein maybind the signal sequence as it initially interacts with thetranslocon and the Sec61p complex (consisting of threeproteins) binds the heavy subunit of the ribosome.The insertion of the signal peptide into the conductingchannel, while the other end of the parent protein isstill attached to ribosomes, is termed “cotranslationalinsertion.” The process of elongation of the remainingportion of the protein probably facilitates passage of thenascent protein across the lipid bilayer as the ribosomesremain attached to the membrane of the ER. Thus, therough (or ribosome-studded) ER is formed. It is importantthat the protein be kept in an unfolded state priorto entering the conducting channel—otherwise, it maynot be able to gain access to the channel.Ribosomes remain attached to the ER during synthesisof signal peptide-containing proteins but are releasedand dissociated into their two types of subunitswhen the process is completed. The signal peptide ishydrolyzed by signal peptidase, located on the luminalside of the ER membrane (Figure 46–4), and then isapparently rapidly degraded by proteases.Cytochrome P450 (Chapter 53), an integral ERmembrane protein, does not completely cross the membrane.Instead, it resides in the membrane with its signalpeptide intact. Its passage through the membrane isprevented by a sequence of amino acids called a halt- orstop-transfer signal.Secretory proteins and proteins destined for membranesdistal to the ER completely traverse the membranebilayer and are discharged into the lumen of theER. N-Glycan chains, if present, are added (Chapter47) as these proteins traverse the inner part of the ERmembrane—a process called “cotranslational glycosylation.”Subsequently, the proteins are found in the

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 5055′AUGSignal codons3′Signal peptideSRPSignal peptidaseRibosome receptorSignal receptorFigure 46–4. Diagram of the signal hypothesis for the transport of secreted proteins across theER membrane. The ribosomes synthesizing a protein move along the messenger RNA specifying theamino acid sequence of the protein. (The messenger is represented by the line between 5′ and 3′.)The codon AUG marks the start of the message for the protein; the hatched lines that follow AUGrepresent the codons for the signal sequence. As the protein grows out from the larger ribosomalsubunit, the signal sequence is exposed and bound by the signal recognition particle (SRP). Translationis blocked until the complex binds to the “docking protein,” also designated SRP-R (representedby the solid bar) on the ER membrane. There is also a receptor (open bar) for the ribosomeitself. The interaction of the ribosome and growing peptide chain with the ER membrane results inthe opening of a channel through which the protein is transported to the interior space of the ER.During translocation, the signal sequence of most proteins is removed by an enzyme called the“signal peptidase,” located at the luminal surface of the ER membrane. The completed protein iseventually released by the ribosome, which then separates into its two components, the large andsmall ribosomal subunits. The protein ends up inside the ER. See text for further details. (Slightlymodified and reproduced, with permission, from Marx JL: Newly made proteins zip through the cell. Science1980;207:164. Copyright © 1980 by the American Association for the Advancement of Science.)lumen of the Golgi apparatus, where further changes inglycan chains occur (Figure 47–9) prior to intracellulardistribution or secretion. There is strong evidence thatthe signal peptide is involved in the process of proteininsertion into ER membranes. Mutant proteins, containingaltered signal peptides in which a hydrophobicamino acid is replaced by a hydrophilic one, are not insertedinto ER membranes. Nonmembrane proteins(eg, α-globin) to which signal peptides have been attachedby genetic engineering can be inserted into thelumen of the ER or even secreted.There is considerable evidence that a second transposonin the ER membrane is involved in retrogradetransport of various molecules from the ER lumen tothe cytosol. These molecules include unfolded or misfoldedglycoproteins, glycopeptides, and oligosaccharides.Some at least of these molecules are degraded inproteasomes. Thus, there is two-way traffic across theER membrane.PROTEINS FOLLOW SEVERAL ROUTESTO BE INSERTED INTO OR ATTACHEDTO THE MEMBRANES OF THEENDOPLASMIC RETICULUMThe routes that proteins follow to be inserted into themembranes of the ER include the following.A. COTRANSLATIONAL INSERTIONFigure 46–5 shows a variety of ways in which proteinsare distributed in the plasma membrane. In particular,the amino terminals of certain proteins (eg, the LDL receptor)can be seen to be on the extracytoplasmic face,whereas for other proteins (eg, the asialoglycoprotein receptor)the carboxyl terminals are on this face. To explainthese dispositions, one must consider the initialbiosynthetic events at the ER membrane. The LDL receptorenters the ER membrane in a manner analogousto a secretory protein (Figure 46–4); it partly traverses

506 / CHAPTER 46NNNCNCCNNEXTRACYTOPLASMICFACEPHOSPHOLIPIDBILAYERCNVarious transporters (eg, glucose)NInfluenza neuraminidaseAsialoglycoprotein receptorTransferrin receptorHLA-DR invariant chainCC CInsulin andIGF-I receptorsCYTOPLASMICFACECNG protein–coupled receptorsLDL receptorHLA-A heavy chainInfluenza hemagglutininFigure 46–5. Variations in the way in which proteins are inserted into membranes. Thisschematic representation, which illustrates a number of possible orientations, shows the segmentsof the proteins within the membrane as α-helices and the other segments as lines. TheLDL receptor, which crosses the membrane once and has its amino terminal on the exterior, iscalled a type I transmembrane protein. The asialoglycoprotein receptor, which also crosses themembrane once but has its carboxyl terminal on the exterior, is called a type II transmembraneprotein. The various transporters indicated (eg, glucose) cross the membrane a number of timesand are called type III transmembrane proteins; they are also referred to as polytopic membraneproteins. (N, amino terminal; C, carboxyl terminal.) (Adapted, with permission, from Wickner WT,Lodish HF: Multiple mechanisms of protein insertion into and across membranes. Science1985;230:400. Copyright © 1985 by the American Association for the Advancement of Science.)the ER membrane, its signal peptide is cleaved, and itsamino terminal protrudes into the lumen. However, it isretained in the membrane because it contains a highlyhydrophobic segment, the halt- or stop-transfer signal.This sequence forms the single transmembrane segmentof the protein and is its membrane-anchoring domain.The small patch of ER membrane in which the newlysynthesized LDL receptor is located subsequently budsoff as a component of a transport vesicle, probably fromthe transitional elements of the ER (Figure 46–2). Asdescribed below in the discussion of asymmetry of proteinsand lipids in membrane assembly, the dispositionof the receptor in the ER membrane is preserved in thevesicle, which eventually fuses with the plasma membrane.In contrast, the asialoglycoprotein receptor possessesan internal insertion sequence, which inserts intothe membrane but is not cleaved. This acts as an anchor,and its carboxyl terminal is extruded through the membrane.The more complex disposition of the transporters(eg, for glucose) can be explained by the factthat alternating transmembrane α-helices act as uncleavedinsertion sequences and as halt-transfer signals,respectively. Each pair of helical segments is inserted as ahairpin. Sequences that determine the structure of aprotein in a membrane are called topogenic sequences.As explained in the legend to Figure 46–5, the abovethree proteins are examples of type I, type II, and typeIII transmembrane proteins.B. SYNTHESIS ON FREE POLYRIBOSOMES& SUBSEQUENT ATTACHMENT TO THEENDOPLASMIC RETICULUM MEMBRANEAn example is cytochrome b 5 , which enters the ERmembrane spontaneously.C. RETENTION AT THE LUMINAL ASPECTOF THE ENDOPLASMIC RETICULUMBY SPECIFIC AMINO ACID SEQUENCESA number of proteins possess the amino acid sequenceKDEL (Lys-Asp-Glu-Leu) at their carboxyl terminal.

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 507This sequence specifies that such proteins will be attachedto the inner aspect of the ER in a relatively loosemanner. The chaperone BiP (see below) is one suchprotein. Actually, KDEL-containing proteins first travelto the Golgi, interact there with a specific KDEL receptorprotein, and then return in transport vesicles to theER, where they dissociate from the receptor.D. RETROGRADE TRANSPORT FROMTHE GOLGI APPARATUSCertain other non-KDEL-containing proteins destinedfor the membranes of the ER also pass to the Golgi andthen return, by retrograde vesicular transport, to the ERto be inserted therein (see below).The foregoing paragraphs demonstrate that a varietyof routes are involved in assembly of the proteins ofthe ER membranes; a similar situation probably holdsfor other membranes (eg, the mitochondrial membranesand the plasma membrane). Precise targeting sequenceshave been identified in some instances (eg,KDEL sequences).The topic of membrane biogenesis is discussed furtherlater in this chapter.PROTEINS MOVE THROUGH CELLULARCOMPARTMENTS TO SPECIFICDESTINATIONSA scheme representing the possible flow of proteinsalong the ER → Golgi apparatus → plasma membraneroute is shown in Figure 46–6. The horizontal arrowsdenote transport steps that may be independent of targetingsignals, whereas the vertical open arrows representsteps that depend on specific signals. Thus, flow ofcertain proteins (including membrane proteins) fromthe ER to the plasma membrane (designated “bulkflow,” as it is nonselective) probably occurs without anytargeting sequences being involved, ie, by default. Onthe other hand, insertion of resident proteins into theER and Golgi membranes is dependent upon specificsignals (eg, KDEL or halt-transfer sequences for theER). Similarly, transport of many enzymes to lysosomesis dependent upon the Man 6-P signal (Chapter 47),and a signal may be involved for entry of proteins intosecretory granules. Table 46–4 summarizes informationon sequences that are known to be involved in targetingvarious proteins to their correct intracellular sites.CHAPERONES ARE PROTEINSTHAT PREVENT FAULTY FOLDING& UNPRODUCTIVE INTERACTIONSOF OTHER PROTEINSExit from the ER may be the rate-limiting step in thesecretory pathway. In this context, it has been foundthat certain proteins play a role in the assembly orproper folding of other proteins without themselvesbeing components of the latter. Such proteins are calledmolecular chaperones; a number of important propertiesof these proteins are listed in Table 46–5, and thenames of some of particular importance in the ER arelisted in Table 46–6. Basically, they stabilize unfoldedLysosomesERcisGolgimedialGolgitransGolgiCellsurfaceSecretorystorage vesiclesFigure 46–6. Flow of membrane proteins from the endoplasmicreticulum (ER) to the cell surface. Horizontal arrows denotesteps that have been proposed to be signal independent andthus represent bulk flow. The open vertical arrows in the boxesdenote retention of proteins that are resident in the membranesof the organelle indicated. The open vertical arrows outside theboxes indicate signal-mediated transport to lysosomes and secretorystorage granules. (Reproduced, with permission, from PfefferSR, Rothman JE: Biosynthetic protein transport and sorting by the endoplasmicreticulum and Golgi. Annu Rev Biochem 1987;56:829.)

508 / CHAPTER 46Table 46–4. Some sequences or compounds thatdirect proteins to specific organelles.Targeting Sequenceor CompoundOrganelle TargetedSignal peptide sequence Membrane of ERAmino terminalLuminal surface of ERKDEL sequence(Lys-Asp-Glu-Leu)Amino terminal sequence Mitochondrial matrix(20–80 residues)NLS 1 (eg, Pro 2 -Lys 2 -Ala- NucleusLys-Val)PTS 1 (eg, Ser-Lys-Leu) PeroxisomeMannose 6-phosphate Lysosome1 NLS, nuclear localization signal; PTS, peroxisomal-matrix targetingsequence.or partially folded intermediates, allowing them time tofold properly, and prevent inappropriate interactions,thus combating the formation of nonfunctional structures.Most chaperones exhibit ATPase activity andbind ADP and ATP. This activity is important for theireffect on folding. The ADP-chaperone complex oftenhas a high affinity for the unfolded protein, which,when bound, stimulates release of ADP with replacementby ATP. The ATP-chaperone complex, in turn,releases segments of the protein that have folded properly,and the cycle involving ADP and ATP binding isrepeated until the folded protein is released.Table 46–5. Some properties of chaperoneproteins.• Present in a wide range of species from bacteria to humans• Many are so-called heat shock proteins (Hsp)• Some are inducible by conditions that cause unfolding ofnewly synthesized proteins (eg, elevated temperature andvarious chemicals)• They bind to predominantly hydrophobic regions of unfoldedand aggregated proteins• They act in part as a quality control or editing mechanismfor detecting misfolded or otherwise defective proteins• Most chaperones show associated ATPase activity, with ATPor ADP being involved in the protein-chaperone interaction• Found in various cellular compartments such as cytosol,mitochondria, and the lumen of the endoplasmic reticulumTable 46–6. Some chaperones and enzymesinvolved in folding that are located in the roughendoplasmic reticulum.• BiP (immunoglobulin heavy chain binding protein)• GRP94 (glucose-regulated protein)• Calnexin• Calreticulin• PDI (protein disulfide isomerase)• PPI (peptidyl prolyl cis-trans isomerase)Several examples of chaperones were introducedabove when the sorting of mitochondrial proteins wasdiscussed. The immunoglobulin heavy chain bindingprotein (BiP) is located in the lumen of the ER. Thisprotein will bind abnormally folded immunoglobulinheavy chains and certain other proteins and preventthem from leaving the ER, in which they are degraded.Another important chaperone is calnexin, a calciumbindingprotein located in the ER membrane. This proteinbinds a wide variety of proteins, including mixedhistocompatibility (MHC) antigens and a variety ofserum proteins. As mentioned in Chapter 47, calnexinbinds the monoglycosylated species of glycoproteinsthat occur during processing of glycoproteins, retainingthem in the ER until the glycoprotein has folded properly.Calreticulin, which is also a calcium-binding protein,has properties similar to those of calnexin; it is notmembrane-bound. Chaperones are not the only proteinsin the ER lumen that are concerned with properfolding of proteins. Two enzymes are present that playan active role in folding. Protein disulfide isomerase(PDI) promotes rapid reshuffling of disulfide bondsuntil the correct set is achieved. Peptidyl prolyl isomerase(PPI) accelerates folding of proline-containingproteins by catalyzing the cis-trans isomerization ofX-Pro bonds, where X is any amino acid residue.TRANSPORT VESICLES ARE KEY PLAYERSIN INTRACELLULAR PROTEIN TRAFFICMost proteins that are synthesized on membraneboundpolyribosomes and are destined for the Golgiapparatus or plasma membrane reach these sites insidetransport vesicles. The precise mechanisms by whichproteins synthesized in the rough ER are inserted intothese vesicles are not known. Those involved in transportfrom the ER to the Golgi apparatus and viceversa—and from the Golgi to the plasma membrane—are mainly clathrin-free, unlike the coated vesicles involvedin endocytosis (see discussions of the LDL receptorin Chapters 25 and 26). For the sake of clarity,the non-clathrin-coated vesicles will be referred to in

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 509this text as transport vesicles. There is evidence thatproteins destined for the membranes of the Golgi apparatuscontain specific signal sequences. On the otherhand, most proteins destined for the plasma membraneor for secretion do not appear to contain specific signals,reaching these destinations by default.The Golgi Apparatus Is Involved inGlycosylation & Sorting of ProteinsThe Golgi apparatus plays two important roles in membranesynthesis. First, it is involved in the processingof the oligosaccharide chains of membrane and otherN-linked glycoproteins and also contains enzymes involvedin O-glycosylation (see Chapter 47). Second, itis involved in the sorting of various proteins prior totheir delivery to their appropriate intracellular destinations.All parts of the Golgi apparatus participate in thefirst role, whereas the trans-Golgi is particularly involvedin the second and is very rich in vesicles. Becauseof their central role in protein transport, considerableresearch has been conducted in recent years concerningthe formation and fate of transport vesicles.A Model of Non-Clathrin-Coated VesiclesInvolves SNAREs & Other FactorsVesicles lie at the heart of intracellular transport ofmany proteins. Recently, significant progress has beenmade in understanding the events involved in vesicleformation and transport. This has transpired because ofthe use of a number of approaches. These include establishmentof cell-free systems with which to studyvesicle formation. For instance, it is possible to observe,by electron microscopy, budding of vesicles from Golgipreparations incubated with cytosol and ATP. The developmentof genetic approaches for studying vesiclesin yeast has also been crucial. The picture is complex,with its own nomenclature (Table 46–7), and involvesa variety of cytosolic and membrane proteins, GTP,ATP, and accessory factors.Based largely on a proposal by Rothman and colleagues,anterograde vesicular transport can be consideredto occur in eight steps (Figure 46–7). The basicconcept is that each transport vesicle bears a unique addressmarker consisting of one or more v-SNARE proteins,while each target membrane bears one or morecomplementary t-SNARE proteins with which theformer interact specifically.Step 1: Coat assembly is initiated when ARF is activatedby binding GTP, which is exchanged forGDP. This leads to the association of GTP-boundARF with its putative receptor (hatched in Figure46–7) in the donor membrane.Table 46–7. Factors involved in the formation ofnon-clathrin-coated vesicles and their transport.• ARF: ADP-ribosylation factor, a GTPase• Coatomer: A family of at least seven coat proteins (α, β, γ, δ,ε, β′, and ζ). Different transport vesicles have different complementsof coat proteins.• SNAP: Soluble NSF attachment factor• SNARE: SNAP receptor• v-SNARE: Vesicle SNARE• t-SNARE: Target SNARE• GTP-γ-S: A nonhydrolyzable analog of GTP, used to test theinvolvement of GTP• NEM: N-Ethylmaleimide, a chemical that alkylates sulfhydrylgroups• NSF: NEM-sensitive factor, an ATPase• Rab proteins: A family of ras-related proteins first observedin rat brain; they are GTPases and are active when GTP isfound• Sec1: A member of a family of proteins that attach tot-SNAREs and are displaced from them by Rab proteins,thereby allowing v-SNARE–t-SNARE interactions to occur.Step 2: Membrane-associated ARF recruits the coatproteins that comprise the coatomer shell from thecytosol, forming a coated bud.Step 3: The bud pinches off in a process involvingacyl-CoA—and probably ATP—to complete theformation of the coated vesicle.Step 4: Coat disassembly (involving dissociation ofARF and coatomer shell) follows hydrolysis ofbound GTP; uncoating is necessary for fusion tooccur.Step 5: Vesicle targeting is achieved via members ofa family of integral proteins, termed v-SNAREs,that tag the vesicle during its budding. v-SNAREspair with cognate t-SNAREs in the target membraneto dock the vesicle.It is presumed that steps 4 and 5 are closely coupledand that step 4 may follow step 5, with ARF and thecoatomer shell rapidly dissociating after docking.Step 6: The general fusion machinery then assembleson the paired SNARE complex; it includes anATPase (NSF; NEM-sensitive factor) and the SNAP(soluble NSF attachment factor) proteins. SNAPsbind to the SNARE (SNAP receptor) complex, enablingNSF to bind.Step 7: Hydrolysis of ATP by NSF is essential forfusion, a process that can be inhibited by NEM (Nethylmaleimide).Certain other proteins and calciumare also required.

510 / CHAPTER 46Coated3 vesicle4t-SNARE21Donormembrane(eg, ER)GTPGDPCoatedbudGTPGTPGTPv-SNAREGTPGTPGTPBFAGDPGDP ARFAcyl-CoAATPCoatomerBuddingGTPGTPGTPGTPGTPGTPGTPGTPNocodazole8GTP-γ-SP iGDPSNAPsSNAPsCa 2+NSFNSF20S fusionparticleATPNEMFusion567Targetmembrane(eg, CGN)Figure 46–7. Model of the steps in a round of anterograde vesicular transport. Thecycle starts in the bottom left-hand side of the figure, where two molecules of ARFare represented as small ovals containing GDP. The steps in the cycle are described inthe text. Most of the abbreviations used are explained in Table 46–7. The roles of Raband Sec1 proteins (see text) in the overall process are not dealt with in this figure.(CGN, cis-Golgi network; BFA, Brefeldin A.) (Adapted from Rothman JE: Mechanisms ofintracellular protein transport. Nature 1994;372:55.) (Courtesy of E Degen.)Step 8: Retrograde transport occurs to restart thecycle. This last step may retrieve certain proteinsor recycle v-SNAREs. Nocodazole, a microtubuledisruptingagent, inhibits this step.Brefeldin A Inhibits the Coating ProcessThe following points expand and clarify the above.(a) To participate in step 1, ARF must first be modifiedby addition of myristic acid (C14:0), employingmyristoyl-CoA as the acyl donor. Myristoylation is oneof a number of enzyme-catalyzed posttranslational modifications,involving addition of certain lipids to specificresidues of proteins, that facilitate the binding of proteinsto the cytosolic surfaces of membranes or vesicles.Others are addition of palmitate, farnesyl, and geranylgeranyl;the two latter molecules are polyisoprenoidscontaining 15 and 20 carbon atoms, respectively.(b) At least three different types of coated vesicleshave been distinguished: COPI, COPII, and clathrincoatedvesicles; the first two are referred to here astransport vesicles. Many other types of vesicles nodoubt remain to be discovered. COPI vesicles are involvedin bidirectional transport from the ER to theGolgi and in the reverse direction, whereas COPII vesiclesare involved mainly in transport in the former direction.Clathrin-containing vesicles are involved intransport from the trans-Golgi network to prelysosomesand from the plasma membrane to endosomes, respectively.Regarding selection of cargo molecules by vesicles,this appears to be primarily a function of the coatproteins of vesicles. Cargo molecules may interact withcoat proteins either directly or via intermediary proteinsthat attach to coat proteins, and they then become enclosedin their appropriate vesicles.(c) The fungal metabolite brefeldin A preventsGTP from binding to ARF in step 1 and thus inhibitsthe entire coating process. In its presence, the Golgi apparatusappears to disintegrate, and fragments are lost.It may do this by inhibiting the guanine nucleotide exchangerinvolved in step 1.(d) GTP--S (a nonhydrolyzable analog of GTPoften used in investigations of the role of GTP in biochemicalprocesses) blocks disassembly of the coat fromcoated vesicles, leading to a build-up of coated vesicles.

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 511(e) A family of Ras-like proteins, called the Rab proteinfamily, are required in several steps of intracellularprotein transport, regulated secretion, and endocytosis.They are small monomeric GTPases that attach to thecytosolic faces of membranes via geranylgeranyl chains.They attach in the GTP-bound state (not shown inFigure 46–7) to the budding vesicle. Another family ofproteins (Sec1) binds to t-SNAREs and prevents interactionwith them and their complementary v-SNAREs.When a vesicle interacts with its target membrane, Rabproteins displace Sec1 proteins and the v-SNAREt-SNAREinteraction is free to occur. It appears thatthe Rab and Sec1 families of proteins regulate the speedof vesicle formation, opposing each other. Rab proteinshave been likened to throttles and Sec1 proteins todampers on the overall process of vesicle formation.(f) Studies using v- and t-SNARE proteins reconstitutedinto separate lipid bilayer vesicles have indicatedthat they form SNAREpins, ie, SNARE complexes thatlink two membranes (vesicles). SNAPs and NSF are requiredfor formation of SNAREpins, but once theyhave formed they can apparently lead to spontaneousfusion of membranes at physiologic temperature, suggestingthat they are the minimal machinery requiredfor membrane fusion.(g) The fusion of synaptic vesicles with the plasmamembrane of neurons involves a series of events similarto that described above. For example, one v-SNARE isdesignated synaptobrevin and two t-SNAREs are designatedsyntaxin and SNAP 25 (synaptosome-associatedprotein of 25 kDa). Botulinum B toxin is one ofthe most lethal toxins known and the most seriouscause of food poisoning. One component of this toxinis a protease that appears to cleave only synaptobrevin,thus inhibiting release of acetylcholine at the neuromuscularjunction and possibly proving fatal, dependingon the dose taken.(h) Although the above model describes nonclathrin-coatedvesicles, it appears likely that many ofthe events described above apply, at least in principle,to clathrin-coated vesicles.THE ASSEMBLY OF MEMBRANESIS COMPLEXThere are many cellular membranes, each with its ownspecific features. No satisfactory scheme describing theassembly of any one of these membranes is available.How various proteins are initially inserted into themembrane of the ER has been discussed above. Thetransport of proteins, including membrane proteins, tovarious parts of the cell inside vesicles has also been described.Some general points about membrane assemblyremain to be addressed.Asymmetry of Both Proteins & Lipids IsMaintained During Membrane AssemblyVesicles formed from membranes of the ER and Golgiapparatus, either naturally or pinched off by homogenization,exhibit transverse asymmetries of both lipidand protein. These asymmetries are maintained duringfusion of transport vesicles with the plasma membrane.The inside of the vesicles after fusion becomes the outsideof the plasma membrane, and the cytoplasmic sideof the vesicles remains the cytoplasmic side of the membrane(Figure 46–8). Since the transverse asymmetry ofthe membranes already exists in the vesicles of the ERwell before they are fused to the plasma membrane, amajor problem of membrane assembly becomes understandinghow the integral proteins are inserted into thelipid bilayer of the ER. This problem was addressedearlier in this chapter.Phospholipids are the major class of lipid in membranes.The enzymes responsible for the synthesis ofphospholipids reside in the cytoplasmic surface of thecisternae of the ER. As phospholipids are synthesized atthat site, they probably self-assemble into thermodynamicallystable bimolecular layers, thereby expandingthe membrane and perhaps promoting the detachmentof so-called lipid vesicles from it. It has been proposedthat these vesicles travel to other sites, donating theirlipids to other membranes; however, little is knownabout this matter. As indicated above, cytosolic proteinsthat take up phospholipids from one membraneand release them to another (ie, phospholipid exchangeproteins) have been demonstrated; they probably play arole in contributing to the specific lipid composition ofvarious membranes.Lipids & Proteins Undergo Turnover atDifferent Rates in Different MembranesIt has been shown that the half-lives of the lipids of theER membranes of rat liver are generally shorter thanthose of its proteins, so that the turnover rates oflipids and proteins are independent. Indeed, differentlipids have been found to have different half-lives.Furthermore, the half-lives of the proteins of thesemembranes vary quite widely, some exhibiting short(hours) and others long (days) half-lives. Thus, individuallipids and proteins of the ER membranes appear tobe inserted into it relatively independently; this is thecase for many other membranes.The biogenesis of membranes is thus a complexprocess about which much remains to be learned. Oneindication of the complexity involved is to consider thenumber of posttranslational modifications that membraneproteins may be subjected to prior to attainingtheir mature state. These include proteolysis, assembly

512 / CHAPTER 46Membrane proteinExterior surfaceTable 46–8. Major features of membraneassembly.LumenIntegralproteinCVesiclemembraneNNCCytoplasmPlasmamembrane• Lipids and proteins are inserted independently into membranes.• Individual membrane lipids and proteins turn over independentlyand at different rates.• Topogenic sequences (eg, signal [amino terminal or internal]and stop-transfer) are important in determining the insertionand disposition of proteins in membranes.• Membrane proteins inside transport vesicles bud off the endoplasmicreticulum on their way to the Golgi; final sortingof many membrane proteins occurs in the trans-Golgi network.• Specific sorting sequences guide proteins to particularorganelles such as lysosomes, peroxisomes, and mitochondria.NNNNCinto multimers, glycosylation, addition of a glycophosphatidylinositol(GPI) anchor, sulfation on tyrosine orcarbohydrate moieties, phosphorylation, acylation, andprenylation—a list that is undoubtedly not complete.Nevertheless, significant progress has been made; Table46–8 summarizes some of the major features of membraneassembly that have emerged to date.Table 46–9. Some disorders due to mutations ingenes encoding proteins involved in intracellularmembrane transport. 1CCFigure 46–8. Fusion of a vesicle with the plasmamembrane preserves the orientation of any integralproteins embedded in the vesicle bilayer. Initially, theamino terminal of the protein faces the lumen, or innercavity, of such a vesicle. After fusion, the amino terminalis on the exterior surface of the plasma membrane.That the orientation of the protein has not been reversedcan be perceived by noting that the other endof the molecule, the carboxyl terminal, is always immersedin the cytoplasm. The lumen of a vesicle andthe outside of the cell are topologically equivalent. (Redrawnand modified, with permission, from Lodish HF,Rothman JE: The assembly of cell membranes. Sci Am[Jan] 1979;240:43.)Disorder 2Protein InvolvedChédiak-Higashi syndrome, Lysosomal trafficking regula-214500 torCombined deficiency of factors ERGIC-53, a mannose-V and VIII, 227300binding lectinHermansky-Pudlak syndrome, AP-3 adaptor complex β3A203300 subunitI-cell disease, 252500N-Acetylglucosamine1-phosphotransferaseOculocerebrorenal syndrome, OCRL-1, an inositol poly-30900 phosphate 5-phosphatase1 Modified from Olkonnen VM, Ikonen E: Genetic defects of intracellular-membranetransport. N Eng J Med 2000;343:1095. Certainrelated conditions not listed here are also described in this publication.I-cell disease is described in Chapter 47. The majority ofthe disorders listed above affect lysosomal function; readersshould consult a textbook of medicine for information on theclinical manifestations of these conditions.2 The numbers after each disorder are the OMIM numbers.

INTRACELLULAR TRAFFIC & SORTING OF PROTEINS / 513Various Disorders Result From Mutationsin Genes Encoding Proteins Involvedin Intracellular TransportSome of these are listed in Table 46–9; the majority affectlysosomal function. A number of other mutationsaffecting intracellular protein transport have been reportedbut are not included here.SUMMARY• Many proteins are targeted to their destinations bysignal sequences. A major sorting decision is madewhen proteins are partitioned between cytosolic andmembrane-bound polyribosomes by virtue of the absenceor presence of a signal peptide.• The pathways of protein import into mitochondria,nuclei, peroxisomes, and the endoplasmic reticulumare described.• Many proteins synthesized on membrane-boundpolyribosomes proceed to the Golgi apparatus andthe plasma membrane in transport vesicles.• A number of glycosylation reactions occur in compartmentsof the Golgi, and proteins are furthersorted in the trans-Golgi network.• Most proteins destined for the plasma membraneand for secretion appear to lack specific signals—adefault mechanism.• The role of chaperone proteins in the folding of proteinsis presented, and a model describing buddingand attachment of transport vesicles to a target membraneis summarized.• Membrane assembly is discussed and shown to becomplex. Asymmetry of both lipids and proteins ismaintained during membrane assembly.• A number of disorders have been shown to be due tomutations in genes encoding proteins involved invarious aspects of protein traffic and sorting.REFERENCESFuller GM, Shields DL: Molecular Basis of Medical Cell Biology.McGraw-Hill, 1998.Gould SJ et al: The peroxisome biogenesis disorders. In: The Metabolicand Molecular Bases of Inherited Disease, 8th ed. ScriverCR et al (editors). McGraw-Hill, 2001.Graham JM, Higgins JA: Membrane Analysis. BIOS Scientific,1997.Griffith J, Sansom C: The Transporter Facts Book. Academic Press,1998.Lodish H et al: Molecular Cell Biology, 4th ed. Freeman, 2000.(Chapter 17 contains comprehensive coverage of protein sortingand organelle biogenesis.)Olkkonen VM, Ikonen E: Genetic defects of intracellular-membranetransport. N Engl J Med 2000;343:1095.Reithmeier RAF: Assembly of proteins into membranes. In: Biochemistryof Lipids, Lipoproteins and Membranes. Vance DE,Vance JE (editors). Elsevier, 1996.Sabatini DD, Adesnik MB: The biogenesis of membranes and organelles.In: The Metabolic and Molecular Bases of InheritedDisease, 8th ed. Scriver CR et al (editors). McGraw-Hill,2001.

Glycoproteins 47Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEGlycoproteins are proteins that contain oligosaccharide(glycan) chains covalently attached to theirpolypeptide backbones. They are one class of glycoconjugateor complex carbohydrates—equivalent termsused to denote molecules containing one or more carbohydratechains covalently linked to protein (to formglycoproteins or proteoglycans) or lipid (to form glycolipids).(Proteoglycans are discussed in Chapter 48 andglycolipids in Chapter 14). Almost all the plasma proteinsof humans—except albumin—are glycoproteins.Many proteins of cellular membranes (Chapter 41)contain substantial amounts of carbohydrate. A numberof the blood group substances are glycoproteins,whereas others are glycosphingolipids. Certain hormones(eg, chorionic gonadotropin) are glycoproteins.A major problem in cancer is metastasis, the phenomenonwhereby cancer cells leave their tissue of origin (eg,the breast), migrate through the bloodstream to somedistant site in the body (eg, the brain), and grow therein an unregulated manner, with catastrophic results forthe affected individual. Many cancer researchers thinkthat alterations in the structures of glycoproteins andother glycoconjugates on the surfaces of cancer cells areimportant in the phenomenon of metastasis.GLYCOPROTEINS OCCUR WIDELY& PERFORM NUMEROUS FUNCTIONSGlycoproteins occur in most organisms, from bacteriato humans. Many viruses also contain glycoproteins,some of which have been much investigated, in part becausethey are very suitable for biosynthetic studies.Numerous proteins with diverse functions are glycoproteins(Table 47–1); their carbohydrate content rangesfrom 1% to over 85% by weight.Many studies have been conducted in an attempt todefine the precise roles oligosaccharide chains play inthe functions of glycoproteins. Table 47–2 summarizesresults from such studies. Some of the functions listedare firmly established; others are still under investigation.OLIGOSACCHARIDE CHAINS ENCODEBIOLOGIC INFORMATIONAn enormous number of glycosidic linkages can be generatedbetween sugars. For example, three different hexosesmay be linked to each other to form over 1000 differenttrisaccharides. The conformations of the sugars inoligosaccharide chains vary depending on their linkagesand proximity to other molecules with which the oligosaccharidesmay interact. It is now established that certainoligosaccharide chains encode considerable biologic informationand that this depends upon their constituentsugars, their sequences, and their linkages. For instance,mannose 6-phosphate residues target newly synthesizedlysosomal enzymes to that organelle (see below).TECHNIQUES ARE AVAILABLEFOR DETECTION, PURIFICATION,& STRUCTURAL ANALYSISOF GLYCOPROTEINSA variety of methods used in the detection, purification,and structural analysis of glycoproteins are listedin Table 47–3. The conventional methods used to purifyproteins and enzymes are also applicable to the purificationof glycoproteins. Once a glycoprotein hasbeen purified, the use of mass spectrometry and highresolutionNMR spectroscopy can often identify thestructures of its glycan chains. Analysis of glycoproteinscan be complicated by the fact that they often exist asglycoforms; these are proteins with identical aminoacid sequences but somewhat different oligosaccharidecompositions. Although linkage details are not stressedin this chapter, it is critical to appreciate that the precisenatures of the linkages between the sugars of glycoproteinsare of fundamental importance in determining thestructures and functions of these molecules.514

GLYCOPROTEINS / 515Table 47–1. Some functions servedby glycoproteins.FunctionGlycoproteinsStructural molecule CollagensLubricant andMucinsprotective agentTransport molecule Transferrin, ceruloplasminImmunologic molecule Immunoglobulins, histocompatibilityantigensHormoneChorionic gonadotropin, thyroidstimulatinghormone (TSH)EnzymeVarious, eg, alkaline phosphataseCell attachment- Various proteins involved in cell-cellrecognition site (eg, sperm-oocyte), virus-cell,bacterium-cell, and hormone-cellinteractionsAntifreezeCertain plasma proteins of coldwater fishInteract with specific Lectins, selectins (cell adhesioncarbohydrates lectins), antibodiesReceptorVarious proteins involved in hormoneand drug actionAffect folding of Calnexin, calreticulincertain proteinsTable 47–2. Some functions of theoligosaccharide chains of glycoproteins. 1• Modulate physicochemical properties, eg, solubility, viscosity,charge, conformation, denaturation, and bindingsites for bacteria and viruses• Protect against proteolysis, from inside and outside of cell• Affect proteolytic processing of precursor proteins tosmaller products• Are involved in biologic activity, eg, of human chorionicgonadotropin (hCG)• Affect insertion into membranes, intracellular migration,sorting and secretion• Affect embryonic development and differentiation• May affect sites of metastases selected by cancer cells1 Adapted from Schachter H: Biosynthetic controls that determinethe branching and heterogeneity of protein-bound oligosaccharides.Biochem Cell Biol 1986;64:163.Table 47–3. Some important methods used tostudy glycoproteins.MethodUsePeriodic acid-Schiff reagent Detects glycoproteins as pinkbands after electrophoretic separation.Incubation of cultured cells Leads to detection of a radiowithglycoproteins as active sugar after electrophoradioactivebands retic separation.Treatment with appropriate Resultant shifts in electrophoendo-or exoglycosidase retic migration help distinguishor phospholipases among proteins with N-glycan,O-glycan, or GPI linkages andalso between high mannoseand complex N-glycans.Sepharose-lectin column To purify glycoproteins or glychromatographycopeptides that bind the particularlectin used.Compositional analysis Identifies sugars that the glyfollowingacid hydrolysis coprotein contains and theirstoichiometry.Mass spectrometryProvides information on molecularmass, composition, sequence,and sometimes branchingof a glycan chain.NMR spectroscopyTo identify specific sugars, theirsequence, linkages, and theanomeric nature of glycosidiclinkages.Methylation (linkage) To determine linkages betweenanalysissugars.Amino acid or cDNA Determination of amino acidsequencingsequence.EIGHT SUGARS PREDOMINATEIN HUMAN GLYCOPROTEINSAbout 200 monosaccharides are found in nature; however,only eight are commonly found in the oligosaccharidechains of glycoproteins (Table 47–4). Most ofthese sugars were described in Chapter 13. N-Acetylneuraminicacid (NeuAc) is usually found at the terminiof oligosaccharide chains, attached to subterminalgalactose (Gal) or N-acetylgalactosamine (GalNAc)residues. The other sugars listed are generally found inmore internal positions. Sulfate is often found in glycoproteins,usually attached to Gal, GalNAc, or GlcNAc.

516 / CHAPTER 47Table 47–4. The principal sugars found in human glycoproteins. Their structures are illustrated inChapter 13.NucleotideSugar Type Abbreviation Sugar CommentsGalactose Hexose Gal UDP-Gal Often found subterminal to NeuAc in N-linked glycoproteins.Also found in core trisaccharide of proteoglycans.Glucose Hexose Glc UDP-Glc Present during the biosynthesis of N-linked glycoproteinsbut not usually present in mature glycoproteins.Present in some clotting factors.Mannose Hexose Man GDP-Man Common sugar in N-linked glycoproteins.N-Acetylneur- Sialic acid (nine NeuAc CMP-NeuAc Often the terminal sugar in both N- and O-linkedaminic acid C atoms) glycoproteins. Other types of sialic acid are alsofound, but NeuAc is the major species found in humans.Acetyl groups may also occur as O-acetylspecies as well as N-acetyl.Fucose Deoxyhexose Fuc GDP-Fuc May be external in both N- and O-linked glycoproteinsor internal, linked to the GlcNAc residue attachedto Asn in N-linked species. Can also occurinternally attached to the OH of Ser (eg, in t-PA andcertain clotting factors).N-Acetylgalactosamine Aminohexose GalNAc UDP-GalNAc Present in both N- and O-linked glycoproteins.N-Acetylglucosamine Aminohexose GlcNAc UDP-GlcNAc The sugar attached to the polypeptide chain viaAsn in N-linked glycoproteins; also found at othersites in the oligosaccharides of these proteins.Many nuclear proteins have GlcNAc attached to theOH of Ser or Thr as a single sugar.Xylose Pentose Xyl UDP-Xyl Xyl is attached to the OH of Ser in many proteoglycans.Xyl in turn is attached to two Gal residues,forming a link trisaccharide. Xyl is also found in t-PAand certain clotting factors.NUCLEOTIDE SUGARS ACTAS SUGAR DONORS IN MANYBIOSYNTHETIC REACTIONSThe first nucleotide sugar to be reported was uridinediphosphate glucose (UDP-Glc); its structure is shownin Figure 18–2. The common nucleotide sugars involvedin the biosynthesis of glycoproteins are listed inTable 47–4; the reasons some contain UDP and othersguanosine diphosphate (GDP) or cytidine monophosphate(CMP) are obscure. Many of the glycosylation reactionsinvolved in the biosynthesis of glycoproteinsutilize these compounds (see below). The anhydro natureof the linkage between the phosphate group andthe sugars is of the high-energy, high-group-transferpotentialtype (Chapter 10). The sugars of these compoundsare thus “activated” and can be transferred tosuitable acceptors provided appropriate transferases areavailable.Most nucleotide sugars are formed in the cytosol,generally from reactions involving the correspondingnucleoside triphosphate. CMP-sialic acids are formedin the nucleus. Formation of uridine diphosphate galactose(UDP-Gal) requires the following two reactions inmammalian tissues:UTP + Glucose 1-phosphateUDP-GlcUDP-GlcPYROPHOS-PHORYLASEUDP-GlcEPIMERASEUDP-Glc + PyrophosphateUDP-Gal

GLYCOPROTEINS / 517Because many glycosylation reactions occur withinthe lumen of the Golgi apparatus, carrier systems (permeases,transporters) are necessary to transport nucleotidesugars across the Golgi membrane. Systemstransporting UDP-Gal, GDP-Man, and CMP-NeuAcinto the cisternae of the Golgi apparatus have been described.They are antiport systems; ie, the influx of onemolecule of nucleotide sugar is balanced by the effluxof one molecule of the corresponding nucleotide (eg,UMP, GMP, or CMP) formed from the nucleotidesugars. This mechanism ensures an adequate concentrationof each nucleotide sugar inside the Golgi apparatus.UMP is formed from UDP-Gal in the aboveprocess as follows:UDP-Gal + ProteinUDPEXO- & ENDOGLYCOSIDASESFACILITATE STUDYOF GLYCOPROTEINSA number of glycosidases of defined specificity haveproved useful in examining structural and functionalaspects of glycoproteins (Table 47–5). These enzymesact at either external (exoglycosidases) or internal (endoglycosidases)positions of oligosaccharide chains. Examplesof exoglycosidases are neuraminidases andgalactosidases; their sequential use removes terminalNeuAc and subterminal Gal residues from most glycoproteins.Endoglycosidases F and H are examples ofthe latter class; these enzymes cleave the oligosaccharidechains at specific GlcNAc residues close to the polypep-GALACTOSYL-TRANSFERASENUCLEOSIDEDIPHOSPHATEPHOSPHATASEProteinUMP + P iGal + UDPTable 47–5. Some glycosidases used to study thestructure and function of glycoproteins. 1EnzymesTypeNeuraminidasesExoglycosidaseGalactosidasesExo- or endoglycosidaseEndoglycosidase FEndoglycosidaseEndoglycosidase HEndoglycosidase1 The enzymes are available from a variety of sources and are oftenspecific for certain types of glycosidic linkages and also for theiranomeric natures. The sites of action of endoglycosidases F and Hare shown in Figure 47–5. F acts on both high-mannose and complexoligosaccharides, whereas H acts on the former.tide backbone (ie, at internal sites; Figure 47–5) and arethus useful in releasing large oligosaccharide chains forstructural analyses. A glycoprotein can be treated withone or more of the above glycosidases to analyze theeffects on its biologic behavior of removal of specificsugars.THE MAMMALIANASIALOGLYCOPROTEIN RECEPTORIS INVOLVED IN CLEARANCEOF CERTAIN GLYCOPROTEINSFROM PLASMA BY HEPATOCYTESExperiments performed by Ashwell and his colleaguesin the early 1970s played an important role in focusingattention on the functional significance of the oligosaccharidechains of glycoproteins. They treated rabbitceruloplasmin (a plasma protein; see Chapter 50) withneuraminidase in vitro. This procedure exposed subterminalGal residues that were normally masked by terminalNeuAc residues. Neuraminidase-treated radioactiveceruloplasmin was found to disappear rapidly fromthe circulation, in contrast to the slow clearance of theuntreated protein. Very significantly, when the Galresidues exposed to treatment with neuraminidase wereremoved by treatment with a galactosidase, the clearancerate of the protein returned to normal. Furtherstudies demonstrated that liver cells contain a mammalianasialoglycoprotein receptor that recognizesthe Gal moiety of many desialylated plasma proteinsand leads to their endocytosis. This work indicated thatan individual sugar, such as Gal, could play an importantrole in governing at least one of the biologic properties(ie, time of residence in the circulation) of certainglycoproteins. This greatly strengthened the conceptthat oligosaccharide chains could contain biologic information.LECTINS CAN BE USED TOPURIFY GLYCOPROTEINS& TO PROBE THEIR FUNCTIONSLectins are carbohydrate-binding proteins that agglutinatecells or precipitate glycoconjugates; a number oflectins are themselves glycoproteins. Immunoglobulinsthat react with sugars are not considered lectins. Lectinscontain at least two sugar-binding sites; proteins with asingle sugar-binding site will not agglutinate cells orprecipitate glycoconjugates. The specificity of a lectin isusually defined by the sugars that are best at inhibitingits ability to cause agglutination or precipitation. Enzymes,toxins, and transport proteins can be classifiedas lectins if they bind carbohydrate. Lectins were firstdiscovered in plants and microbes, but many lectins of

518 / CHAPTER 47animal origin are now known. The mammalian asialoglycoproteinreceptor described above is an importantexample of an animal lectin. Some important lectins arelisted in Table 47–6. Much current research is centeredon the roles of various animal lectins (eg, the selectins)in cell-cell interactions that occur in pathologic conditionssuch as inflammation and cancer metastasis (seebelow).Numerous lectins have been purified and are commerciallyavailable; three plant lectins that have beenwidely used experimentally are listed in Table 47–7.Among many uses, lectins have been employed to purifyspecific glycoproteins, as tools for probing the glycoproteinprofiles of cell surfaces, and as reagents forgenerating mutant cells deficient in certain enzymes involvedin the biosynthesis of oligosaccharide chains.THERE ARE THREE MAJOR CLASSESOF GLYCOPROTEINSBased on the nature of the linkage between their polypeptidechains and their oligosaccharide chains, glycoproteinscan be divided into three major classes (Figure47–1): (1) those containing an O-glycosidic linkage (ie,Table 47–6. Some important lectins.LectinsExamples or CommentsLegume lectins Concanavalin A, pea lectinWheat germ Widely used in studies of surfaces of noragglutininmal cells and cancer cellsRicinCytotoxic glycoprotein derived from seedsof the castor plantBacterial toxins Heat-labile enterotoxin of E coli andcholera toxinInfluenza virus Responsible for host-cell attachment andhemagglutinin membrane fusionC-type lectins Characterized by a Ca 2+ -dependent carbohydraterecognition domain (CRD); includesthe mammalian asialoglycoproteinreceptor, the selectins, and the mannosebindingproteinS-type lectins β-Galactoside-binding animal lectins withroles in cell-cell and cell-matrix interactionsP-type lectins Mannose 6-P receptorl-type lectins Members of the immunoglobulin superfamily,eg, sialoadhesin mediating adhesionof macrophages to various cellsO-linked), involving the hydroxyl side chain of serineor threonine and a sugar such as N-acetylgalactosamine(GalNAc-Ser[Thr]); (2) those containing an N-glycosidiclinkage (ie, N-linked), involving the amide nitrogenof asparagine and N-acetylglucosamine (GlcNAc-Asn); and (3) those linked to the carboxyl terminalamino acid of a protein via a phosphoryl-ethanolaminemoiety joined to an oligosaccharide (glycan), which inturn is linked via glucosamine to phosphatidylinositol(PI). This latter class is referred to as glycosylphosphatidylinositol-anchored(GPI-anchored, or GPIlinked)glycoproteins. Other minor classes of glycoproteinsalso exist.The number of oligosaccharide chains attached toone protein can vary from one to 30 or more, with thesugar chains ranging from one or two residues inlength to much larger structures. Many proteins containmore than one type of linkage; for instance, glycophorin,an important red cell membrane glycoprotein(Chapter 52), contains both O- and N-linkedoligosaccharides.GLYCOPROTEINS CONTAIN SEVERALTYPES OF O-GLYCOSIDIC LINKAGESAt least four subclasses of O-glycosidic linkages arefound in human glycoproteins: (1) The GalNAc-Ser(Thr) linkage shown in Figure 47–1 is the predominantlinkage. Two typical oligosaccharide chains foundin members of this subclass are shown in Figure 47–2.Usually a Gal or a NeuAc residue is attached to theGalNAc, but many variations in the sugar compositionsand lengths of such oligosaccharide chains are found.This type of linkage is found in mucins (see below).(2) Proteoglycans contain a Gal-Gal-Xyl-Ser trisaccharide(the so-called link trisaccharide). (3) Collagenscontain a Gal-hydroxylysine (Hyl) linkage. (Subclasses[2] and [3] are discussed further in Chapter 48.)(4) Many nuclear proteins (eg, certain transcriptionfactors) and cytosolic proteins contain side chains consistingof a single GlcNAc attached to a serine or threonineresidue (GlcNAc-Ser[Thr]).Table 47–7. Three plant lectins and the sugarswith which they interact. 1Lectin Abbreviation SugarsConcanavalin A ConA Man and GlcSoybean lectinGal and GalNAcWheat germ agglutinin WGA Glc and NeuAc1 In most cases, lectins show specificity for the anomeric nature ofthe glycosidic linkage (α or β); this is not indicated in the table.

GLYCOPROTEINS / 519AOHCHCH 2 OHCHOHCHHC NH 2CH 3Ethanolamine P MannoseOαProteinHC O CH 2 CGlycineHCSerEthanolamineNPC OMannoseMannoseBCH 2 OHGlucosamineHCHOCOHCOHCH HβOC N CAsnCH 2CPI-PLCPInositolAdditional fatty acidPlasmamembraneHHNCCH 3OFigure 47–1. Depictions of (A) an O-linkage (N-acetylgalactosamine to serine); (B) an N-linkage (N-acetylglucosamineto asparagine) and (C) a glycosylphosphatidylinositol (GPI) linkage. The GPI structure shown is thatlinking acetylcholinesterase to the plasma membrane of the human red blood cell. The carboxyl terminal aminoacid is glycine joined in amide linkage via its COOH group to the NH 2 group of phosphorylethanolamine, whichin turn is joined to a mannose residue. The core glycan contains three mannose and one glucosamine residues.The glucosamine is linked to inositol, which is attached to phosphatidic acid. The site of action of PI-phospholipaseC (PI-PLC) is indicated. The structure of the core glycan is shown in the text. This particular GPI contains anextra fatty acid attached to inositol and also an extra phosphorylethanolamine moiety attached to the middle ofthe three mannose residues. Variations found among different GPI structures include the identity of the carboxylterminal amino acid, the molecules attached to the mannose residues, and the precise nature of the lipid moiety.ANeuAcBGalα 2,6β 1,3α 2,3NeuAcGalNAcGalNAcα 2,6NeuAcSer(Thr)Ser(Thr)Figure 47–2. Structures of two O-linked oligosaccharidesfound in (A) submaxillary mucins and (B) fetuinand in the sialoglycoprotein of the membrane ofhuman red blood cells. (Modified and reproduced, withpermission, from Lennarz WJ: The Biochemistry of Glycoproteinsand Proteoglycans. Plenum Press, 1980.)Mucins Have a High Content of O-LinkedOligosaccharides & Exhibit RepeatingAmino Acid SequencesMucins are glycoproteins with two major characteristics:(1) a high content of O-linked oligosaccharides(the carbohydrate content of mucins is generally morethan 50%); and (2) the presence of repeating aminoacid sequences (tandem repeats) in the center of theirpolypeptide backbones, to which the O-glycan chainsare attached in clusters (Figure 47–3). These sequencesare rich in serine, threonine, and proline. Although O-glycans predominate, mucins often contain a numberof N-glycan chains. Both secretory and membraneboundmucins occur. The former are found in themucus present in the secretions of the gastrointestinal,respiratory, and reproductive tracts. Mucus consists ofabout 94% water and 5% mucins, with the remainder

520 / CHAPTER 47NO-glycan chainTandem repeat sequenceN-glycan chainFigure 47–3. Schematic diagram of a mucin. O-glycanchains are shown attached to the central region ofthe extended polypeptide chain and N-glycan chains tothe carboxyl terminal region. The narrow rectanglesrepresent a series of tandem repeat amino acid sequences.Many mucins contain cysteine residues whoseSH groups form interchain linkages; these are notshown in the figure. (Adapted from Strous GJ, Dekker J:Mucin-type glycoproteins. Crit Rev Biochem Mol Biol1992;27:57.)being a mixture of various cell molecules, electrolytes,and remnants of cells. Secretory mucins generally havean oligomeric structure and thus often have a very highmolecular mass. The oligomers are composed ofmonomers linked by disulfide bonds. Mucus exhibits ahigh viscosity and often forms a gel. These qualities arefunctions of its content of mucins. The high content ofO-glycans confers an extended structure on mucins.This is in part explained by steric interactions betweentheir GalNAc moieties and adjacent amino acids, resultingin a chain-stiffening effect so that the conformationsof mucins often become those of rigid rods. Intermolecularnoncovalent interactions between varioussugars on neighboring glycan chains contribute to gelformation. The high content of NeuAc and sulfateresidues found in many mucins confers a negativecharge on them. With regard to function, mucins helplubricate and form a protective physical barrier onepithelial surfaces. Membrane-bound mucins participatein various cell-cell interactions (eg, involving selectins;see below). The density of oligosaccharidechains makes it difficult for proteases to approach theirpolypeptide backbones, so that mucins are often resistantto their action. Mucins also tend to “mask” certainsurface antigens. Many cancer cells form excessiveamounts of mucins; perhaps the mucins may mask certainsurface antigens on such cells and thus protect thecells from immune surveillance. Mucins also carry cancer-specificpeptide and carbohydrate epitopes (an epitopeis a site on an antigen recognized by an antibody,also called an antigenic determinant). Some of theseCepitopes have been used to stimulate an immune responseagainst cancer cells.The genes encoding the polypeptide backbones of anumber of mucins derived from various tissues (eg,pancreas, small intestine, trachea and bronchi, stomach,and salivary glands) have been cloned and sequenced.These studies have revealed new information about thepolypeptide backbones of mucins (size of tandem repeats,potential sites of N-glycosylation, etc) and ultimatelyshould reveal aspects of their genetic control.Some important properties of mucins are summarizedin Table 47–8.The Biosynthesis of O-LinkedGlycoproteins Uses Nucleotide SugarsThe polypeptide chains of O-linked and other glycoproteinsare encoded by mRNA species; because mostglycoproteins are membrane-bound or secreted, theyare generally translated on membrane-bound polyribosomes(Chapter 38). Hundreds of different oligosaccharidechains of the O-glycosidic type exist. These glycoproteinsare built up by the stepwise donation ofsugars from nucleotide sugars, such as UDP-GalNAc,UDP-Gal, and CMP-NeuAc. The enzymes catalyzingthis type of reaction are membrane-bound glycoproteinglycosyltransferases. Generally, synthesis of onespecific type of linkage requires the activity of a correspondinglyspecific transferase. The factors that determinewhich specific serine and threonine residues areglycosylated have not been identified but are probablyfound in the peptide structure surrounding the glycosylationsite. The enzymes assembling O-linked chains arelocated in the Golgi apparatus, sequentially arranged inan assembly line with terminal reactions occurring inthe trans-Golgi compartments.The major features of the biosynthesis of O-linkedglycoproteins are summarized in Table 47–9.Table 47–8. Some properties of mucins.• Found in secretions of the gastrointestinal, respiratory,and reproductive tracts and also in membranes of variouscells.• Exhibit high content of O-glycan chains, usually containingNeuAc.• Contain repeating amino acid sequences rich in serine, threonine,and proline.• Extended structure contributes to their high viscoelasticity.• Form protective physical barrier on epithelial surfaces, areinvolved in cell-cell interactions, and may contain or maskcertain surface antigens.

GLYCOPROTEINS / 521Table 47–9. Summary of main featuresof O-glycosylation.• Involves a battery of membrane-bound glycoprotein glycosyltransferasesacting in a stepwise manner; each transferaseis generally specific for a particular type of linkage.• The enzymes involved are located in various subcompartmentsof the Golgi apparatus.• Each glycosylation reaction involves the appropriatenucleotide-sugar.• Dolichol-P-P-oligosaccharide is not involved, nor are glycosidases;and the reactions are not inhibited by tunicamycin.• O-Glycosylation occurs posttranslationally at certain Ser andThr residues.N-LINKED GLYCOPROTEINS CONTAINAN Asn-GlcNAc LINKAGEN-Linked glycoproteins are distinguished by the presenceof the Asn-GlcNAc linkage (Figure 47–1). It is themajor class of glycoproteins and has been much studied,since the most readily accessible glycoproteins (eg,plasma proteins) mainly belong to this group. It includesboth membrane-bound and circulating glycoproteins.The principal difference between this and theprevious class, apart from the nature of the amino acidto which the oligosaccharide chain is attached (Asn versusSer or Thr), concerns their biosynthesis.Complex, Hybrid, & High-MannoseAre the Three Major Classesof N-Linked OligosaccharidesThere are three major classes of N-linked oligosaccharides:complex, hybrid, and high-mannose (Figure47–4). Each type shares a common pentasaccharide,Man 3 GlcNAc 2 —shown within the boxed area in Figure47–4 and depicted also in Figure 47–5—but they differin their outer branches. The presence of the commonpentasaccharide is explained by the fact that all threeclasses share an initial common mechanism of biosynthesis.Glycoproteins of the complex type generallycontain terminal NeuAc residues and underlying Galand GlcNAc residues, the latter often constituting thedisaccharide N-acetyllactosamine. Repeating N-acetyllactosamineunits—[Galβ1–3/4GlcNAcβ1–3] n (poly-N-acetyllactosaminoglycans)—are often found on N-linked glycan chains. I/i blood group substances belongto this class. The majority of complex-type oligosaccharidescontain two, three, or four outer branches (Figure47–4), but structures containing five branches have alsobeen described. The oligosaccharide branches are oftenreferred to as antennae, so that bi-, tri-, tetra-, andpenta-antennary structures may all be found. A bewilderingnumber of chains of the complex type exist, andthat indicated in Figure 47–4 is only one of many.Other complex chains may terminate in Gal or Fuc.High-mannose oligosaccharides typically have two tosix additional Man residues linked to the pentasaccharidecore. Hybrid molecules contain features of both ofthe two other classes.The Biosynthesis of N-LinkedGlycoproteins InvolvesDolichol-P-P-OligosaccharideLeloir and his colleagues described the occurrence ofa dolichol-pyrophosphate-oligosaccharide (Dol-P-Poligosaccharide),which subsequent research showedto play a key role in the biosynthesis of N-linked glycoproteins.The oligosaccharide chain of this compoundgenerally has the structure R-GlcNAc 2 Man 9 Glc 3 (R =Dol-P-P). The sugars of this compound are first assembledon the Dol-P-P backbone, and the oligosaccharidechain is then transferred en bloc to suitable Asn residuesof acceptor apoglycoproteins during their synthesison membrane-bound polyribosomes. All N-glycanshave a common pentasaccharide core structure (Figure47–5).To form high-mannose chains, only the Glcresidues plus certain of the peripheral Man residues areremoved. To form an oligosaccharide chain of the complextype, the Glc residues and four of the Manresidues are removed by glycosidases in the endoplasmicreticulum and Golgi. The sugars characteristic ofcomplex chains (GlcNAc, Gal, NeuAc) are added bythe action of individual glycosyltransferases located inthe Golgi apparatus. The phenomenon whereby theglycan chains of N-linked glycoproteins are first partiallydegraded and then in some cases rebuilt is referredto as oligosaccharide processing. Hybrid chains areformed by partial processing, forming complex chainson one arm and Man structures on the other arm.Thus, the initial steps involved in the biosynthesis ofthe N-linked glycoproteins differ markedly from thoseinvolved in the biosynthesis of the O-linked glycoproteins.The former involves Dol-P-P-oligosaccharide; thelatter, as described earlier, does not.The process of N-glycosylation can be broken downinto two stages: (1) assembly of Dol-P-P-oligosaccharideand transfer of the oligosaccharide; and (2) processingof the oligosaccharide chain.A. ASSEMBLY & TRANSFER OFDOLICHOL-P-P-OLIGOSACCHARIDEPolyisoprenol compounds exist in both bacteria andeukaryotic cells. They participate in the synthesis ofbacterial polysaccharides and in the biosynthesis of N-

522 / CHAPTER 47α2,3 or 2,6Sialic acidSialic acidα2,3 or 2,6±GlcNAc±Fucβ1,4α1,6Gal Gal Gal Man Man Manβ1,4 β1,4 β1,4GlcNAcGlcNAcGlcNAcGlcNAcβ1,4 β1,4 β1,4GlcNAcGlcNAc GlcNAc Man Man Man Manβ1,2 β1,2 β1,2α1,3 α1,6Man Man Man Man Man Manα1,6α1,3 α1,6α1,3β1,4 α1,3 α1,6ManManGlcNAc Manβ1,4β1,4β1,4GlcNAcα1,2α1,2α1,2 α1,2GlcNAcManα1,3 α1,6AsnComplexAsnHybridAsnHigh-mannoseFigure 47–4. Structures of the major types of asparagine-linked oligosaccharides. The boxed area enclosesthe pentasaccharide core common to all N-linked glycoproteins. (Reproduced, with permission,from Kornfeld R, Kornfeld S: Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem1985;54:631.)linked glycoproteins and GPI anchors. The polyisoprenolused in eukaryotic tissues is dolichol, which is,next to rubber, the longest naturally occurring hydrocarbonmade up of a single repeating unit. Dolichol iscomposed of 17–20 repeating isoprenoid units (Figure47–6).Before it participates in the biosynthesis of Dol-P-Poligosaccharide,dolichol must first be phosphorylatedto form dolichol phosphate (Dol-P) in a reaction catalyzedby dolichol kinase and using ATP as the phosphatedonor.Dolichol-P-P-GlcNAc (Dol-P-P-GlcNAc) is thekey lipid that acts as an acceptor for other sugars in theassembly of Dol-P-P-oligosaccharide. It is synthesizedManα1,6Manα1,3Manβ1,4GlcNAcβ1,4Endoglycosidase HEndoglycosidase FGlcNAcAsnFigure 47–5. Schematic diagram of the pentasaccharidecore common to all N-linked glycoproteins andto which various outer chains of oligosaccharides maybe attached. The sites of action of endoglycosidases Fand H are also indicated.in the membranes of the endoplasmic reticulum fromDol-P and UDP-GlcNAc in the following reaction,catalyzed by GlcNAc-P transferase:Dol- P + UDP - GlcNAc → Dol- P - P - GlcNAc+ UMPThe above reaction—which is the first step in the assemblyof Dol-P-P-oligosaccharide—and the other laterreactions are summarized in Figure 47–7. The essentialfeatures of the subsequent steps in the assembly of Dol-P-P-oligosaccharide are as follows:(1) A second GlcNAc residue is added to the first,again using UDP-GlcNAc as the donor.(2) Five Man residues are added, using GDP-mannoseas the donor.(3) Four additional Man residues are next added,using Dol-P-Man as the donor. Dol-P-Man isformed by the following reaction:Dol-P + GDP -Man → Dol-P -Man + GDP(4) Finally, the three peripheral glucose residues aredonated by Dol-P-Glc, which is formed in a reactionanalogous to that just presented except thatDol-P and UDP-Glc are the substrates.

GLYCOPROTEINS / 523Figure 47–6. The structure of dolichol.The phosphate in dolichol phosphate is attachedto the primary alcohol group at theleft-hand end of the molecule. The groupwithin the brackets is an isoprene unit (n =17–20 isoprenoid units).CH 3HCH 3HO CH 2 CH 2 C CH 2 CH 2 CH C CH 2 CH 2nCHCH 3CCH 3It should be noted that the first seven sugars (twoGlcNAc and five Man residues) are donated by nucleotidesugars, whereas the last seven sugars (four Manand three Glc residues) added are donated by dolichol-P-sugars. The net result is assembly of the compoundillustrated in Figure 47–8 and referred to in shorthandas Dol-P-P-GlcNAc 2 Man 9 Glc 3 .The oligosaccharide linked to dolichol-P-P is transferreden bloc to form an N-glycosidic bond with oneor more specific Asn residues of an acceptor proteinemerging from the luminal surface of the membrane ofthe endoplasmic reticulum. The reaction is catalyzed byoligosaccharide:protein transferase, a membraneassociatedenzyme complex. The transferase will recognizeand transfer any substrate with the general structureDol-P-P-(GlcNAc) 2 -R, but it has a strongpreference for the Dol-P-P-GlcNAc 2 Man 9 Glc 3 structure.Glycosylation occurs at the Asn residue of an Asn-X-Ser/Thr tripeptide sequence, where X is any aminoacid except proline, aspartic acid, or glutamic acid. Atripeptide site contained within a β turn is favored.Only about one-third of the Asn residues that are potentialacceptor sites are actually glycosylated, suggestingthat factors other than the tripeptide are also important.The acceptor proteins are of both the secretoryand integral membrane class. Cytosolic proteins arerarely glycosylated. The transfer reaction and subsequentprocesses in the glycosylation of N-linked glycoproteins,along with their subcellular locations, are depictedin Figure 47–9. The other product of theoligosaccharide:protein transferase reaction is dolichol-P-P, which is subsequently converted to dolichol-P by aUDP-GIcNAcDol-PUMPTunicamycinUDP-GIcNAcGIcNAc P P DolMMMMMM (GIcNAc) 2 P P DolUDPG G GM M MP DolGIcNAc GIcNAc P P DolGDP-MM P Dol and G P Dol(M) (GIcNAc) P P DolGDP 62P DolM GIcNAc GIcNAc P P DolMM P DolM (GIcNAc) 2 P P Dol(GDP-M) M M M4 (GDP) 4Figure 47–7. Pathway of biosynthesis of dolichol-P-P-oligosaccharide. The specific linkages formed are indicatedin Figure 47–8. Note that the first five internal mannose residues are donated by GDP-mannose, whereasthe more external mannose residues and the glucose residues are donated by dolichol-P-mannose and dolichol-P-glucose. (UDP, uridine diphosphate; Dol, dolichol; P, phosphate; UMP, uridine monophosphate; GDP, guanosinediphosphate; M, mannose; G, glucose.)

524 / CHAPTER 47Man α1,2 GlcNAc P P DolicholManα1,6Glc α1,2 Glc α1,3ManMan α1,2α1,6α1,3Manβ1,4Man GlcNAcβ1,4α1,2 α1,3Glc α1,3 Man α1,2 Man ManFigure 47–8. Structure of dolichol-P-P-oligosaccharide. (Reproduced, with permission, from Lennarz WJ:The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, 1980.)αphosphatase. The dolichol-P can serve again as an acceptorfor the synthesis of another molecule of Dol-P-P-oligosaccharide.B. PROCESSING OF THE OLIGOSACCHARIDE CHAIN1. Early phase—The various reactions involved are indicatedin Figure 47–9. The oligosaccharide:proteintransferase catalyzes reaction 1 (see above). Reactions 2and 3 involve the removal of the terminal Glc residueby glucosidase I and of the next two Glc residues byglucosidase II, respectively. In the case of highmannoseglycoproteins, the process may stop here, orup to four Man residues may also be removed. However,to form complex chains, additional steps are necessary,as follows. Four external Man residues are removedin reactions 4 and 5 by at least two differentmannosidases. In reaction 6, a GlcNAc residue is addedto the Man residue of the Manα1–3 arm by GlcNActransferase I. The action of this latter enzyme permitsthe occurrence of reaction 7, a reaction catalyzed by yetanother mannosidase (Golgi α-mannosidase II) andwhich results in a reduction of the Man residues to thecore number of three (Figure 47–5).An important additional pathway is indicated in reactionsI and II of Figure 47–9. This involves enzymesdestined for lysosomes. Such enzymes are targeted tothe lysosomes by a specific chemical marker. In reactionI, a residue of GlcNAc-1-P is added to carbon 6 of oneor more specific Man residues of these enzymes. Thereaction is catalyzed by a GlcNAc phosphotransferase,which uses UDP-GlcNAc as the donor and generatesUMP as the other product:UDP-GlcNAc + ManGlcNAcPHOSPHO-TRANSFERASEProteinGlcNAc-1-P-6-Man Protein + UMPIn reaction II, the GlcNAc is removed by the action ofa phosphodiesterase, leaving the Man residues phosphorylatedin the 6 position:GlcNAc-1-P-6-ManProteinPHOSPHO-DIESTERASEP-6-ManProtein + GlcNAcMan 6-P receptors, located in the Golgi apparatus,bind the Man 6-P residue of these enzymes and directthem to the lysosomes. Fibroblasts from patients withI-cell disease (see below) are severely deficient in theactivity of the GlcNAc phosphotransferase.2. Late phase—To assemble a typical complexoligosaccharide chain, additional sugars must be addedto the structure formed in reaction 7. Hence, in reaction8, a second GlcNAc is added to the peripheralMan residue of the other arm of the bi-antennary structureshown in Figure 47–9; the enzyme catalyzing thisstep is GlcNAc transferase II. Reactions 9, 10, and 11involve the addition of Fuc, Gal, and NeuAc residues atthe sites indicated, in reactions catalyzed by fucosyl,galactosyl, and sialyl transferases, respectively. The assemblyof poly-N-acetyllactosamine chains requires additionalGlcNAc transferases.The Endoplasmic Reticulum& Golgi Apparatus Are theMajor Sites of GlycosylationAs indicated in Figure 47–9, the endoplasmic reticulumand the Golgi apparatus are the major sites involved inglycosylation processes. The assembly of Dol-P-Poligosaccharideoccurs on both the cytoplasmic and luminalsurfaces of the ER membranes. Addition of theoligosaccharide to protein occurs in the rough endoplasmicreticulum during or after translation. Removalof the Glc and some of the peripheral Man residues alsooccurs in the endoplasmic reticulum. The Golgi apparatusis composed of cis, medial, and trans cisternae;these can be separated by appropriate centrifugationprocedures. Vesicles containing glycoproteins appear tobud off in the endoplasmic reticulum and are transportedto the cis Golgi. Various studies have shown

GLYCOPROTEINS / 525ROUGH ENDOPLASMIC RETICULUM1 23 4PPDolGOLGI APPARATUSP– –P II –P– –P–I5cis6 7 8 9medialUDP-trans10 11UDP-UDP-UDP-GDP-CMP-ExitFigure 47–9. Schematic pathway of oligosaccharide processing. The reactions are catalyzedby the following enzymes: 1 , oligosaccharide:protein transferase; 2 , α-glucosidase I;3 , α-glucosidase II; 4 , endoplasmic reticulum α1,2-mannosidase; l , N-acetylglucosaminylphosphotransferase;ll , N-acetylglucosamine-1-phosphodiester α-N-acetylglucosaminidase;5 , Golgi apparatus α-mannosidase I; 6 , N-acetylglucosaminyltransferase I;7 , Golgi apparatus α-mannosidase II; 8 , N-acetylglucosaminyltransferase II; 9 , fucosyltransferase;10, galactosyltransferase; 11 , sialyltransferase. The thick arrows indicate various nucleotidesugars involved in the oveall scheme. (Solid square, N-acetylglucosamine; open circle,mannose; solid triangle, glucose; open triangle, fucose; solid circle, galactose; soliddiamond, sialic acid.) (Reproduced, with permission, from Kornfeld R, Kornfeld S: Assembly ofasparagine-linked oligosaccharides. Annu Rev Biochem 1985;54:631.)that the enzymes involved in glycoprotein processingshow differential locations in the cisternae of the Golgi.As indicated in Figure 47–9, Golgi α-mannosidase I(catalyzing reaction 5) is located mainly in the cisGolgi, whereas GlcNAc transferase I (catalyzing reaction6) appears to be located in the medial Golgi, andthe fucosyl, galactosyl, and sialyl transferases (catalyzingreactions 9, 10, and 11) are located mainly in the transGolgi. The major features of the biosynthesis of N-linked glycoproteins are summarized in Table 47–10and should be contrasted with those previously listed(Table 47–9) for O-linked glycoproteins.

526 / CHAPTER 47Table 47–10. Summary of main features ofN-glycosylation.• The oligosaccharide Glc 3 Man 9 (GIcNAc) 2 is transferred fromdolichol-P-P-oligosaccharide in a reaction catalyzed byoligosaccharide:protein transferase, which is inhibited bytunicamycin.• Transfer occurs to specific Asn residues in the sequenceAsn-X-Ser/Thr, where X is any residue except Pro, Asp, orGlu.• Transfer can occur cotranslationally in the endoplasmicreticulum.• The protein-bound oligosaccharide is then partiallyprocessed by glucosidases and mannosidases; if no additionalsugars are added, this results in a high-mannosechain.• If processing occurs down to the core heptasaccharide(Man 5 [GlcNAc] 2 ), complex chains are synthesized by the additionof GlcNAc, removal of two Man, and the stepwise additionof individual sugars in reactions catalyzed by specifictransferases (eg, GlcNAc, Gal, NeuAc transferases) that employappropriate nucleotide sugars.Some Glycan IntermediatesFormed During N-GlycosylationHave Specific FunctionsThe following are a number of specific functions of N-glycan chains that have been established or are underinvestigation. (1) The involvement of the mannose 6-Psignal in targeting of certain lysosomal enzymes is clear(see above and discussion of I-cell disease, below). (2) Itis likely that the large N-glycan chains present on newlysynthesized glycoproteins may assist in keeping theseproteins in a soluble state inside the lumen of the endoplasmicreticulum. (3) One species of N-glycan chainshas been shown to play a role in the folding and retentionof certain glycoproteins in the lumen of the endoplasmicreticulum. Calnexin is a protein present in theendoplasmic reticulum membrane that acts as a “chaperone”(Chapter 46). It has been found that calnexinwill bind specifically to a number of glycoproteins (eg,the influenza virus hemagglutinin [HA]) that possess themonoglycosylated core structure. This species is theproduct of reaction 2 shown in Figure 47–9 but fromwhich the terminal glucose residue has been removed,leaving only the innermost glucose attached. The releaseof fully folded HA from calnexin requires the enzymaticremoval of this last glucosyl residue by α-glucosidaseII. In this way, calnexin retains certain partlyfolded (or misfolded) glycoproteins and releases themwhen proper folding has occurred; it is thus an importantcomponent of the quality control systems operatingin the lumen of the ER. The soluble protein calreticulinappears to play a similar function.Several Factors Regulate the Glycosylationof GlycoproteinsIt is evident that glycosylation of glycoproteins is acomplex process involving a large number of enzymes.One index of its complexity is that more than ten distinctGlcNAc transferases involved in glycoproteinbiosynthesis have been reported, and many others aretheoretically possible. Multiple species of the other glycosyltransferases(eg, sialyltransferases) also exist. Controllingfactors of the first stage of N-linked glycoproteinbiosynthesis (ie, oligosaccharide assembly andtransfer) include (1) the presence of suitable acceptorsites in proteins, (2) the tissue level of Dol-P, and (3) theactivity of the oligosaccharide:protein transferase.Some factors known to be involved in the regulationof oligosaccharide processing are listed in Table47–11. Two of the points listed merit further comment.First, species variations among processing enzymeshave assumed importance in relation to productionof glycoproteins of therapeutic use by means ofrecombinant DNA technology. For instance, recombinanterythropoietin (epoetin alfa; EPO) is sometimesadministered to patients with certain types of chronicanemia in order to stimulate erythropoiesis. The halflifeof EPO in plasma is influenced by the nature of itsglycosylation pattern, with certain patterns being associatedwith a short half-life, appreciably limiting its periodof therapeutic effectiveness. It is thus important toharvest EPO from host cells that confer a pattern ofglycosylation consistent with a normal half-life inplasma. Second, there is great interest in analysis of theactivities of glycoprotein-processing enzymes in varioustypes of cancer cells. These cells have often been foundto synthesize different oligosaccharide chains (eg, theyoften exhibit greater branching) from those made incontrol cells. This could be due to cancer cells containingdifferent patterns of glycosyltransferases from thoseexhibited by corresponding normal cells, due to specificgene activation or repression. The differences inoligosaccharide chains could affect adhesive interactionsbetween cancer cells and their normal parent tissuecells, contributing to metastasis. If a correlation couldbe found between the activity of particular processingenzymes and the metastatic properties of cancer cells,this could be important as it might permit synthesis ofdrugs to inhibit these enzymes and, secondarily, metastasis.The genes encoding many glycosyltransferases havealready been cloned, and others are under study.Cloning has revealed new information on both proteinand gene structures. The latter should also cast light on

GLYCOPROTEINS / 527Table 47–11. Some factors affecting the activitiesof glycoprotein processing enzymes.FactorCommentCell type Different cell types contain different profilesof processing enzymes.Previous enzyme Certain glycosyltransferases will only acton an oligosaccharide chain if it has alreadybeen acted upon by another processingenzyme. 1Development The cellular profile of processing enzymesmay change during development if theirgenes are turned on or off.Intracellular For instance, if an enzyme is destined forlocation insertion into the membrane of the ER(eg, HMG-CoA reductase), it may neverencounter Golgi-located processingenzymes.ProteinDifferences in conformation of differentconformation proteins may facilitate or hinder access ofprocessing enzymes to identical oligosaccharidechains.SpeciesSame cells (eg, fibroblasts) from differentspecies may exhibit different patterns ofprocessing enzymes.CancerCancer cells may exhibit processing enzymesdifferent from those of correspondingnormal cells.1 For example, prior action of GlcNAc transferase I is necessary forthe action of Golgi α-mannosidase II.the mechanisms involved in their transcriptional control,and gene knockout studies are being used to evaluatethe biologic importance of various glycosyltransferases.Tunicamycin InhibitsN- but Not O-GlycosylationA number of compounds are known to inhibit variousreactions involved in glycoprotein processing. Tunicamycin,deoxynojirimycin, and swainsonine arethree such agents. The reactions they inhibit are indicatedin Table 47–12. These agents can be used experimentallyto inhibit various stages of glycoproteinbiosynthesis and to study the effects of specific alterationsupon the process. For instance, if cells are grownin the presence of tunicamycin, no glycosylation oftheir normally N-linked glycoproteins will occur. Incertain cases, lack of glycosylation has been shown toTable 47–12. Three inhibitors of enzymesinvolved in the glycosylation of glycoproteins andtheir sites of action.InhibitorSite of ActionTunicamycin Inhibits GlcNAc-P transferase, the enzymecatalyzing addition of GlcNAc to dolichol-P, the first step in the biosynthesis ofoligosaccharide-P-P-dolicholDeoxynojirimycin Inhibitor of glucosidases I and IISwainsonine Inhibitor of mannosidase IIincrease the susceptibility of these proteins to proteolysis.Inhibition of glycosylation does not appear to havea consistent effect upon the secretion of glycoproteinsthat are normally secreted. The inhibitors of glycoproteinprocessing listed in Table 47–12 do not affect thebiosynthesis of O-linked glycoproteins. The extensionof O-linked chains can be prevented by GalNAcbenzyl.This compound competes with natural glycoproteinsubstrates and thus prevents chain growth beyondGalNAc.SOME PROTEINS ARE ANCHOREDTO THE PLASMA MEMBRANEBY GLYCOSYLPHOSPHATIDYL-INOSITOL STRUCTURESGlycosylphosphatidylinositol (GPI)-linked glycoproteinscomprise the third major class of glycoprotein.The GPI structure (sometimes called a “sticky foot”)involved in linkage of the enzyme acetylcholinesterase(ACh esterase) to the plasma membrane of the redblood cell is shown in Figure 47–1. GPI-linked proteinsare anchored to the outer leaflet of the plasmamembrane by the fatty acids of phosphatidylinositol(PI). The PI is linked via a GlcNH 2 moiety to a glycanchain that contains various sugars (eg, Man, GlcNH 2 ).In turn, the oligosaccharide chain is linked via phosphorylethanolaminein an amide linkage to the carboxylterminal amino acid of the attached protein. Thecore of most GPI structures contains one molecule ofphosphorylethanolamine, three Man residues, one moleculeof GlcNH 2 , and one molecule of phosphatidylinositol,as follows:Ethanolamine - phospho → 6Manα1→2Manα1→ 6Manα1→ GlcNα1→6 — myo - inositol-1- phospholipid

528 / CHAPTER 47Additional constituents are found in many GPI structures;for example, that shown in Figure 47–1 containsan extra phosphorylethanolamine attached to the middleof the three Man moieties of the glycan and an extrafatty acid attached to GlcNH 2 . The functional significanceof these variations among structures is not understood.This type of linkage was first detected by the useof bacterial PI-specific phospholipase C (PI-PLC),which was found to release certain proteins from theplasma membrane of cells by splitting the bond indicatedin Figure 47–1. Examples of some proteins thatare anchored by this type of linkage are given in Table47–13. At least three possible functions of this type oflinkage have been suggested: (1) The GPI anchor mayallow greatly enhanced mobility of a protein in theplasma membrane compared with that observed for aprotein that contains transmembrane sequences. This isperhaps not surprising, as the GPI anchor is attachedonly to the outer leaflet of the lipid bilayer, so that it isfreer to diffuse than a protein anchored via both leafletsof the bilayer. Increased mobility may be important in facilitatingrapid responses to appropriate stimuli. (2) SomeGPI anchors may connect with signal transductionpathways. (3) It has been shown that GPI structures cantarget certain proteins to apical domains of the plasmamembrane of certain epithelial cells. The biosynthesis ofGPI anchors is complex and begins in the endoplasmicreticulum. The GPI anchor is assembled independentlyby a series of enzyme-catalyzed reactions and then transferredto the carboxyl terminal end of its acceptor protein,accompanied by cleavage of the preexisting carboxylterminal hydrophobic peptide from that protein.This process is sometimes called glypiation. An acquireddefect in an early stage of the biosynthesis of theGPI structure has been implicated in the causation ofparoxysmal nocturnal hemoglobinuria (see below).GLYCOPROTEINS ARE INVOLVEDIN MANY BIOLOGIC PROCESSES& IN MANY DISEASESAs listed in Table 47–1, glycoproteins have many differentfunctions; some have already been addressed inthis chapter and others are described elsewhere in thisTable 47–13. Some GPI-linked proteins.• Acetylcholinesterase (red cell membrane)• Alkaline phosphatase (intestinal, placental)• Decay-accelerating factor (red cell membrane)• 5′-Nucleotidase (T lymphocytes, other cells)• Thy-1 antigen (brain, T lymphocytes)• Variable surface glycoprotein (Trypanosoma brucei)text (eg, transport molecules, immunologic molecules,and hormones). Here, their involvement in two specificprocesses—fertilization and inflammation—will bebriefly described. In addition, the bases of a number ofdiseases that are due to abnormalities in the synthesisand degradation of glycoproteins will be summarized.Glycoproteins Are Importantin FertilizationTo reach the plasma membrane of an oocyte, a spermhas to traverse the zona pellucida (ZP), a thick, transparent,noncellular envelope that surrounds the oocyte.The zona pellucida contains three glycoproteins of interest,ZP1–3. Of particular note is ZP3, an O-linked glycoproteinthat functions as a receptor for the sperm. Aprotein on the sperm surface, possibly galactosyl transferase,interacts specifically with oligosaccharide chains ofZP3; in at least certain species (eg, the mouse), this interaction,by transmembrane signaling, induces the acrosomalreaction, in which enzymes such as proteases andhyaluronidase and other contents of the acrosome of thesperm are released. Liberation of these enzymes helpsthe sperm to pass through the zona pellucida and reachthe plasma membrane (PM) of the oocyte. In hamsters,it has been shown that another glycoprotein, PH-30, isimportant in both the binding of the PM of the sperm tothe PM of the oocyte and also in the subsequent fusionof the two membranes. These interactions enable thesperm to enter and thus fertilize the oocyte. It may bepossible to inhibit fertilization by developing drugs orantibodies that interfere with the normal functions ofZP3 and PH-30 and which would thus act as contraceptiveagents.Selectins Play Key Roles in Inflammation& in Lymphocyte HomingLeukocytes play important roles in many inflammatoryand immunologic phenomena. The first steps in manyof these phenomena are interactions between circulatingleukocytes and endothelial cells prior to passage ofthe former out of the circulation. Work done to identifyspecific molecules on the surfaces of the cells involvedin such interactions has revealed that leukocytesand endothelial cells contain on their surfaces specificlectins, called selectins, that participate in their intercellularadhesion. Features of the three major classes ofselectins are summarized in Table 47–14. Selectins aresingle-chain Ca 2+ -binding transmembrane proteins thatcontain a number of domains (Figure 47–10). Theiramino terminal ends contain the lectin domain, whichis involved in binding to specific carbohydrate ligands.The adhesion of neutrophils to endothelial cells ofpostcapillary venules can be considered to occur in four

Table 47–14. Some molecules involved in leukocyte-endothelial cell interactions. 1GLYCOPROTEINS / 529Molecule Cell LigandsSelectinsL-selectin PMN, lymphs CD34, Gly-CAM-1 2Sialyl-Lewis x and othersP-selectin EC, platelets P-selectin glycoprotein ligand-1 (PSGL-1)Sialyl-Lewis x and othersE-selectin EC Sialyl-Lewis x and othersIntegrinsLFA-1 PMN, lymphs ICAM-1, ICAM-2 (CD11a/CD18)Mac-1 PMN ICAM-1 and others (CD11b/CD18)Immunoglobulin superfamilyICAM-1 Lymphs, EC LFA-1, Mac-1ICAM-2 Lymphs, EC LFA-1PECAM-1 EC, PMN, lymphs Various platelets1 Modified from Albelda SM, Smith CW, Ward PA: Adhesion molecules and inflammatory injury. FASEB J 1994;8:504.2 These are ligands for lymphocyte L-selectin; the ligands for neutrophil L-selectin have not been identified.Key: PMN, polymorphonuclear leukocytes; EC, endothelial cell; lymphs, lymphocytes; CD, cluster of differentiation; ICAM, intercellular adhesionmolecule; LFA-1, lymphocyte function-associated antigen-1; PECAM-1, platelet endothelial cell adhesion cell molecule-1.stages, as shown in Figure 47–11. The initial baselinestage is succeeded by slowing or rolling of the neutrophils,mediated by selectins. Interactions betweenL-selectin on the neutrophil surface and CD34 andGlyCAM-1 or other glycoproteins on the endothelialsurface are involved. These particular interactions areinitially short-lived, and the overall binding is of relativelylow affinity, permitting rolling. However, duringNH 2L-selectinLectinEGF 1 2COOHFigure 47–10. Schematic diagram of the structureof human L-selectin. The extracellular portion containsan amino terminal domain homologous to C-typelectins and an adjacent epidermal growth factor-likedomain. These are followed by a variable number ofcomplement regulatory-like modules (numbered circles)and a transmembrane sequence (black diamond).A short cytoplasmic sequence (open rectangle) is at thecarboxyl terminal. The structures of P- and E-selectinare similar to that shown except that they contain morecomplement-regulatory modules. The numbers ofamino acids in L-, P-, and E- selectins, as deduced fromthe cDNA sequences, are 385, 789, and 589, respectively.(Reproduced, with permission, from Bevilacqua MP,Nelson RM: Selectins. J Clin Invest 1993;91:370.)this stage, activation of the neutrophils by variouschemical mediators (discussed below) occurs, resultingin a change of shape of the neutrophils and firm adhesionof these cells to the endothelium. An additional setof adhesion molecules is involved in firm adhesion,namely, LFA-1 and Mac-1 on the neutrophils andICAM-1 and ICAM-2 on endothelial cells. LFA-1 andMac-1 are CD11/CD18 integrins (see Chapter 52 for adiscussion of integrins), whereas ICAM-1 and ICAM-2are members of the immunoglobulin superfamily. Thefourth stage is transmigration of the neutrophils acrossthe endothelial wall. For this to occur, the neutrophilsinsert pseudopods into the junctions between endothelialcells, squeeze through these junctions, cross thebasement membrane, and then are free to migrate inthe extravascular space. Platelet-endothelial cell adhesionmolecule-1 (PECAM-1) has been found to be localizedat the junctions of endothelial cells and thusmay have a role in transmigration. A variety of biomoleculeshave been found to be involved in activation ofneutrophil and endothelial cells, including tumornecrosis factor α, various interleukins, platelet activatingfactor (PAF), leukotriene B 4 , and certain complementfragments. These compounds stimulate varioussignaling pathways, resulting in changes in cell shapeand function, and some are also chemotactic. One importantfunctional change is recruitment of selectins tothe cell surface, as in some cases selectins are stored ingranules (eg, in endothelial cells and platelets).

530 / CHAPTER 47ABCDBaselineRollingActivationandfirm adhesionTransmigrationThe precise chemical nature of some of the ligandsinvolved in selectin-ligand interactions has been determined.All three selectins bind sialylated and fucosylatedoligosaccharides, and in particular all three bindsialyl-Lewis X (Figure 47–12), a structure present onboth glycoproteins and glycolipids. Whether this compoundis the actual ligand involved in vivo is not established.Sulfated molecules, such as the sulfatides (Chapter14), may be ligands in certain instances. This basicknowledge is being used in attempts to synthesize compoundsthat block selectin-ligand interactions and thusmay inhibit the inflammatory response. Approaches includeadministration of specific monoclonal antibodiesor of chemically synthesized analogs of sialyl-Lewis X ,both of which bind selectins. Cancer cells often exhibitsialyl-Lewis X and other selectin ligands on their surfaces.It is thought that these ligands play a role in theinvasion and metastasis of cancer cells.Abnormalities in the Synthesis ofGlycoproteins Underlie Certain DiseasesTable 47–15 lists a number of conditions in which abnormalitiesin the synthesis of glycoproteins are of im-Figure 47–11. Schematic diagram of neutrophilendothelialcell interactions. A: Baseline conditions:Neutrophils do not adhere to the vessel wall. B: The firstevent is the slowing or rolling of the neutrophils withinthe vessel (venule) mediated by selectins. C: Activationoccurs, resulting in neutrophils firmly adhering to thesurfaces of endothelial cells and also assuming a flattenedshape. This requires interaction of activatedCD18 integrins on neutrophils with ICAM-1 on the endothelium.D: The neutrophils then migrate throughthe junctions of endothelial cells into the interstitial tissue;this requires involvement of PECAM-1. Chemotaxisis also involved in this latter stage. (Reproduced, withpermission, from Albelda SM, Smith CW, Ward PA: Adhesionmolecules and inflammatory injury. FASEB J1994;8;504.)NeuAcα2 3Galβ1 4GlcNAcα 1–3FucFigure 47–12. Schematic representation of thestructure of sialyl-Lewis X .Table 47–15. Some diseases due to or involvingabnormalities in the biosynthesis ofglycoproteins.DiseaseAbnormalityCancerIncreased branching of cell surfaceglycans or presentation of selectin ligandsmay be important in metastasis.Congenital disorders See Table 47–16.of glycosylation 1HEMPAS 2 (MIM Abnormalities in certain enzymes (eg,224100) mannosidase II and others) involved inthe biosynthesis of N-glycans, particularlyaffecting the red blood cell membrane.Leukocyte adhesion Probably mutations affecting a Golgideficiency,type II located GDP-fucose transporter, re-(MIM 266265) sulting in defective fucosylation.Paroxysmal nocturnal Acquired defect in biosynthesis of thehemoglobinuria GPI 3 structures of decay accelerating(MIM 311770) factor (DAF) and CD59.I-cell diseaseDeficiency of GlcNAc phosphotrans-(MIM 252500) ferase, resulting in abnormal targetingof certain lysosomal enzymes.1 The MIM number for congenital disorder of glycosylation type Iais 212065.2 Hereditary erythroblastic multinuclearity with a positive acidifiedserum lysis test (congenital dyserythropoietic anemia type II). Thisis a relatively mild form of anemia. It reflects at least in part thepresence in the red cell membranes of various glycoproteins withabnormal N-glycan chains, which contribute to the susceptibilityto lysis.3 Glycosylphosphatidylinositol.

GLYCOPROTEINS / 531portance. As mentioned above, many cancer cells exhibitdifferent profiles of oligosaccharide chains ontheir surfaces, some of which may contribute to metastasis.The congenital disorders of glycosylation(CDG) are a group of disorders of considerable currentinterest. The major features of these conditions aresummarized in Table 47–16. Leukocyte adhesion deficiency(LAD) II is a rare condition probably due tomutations affecting the activity of a Golgi-locatedGDP-fucose transporter. It can be considered a congenitaldisorder of glycosylation. The absence of fucosylatedligands for selectins leads to a marked decrease inneutrophil rolling. Subjects suffer life-threatening, recurrentbacterial infections and also psychomotor andmental retardation. The condition appears to respondto oral fucose. Hereditary erythroblastic multinuclearitywith a positive acidified lysis test (HEMPAS)—congenital dyserythropoietic anemia type II—is anotherdisorder due to abnormalities in the processing ofN-glycans. Some cases have been claimed to be due todefects in alpha–mannosidase II. I-cell disease is discussedfurther below. Paroxysmal nocturnal hemoglobinuriais an acquired mild anemia characterized bythe presence of hemoglobin in urine due to hemolysisof red cells, particularly during sleep. This latter phenomenonmay reflect a slight drop in plasma pH duringsleep, which increases susceptibility to lysis by thecomplement system (Chapter 50). The basic defect inparoxysmal nocturnal hemoglobinuria is the acquisitionTable 47–16. Major features of the congenitaldisorders of glycosylation.• Autosomal recessive disorders• Multisystem disorders that have probably not been recognizedin the past• Generally affect the central nervous system, resulting inpsychomotor retardation and other features• Type I disorders are due to mutations in genes encoding enzymes(eg, phosphomannomutase-2 [PMM-2], causing CDGIa) involved in the synthesis of dolichol-P-P-oligosaccharide• Type II disorders are due to mutations in genes encodingenzymes (eg, GlcNAc transferase-2, causing CDG IIa) involvedin the processing of N-glycan chains• About 11 distinct disorders have been recognized• Isoelectric focusing of transferrin is a useful biochemical testfor assisting in the diagnosis of these conditions; truncationof the oligosaccharide chains of this protein alters itsisolectric focusing pattern• Oral mannose has proved of benefit in the treatment ofCDG IaKey: CDG, congenital disorder of glycosylation.of somatic mutations in the PIG-A (for phosphatidylinositolglycan class A) gene of certain hematopoieticcells. The product of this gene appears to be the enzymethat links glucosamine to phosphatidylinositol inthe GPI structure (Figure 47–1). Thus, proteins thatare anchored by a GPI linkage are deficient in the redcell membrane. Two proteins are of particular interest:decay accelerating factor (DAF) and another proteindesignated CD59. They normally interact with certaincomponents of the complement system (Chapter 50) toprevent the hemolytic actions of the latter. However,when they are deficient, the complement system can acton the red cell membrane to cause hemolysis. Paroxysmalnocturnal hemoglobinuria can be diagnosed relativelysimply, as the red cells are much more sensitive tohemolysis in normal serum acidified to pH 6.2 (Ham’stest); the complement system is activated under theseconditions, but normal cells are not affected. Figure47–13 summarizes the etiology of paroxysmal nocturnalhemoglobinuria.I-Cell Disease Results From FaultyTargeting of Lysosomal EnzymesAs indicated above, Man 6-P serves as a chemicalmarker to target certain lysosomal enzymes to that organelle.Analysis of cultured fibroblasts derived frompatients with I-cell (inclusion cell) disease played a largepart in revealing the above role of Man 6-P. I-cell diseaseis an uncommon condition characterized by severeprogressive psychomotor retardation and a variety ofphysical signs, with death often occurring in the firstdecade. Cultured cells from patients with I-cell diseasewere found to lack almost all of the normal lysosomalenzymes; the lysosomes thus accumulate many differentAcquired mutations in the PIG-A geneof certain hematopoietic cellsDefective synthesis of the GlcNH 2 -PIlinkage of GPI anchorsDecreased amounts in the red blood membrane ofGPI-anchored proteins, with decay accelerating factor(DAF) and CD59 being of especial importanceCertain components of the complement systemare not opposed by DAF and CD59, resultingin complement-mediated lysis of red cellsFigure 47–13. Scheme of causation of paroxysmalnocturnal hemoglobinuria (MIM 311770).

532 / CHAPTER 47Mutations in DNAMutant GlcNAc phosphotransferaseLack of normal transfer of GlcNAc 1-Pto specific mannose residues of certain enzymesdestined for lysosomesThese enzymes consequently lack Man 6-Pand are secreted from cells (eg, into the plasma)rather than targeted to lysosomesLysosomes are thus deficient in certain hydrolases, donot function properly, and accumulate partly digestedcellular material, manifesting as inclusion bodiesFigure 47–14. Summary of the causation of I-celldisease (MIM 252500).types of undegraded molecules, forming inclusion bodies.Samples of plasma from patients with the diseasewere observed to contain very high activities of lysosomalenzymes; this suggested that the enzymes werebeing synthesized but were failing to reach their properintracellular destination and were instead being secreted.Cultured cells from patients with the diseasewere noted to take up exogenously added lysosomal enzymesobtained from normal subjects, indicating thatthe cells contained a normal receptor on their surfacesfor endocytic uptake of lysosomal enzymes. In addition,this finding suggested that lysosomal enzymes from patientswith I-cell disease might lack a recognitionmarker. Further studies revealed that lysosomal enzymesfrom normal individuals carried the Man 6-Precognition marker described above, which interactedwith a specific intracellular protein, the Man 6-P receptor.Cultured cells from patients with I-cell disease werethen found to be deficient in the activity of the cisGolgi-located GlcNAc phosphotransferase, explaininghow their lysosomal enzymes failed to acquire the Man6-P marker. It is now known that there are two Man6-P receptor proteins, one of high (275 kDa) and oneof low (46 kDa) molecular mass. These proteins arelectins, recognizing Man 6-P. The former is cationindependentand also binds IGF-II (hence it is namedthe Man 6-P–IGF-II receptor), whereas the latter iscation-dependent in some species and does not bindIGF-II. It appears that both receptors function in theintracellular sorting of lysosomal enzymes into clathrincoatedvesicles, which occurs in the trans Golgi subsequentto synthesis of Man 6-P in the cis Golgi. Thesevesicles then leave the Golgi and fuse with a prelysosomalcompartment. The low pH in this compartmentcauses the lysosomal enzymes to dissociate from theirreceptors and subsequently enter into lysosomes. Thereceptors are recycled and reused. Only the smaller receptorfunctions in the endocytosis of extracellular lysosomalenzymes, which is a minor pathway for lysosomallocation. Not all cells employ the Man 6-P receptor totarget their lysosomal enzymes (eg, hepatocytes use adifferent but undefined pathway); furthermore, not alllysosomal enzymes are targeted by this mechanism.Thus, biochemical investigations of I-cell disease notonly led to elucidation of its basis but also contributedsignificantly to knowledge of how newly synthesizedproteins are targeted to specific organelles, in this casethe lysosome. Figure 47–14 summarizes the causationof I-cell disease.Pseudo-Hurler polydystrophy is another geneticdisease closely related to I-cell disease. It is a mildercondition, and patients may survive to adulthood.Studies have revealed that the GlcNAc phosphotransferaseinvolved in I-cell disease has several domains, includinga catalytic domain and a domain that specificallyrecognizes and interacts with lysosomal enzymes.It has been proposed that the defect in pseudo-Hurlerpolydystrophy lies in the latter domain, and the retentionof some catalytic activity results in a milder condition.Genetic Deficiencies of GlycoproteinLysosomal Hydrolases Cause DiseasesSuch as α-MannosidosisGlycoproteins, like most other biomolecules, undergoboth synthesis and degradation (ie, turnover). Degradationof the oligosaccharide chains of glycoproteins involvesa battery of lysosomal hydrolases, includingα-neuraminidase, β-galactosidase, β-hexosaminidase,α- and β-mannosidases, α-N-acetylgalactosaminidase,α-fucosidase, endo-β-N-acetylglucosaminidase, and aspartylglucosaminidase.The sites of action of the lasttwo enzymes are indicated in the legend to Figure47–5. Genetically determined defects of the activities ofthese enzymes can occur, resulting in abnormal degradationof glycoproteins. The accumulation in tissues ofsuch abnormally degraded glycoproteins can lead tovarious diseases. Among the best-recognized of thesediseases are mannosidosis, fucosidosis, sialidosis, aspartylglycosaminuria,and Schindler disease, due respectivelyto deficiencies of α-mannosidase, α-fucosidase,α-neuraminidase, aspartylglucosaminidase, andα-N-acetyl-galactosaminidase. These diseases, whichare relatively uncommon, have a variety of manifestations;some of their major features are listed in Table47–17. The fact that patients affected by these disordersall show signs referable to the central nervous sys-

GLYCOPROTEINS / 533Table 47–17. Major features of some diseases(eg, α-mannosidosis, β-mannosidosis, fucosidosis,sialidosis, aspartylglycosaminuria, and Schindlerdisease) due to deficiencies of glycoproteinhydrolases. 1• Usually exhibit mental retardation or other neurologic abnormalities,and in some disorders coarse features or visceromegaly(or both)• Variations in severity from mild to rapidly progressive• Autosomal recessive inheritance• May show ethnic distribution (eg, aspartylglycosaminuria iscommon in Finland)• Vacuolization of cells observed by microscopy in somedisorders• Presence of abnormal degradation products (eg, oligosaccharidesthat accumulate because of the enzymedeficiency) in urine, detectable by TLC and characterizableby GLC-MS• Definitive diagnosis made by assay of appropriate enzyme,often using leukocytes• Possibility of prenatal diagnosis by appropriate enzymeassays• No definitive treatment at present1 MIM numbers: α-mannosidosis, 248500; β-mannosidosis,248510; fucosidosis, 230000; sialidosis, 256550; aspartylglycosaminuria,208400; Schindler disease, 104170.tem reflects the importance of glycoproteins in the developmentand normal function of that system.From the above, it should be apparent that glycoproteinsare involved in a wide variety of biologicprocesses and diseases. Glycoproteins play direct or indirectroles in a number of other diseases, as shown inthe following examples.(1) The influenza virus possesses a neuraminidasethat plays a key role in elution of newly synthesizedprogeny from infected cells. If this process is inhibited,spread of the virus is markedly diminished. Inhibitorsof this enzyme are now available for use in treating patientswith influenza.(2) HIV-1, thought by many to be the causativeagent of AIDS, attaches to cells via one of its surfaceglycoproteins, gp120.(3) Rheumatoid arthritis is associated with an alterationin the glycosylation of circulating immunoglobulin-γ(IgG) molecules (Chapter 50), suchthat they lack galactose in their Fc regions and terminatein GlcNAc. Mannose-binding protein (not to beconfused with the mannose-6-P receptor), a C-lectinsynthesized by liver cells and secreted into the circulation,binds mannose, GlcNAc, and certain other sugars.It can thus bind the agalactosyl IgG molecules, whichsubsequently activate the complement system, contributingto chronic inflammation in the synovial membranesof joints. This protein can also bind the abovesugars when they are present on the surfaces of certainbacteria, fungi, and viruses, preparing these pathogensfor opsonization or for destruction by the complementsystem. This is an example of innate immunity, not involvingimmunoglobulins. Deficiency of this protein inyoung infants, due to mutation, renders them very susceptibleto recurrent infections.Other disorders in which glycoproteins have beenimplicated include hepatitis B and C, Creutzfeldt-Jakob disease, and diarrheas due to a number of bacterialenterotoxins. It is hoped that basic studies of glycoproteinsand other glycoconjugates (ie, the field ofglycobiology) will lead to effective treatments for diseasesin which these molecules are involved. Already, atleast two disorders have been found to respond to oralsupplements of sugars.The fantastic progress made in relation to thehuman genome has stimulated intense interest in bothgenomics and proteomics. It is anticipated that the paceof research in glycomics—characterization of the entirecomplement of sugar chains found in cells (the glycome)—willalso accelerate markedly. For a number ofreasons, this field will prove more challenging than eithergenomics or proteomics. These reasons include thecomplexity of the structures of oligosaccharide chainsdue to linkage variations—in contrast to the generallyuniform nature of the linkages between nucleotides andbetween amino acids. There are also significant variationsin oligosaccharide structures among cells and atdifferent stages of development. In addition, no simpletechnique exists for amplifying oligosaccharides, comparableto the PCR reaction. Despite these and otherproblems, it seems certain that research in this area willuncover many new important biologic interactions thatare sugar-dependent and will provide targets for drugand other therapies.SUMMARY• Glycoproteins are widely distributed proteins—withdiverse functions—that contain one or more covalentlylinked carbohydrate chains.• The carbohydrate components of a glycoproteinrange from 1% to more than 85% of its weight andmay be simple or very complex in structure.• At least certain of the oligosaccharide chains of glycoproteinsencode biologic information; they are alsoimportant to glycoproteins in modulating their solubilityand viscosity, in protecting them against proteolysis,and in their biologic actions.

534 / CHAPTER 47• The structures of many oligosaccharide chains can beelucidated by gas-liquid chromatography, mass spectrometry,and high-resolution NMR spectrometry.• Glycosidases hydrolyze specific linkages in oligosaccharidesand are used to explore both the structuresand functions of glycoproteins.• Lectins are carbohydrate-binding proteins involvedin cell adhesion and other biologic processes.• The major classes of glycoproteins are O-linked (involvingan OH of serine or threonine), N-linked (involvingthe N of the amide group of asparagine), andglycosylphosphatidylinositol (GPI)-linked.• Mucins are a class of O-linked glycoproteins that aredistributed on the surfaces of epithelial cells of therespiratory, gastrointestinal, and reproductive tracts.• The Golgi apparatus plays a major role in glycosylationreactions involved in the biosynthesis of glycoproteins.• The oligosaccharide chains of O-linked glycoproteinsare synthesized by the stepwise addition of sugars donatedby nucleotide sugars in reactions catalyzed byindividual specific glycoprotein glycosyltransferases.• In contrast, the biosynthesis of N-linked glycoproteinsinvolves a specific dolichol-P-P-oligosaccharideand various glycosidases. Depending on the glycosidasesand precursor proteins synthesized by a tissue,it can synthesize complex, hybrid, or high-mannosetypes of N-linked oligosaccharides.• Glycoproteins are implicated in many biologicprocesses. For instance, they have been found to playkey roles in fertilization and inflammation.• A number of diseases involving abnormalities in thesynthesis and degradation of glycoproteins have beenrecognized. Glycoproteins are also involved in manyother diseases, including influenza, AIDS, andrheumatoid arthritis.• Developments in the new field of glycomics are likelyto provide much new information on the roles ofsugars in health and disease and also indicate targetsfor drug and other types of therapies.REFERENCESBrockhausen I, Kuhns W: Glycoproteins and Human Disease. Chapman& Hall, 1997.Kornfeld R, Kornfeld S: Assembly of asparagine-linked oligosaccharides.Annu Rev Biochem 1985;54:631.Lehrman MA Oligosaccharide-based information in endoplasmicreticulum quality control and other biological systems. J BiolChem. 2001;276:8623.Perkel JM: Glycobiology goes to the ball. The Scientist 2002;16:32.Roseman S: Reflections on glycobiology. J Biol Chem 2001;276:41527.Schachter H: The clinical relevance of glycobiology. J Clin Invest2001;108:1579.Schwartz NB, Domowicz M: Chondrodysplasias due to proteoglycandefects. Glycobiology 2002;12:57R.Science 2001;21(5512):2263. (This issue contains a special sectionentitled Carbohydrates and Glycobiology. It contains articleson the synthesis, structural determination, and functions ofsugar-containing molecules and the roles of glycosylation inthe immune system).Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001.(Various chaptersin this text give in-depth coverage of topics such as I-cell diseaseand disorders of glycoprotein degradation.)Spiro RG: Protein glycosylation: nature, distribution, enzymaticformation, and disease implications of glycopeptide bonds.Glycobiology 2002;12:43R.Varki A et al (editors): Essentials of Glycobiology. Cold Spring HarborLaboratory Press, 1999.Vestweber W, Blanks JE: Mechanisms that regulate the function ofthe selectins and their ligands. Physiol Rev 1999;79:181.

The Extracellular Matrix 48Robert K. Murray, MD, PhD, & Frederick W. Keeley, PhDBIOMEDICAL IMPORTANCEMost mammalian cells are located in tissues where theyare surrounded by a complex extracellular matrix(ECM) often referred to as “connective tissue.” TheECM contains three major classes of biomolecules: (1)the structural proteins, collagen, elastin, and fibrillin;(2) certain specialized proteins such as fibrillin, fibronectin,and laminin; and (3) proteoglycans, whosechemical natures are described below. The ECM hasbeen found to be involved in many normal and pathologicprocesses—eg, it plays important roles in development,in inflammatory states, and in the spread of cancercells. Involvement of certain components of theECM has been documented in both rheumatoid arthritisand osteoarthritis. Several diseases (eg, osteogenesisimperfecta and a number of types of the Ehlers-Danlossyndrome) are due to genetic disturbances of the synthesisof collagen. Specific components of proteoglycans(the glycosaminoglycans; GAGs) are affected inthe group of genetic disorders known as the mucopolysaccharidoses.Changes occur in the ECM duringthe aging process. This chapter describes the basicbiochemistry of the three major classes of biomoleculesfound in the ECM and illustrates their biomedical significance.Major biochemical features of two specializedforms of ECM—bone and cartilage—and of a numberof diseases involving them are also briefly considered.COLLAGEN IS THE MOST ABUNDANTPROTEIN IN THE ANIMAL WORLDCollagen, the major component of most connective tissues,constitutes approximately 25% of the protein ofmammals. It provides an extracellular framework for allmetazoan animals and exists in virtually every animaltissue. At least 19 distinct types of collagen made up of30 distinct polypeptide chains (each encoded by a separategene) have been identified in human tissues. Althoughseveral of these are present only in small proportions,they may play important roles in determiningthe physical properties of specific tissues. In addition, anumber of proteins (eg, the C1q component of thecomplement system, pulmonary surfactant proteinsSP-A and SP-D) that are not classified as collagens havecollagen-like domains in their structures; these proteinsare sometimes referred to as “noncollagen collagens.”Table 48–1 summarizes the types of collagens foundin human tissues; the nomenclature used to designatetypes of collagen and their genes is described in thefootnote.The 19 types of collagen mentioned above can besubdivided into a number of classes based primarily onthe structures they form (Table 48–2). In this chapter,we shall be primarily concerned with the fibril-formingcollagens I and II, the major collagens of skin and boneand of cartilage, respectively. However, mention will bemade of some of the other collagens.COLLAGEN TYPE I IS COMPOSEDOF A TRIPLE HELIX STRUCTURE& FORMS FIBRILSAll collagen types have a triple helical structure. Insome collagens, the entire molecule is triple helical,whereas in others the triple helix may involve only afraction of the structure. Mature collagen type I, containingapproximately 1000 amino acids, belongs to theformer type; in it, each polypeptide subunit or alphachain is twisted into a left-handed helix of threeresidues per turn (Figure 48–1). Three of these alphachains are then wound into a right-handed superhelix,forming a rod-like molecule 1.4 nm in diameter andabout 300 nm long. A striking characteristic of collagenis the occurrence of glycine residues at every third positionof the triple helical portion of the alpha chain.This is necessary because glycine is the only amino acidsmall enough to be accommodated in the limited spaceavailable down the central core of the triple helix. Thisrepeating structure, represented as (Gly-X-Y) n, is an absoluterequirement for the formation of the triple helix.While X and Y can be any other amino acids, about100 of the X positions are proline and about 100 of theY positions are hydroxyproline. Proline and hydroxyprolineconfer rigidity on the collagen molecule. Hydroxyprolineis formed by the posttranslational hydroxylationof peptide-bound proline residues catalyzedby the enzyme prolyl hydroxylase, whose cofactors areascorbic acid (vitamin C) and α-ketoglutarate. Lysines535

536 / CHAPTER 48Table 48–1. Types of collagen and their genes. 1,2Type Genes TissueI COL1A1, COL1A2 Most connective tissues,including boneII COL2A1 Cartilage, vitreous humorIII COL3A1 Extensible connective tissuessuch as skin, lung, and thevascular systemIV COL4A1–COL4A6 Basement membranesV COL5A1–COL5A3 Minor component in tissuescontaining collagen IVI COL6A1–COL6A3 Most connective tissuesVII COL7A1 Anchoring fibrilsVIII COL8A1–COL8A2 Endothelium, other tissuesIX COL9A1–COL9A3 Tissues containing collagen IIX COL10A1 Hypertrophic cartilageXI COL11A1, COL11A2, Tissues containing collagen IICOL2A1XII COL12A1 Tissues containing collagen IXIII COL13A1 Many tissuesXIV COL14A1 Tissues containing collagen IXV COL15A1 Many tissuesXVI COL16A1 Many tissuesXVII COL17A1 Skin hemidesmosomesXVIII COL18A1 Many tissues (eg, liver, kidney)XIX COL19A1 Rhabdomyosarcoma cells1 Adapted slightly from Prockop DJ, Kivirrikko KI: Collagens: molecularbiology, diseases, and potentials for therapy. Annu RevBiochem 1995;64:403.2 The types of collagen are designated by Roman numerals. Constituentprocollagen chains, called proα chains, are numberedusing Arabic numerals, followed by the collagen type in parentheses.For instance, type I procollagen is assembled from twoproα1(I) and one proα2(I) chain. It is thus a heterotrimer, whereastype 2 procollagen is assembled from three proα1(II) chains andis thus a homotrimer. The collagen genes are named according tothe collagen type, written in Arabic numerals for the gene symbol,followed by an A and the number of the proα chain that theyencode. Thus, the COL1A1 and COL1A2 genes encode the α1 andα2 chains of type I collagen, respectively.Table 48–2. Classification of collagens, basedprimarily on the structures that they form. 1Fibril-formingNetwork-likeFACITs 2Beaded filamentsAnchoring fibrilsClassTransmembrane domainOthersTypeI, II, III, V, and XIIV, VIII, XIX, XII, XIV, XVI, XIXVIVIIXIII, XVIIXV, XVIII1 Based on Prockop DJ, Kivirrikko KI: Collagens: molecular biology,diseases, and potentials for therapy. Annu Rev Biochem1995;64:403.2 FACITs = fibril-associated collagens with interrupted triplehelices.FibrilMoleculeTriple helixAlpha chainAmino acidsequence67 nm300 nm1.4 nm– Gly – X – Y – Gly – X – Y – Gly – X – Y –Figure 48–1. Molecular features of collagen structurefrom primary sequence up to the fibril. (Slightlymodified and reproduced, with permission, from Eyre DR:Collagen: Molecular diversity in the body’s protein scaffold.Science 1980;207:1315. Copyright © 1980 by theAmerican Association for the Advancement of Science.)

THE EXTRACELLULAR MATRIX / 537in the Y position may also be posttranslationally modifiedto hydroxylysine through the action of lysyl hydroxylase,an enzyme with similar cofactors. Some ofthese hydroxylysines may be further modified by theaddition of galactose or galactosyl-glucose through anO-glycosidic linkage, a glycosylation site that isunique to collagen.Collagen types that form long rod-like fibers in tissuesare assembled by lateral association of these triplehelical units into a “quarter staggered” alignment suchthat each is displaced longitudinally from its neighborby slightly less than one-quarter of its length (Figure48–1, upper part). This arrangement is responsible forthe banded appearance of these fibers in connective tissues.Collagen fibers are further stabilized by the formationof covalent cross-links, both within and betweenthe triple helical units. These cross-links form throughthe action of lysyl oxidase, a copper-dependent enzymethat oxidatively deaminates the ε-amino groups ofcertain lysine and hydroxylysine residues, yielding reactivealdehydes. Such aldehydes can form aldol condensationproducts with other lysine- or hydroxylysinederivedaldehydes or form Schiff bases with theε-amino groups of unoxidized lysines or hydroxylysines.These reactions, after further chemical rearrangements,result in the stable covalent cross-linksthat are important for the tensile strength of the fibers.Histidine may also be involved in certain cross-links.Several collagen types do not form fibrils in tissues(Table 48–2). They are characterized by interruptionsof the triple helix with stretches of protein lacking Gly-X-Y repeat sequences. These non-Gly-X-Y sequencesresult in areas of globular structure interspersed in thetriple helical structure.Type IV collagen, the best-characterized example ofa collagen with discontinuous triple helices, is an importantcomponent of basement membranes, where itforms a mesh-like network.Collagen Undergoes ExtensivePosttranslational ModificationsNewly synthesized collagen undergoes extensive posttranslationalmodification before becoming part of amature extracellular collagen fiber (Table 48–3). Likemost secreted proteins, collagen is synthesized on ribosomesin a precursor form, preprocollagen, which containsa leader or signal sequence that directs thepolypeptide chain into the lumen of the endoplasmicreticulum. As it enters the endoplasmic reticulum, thisleader sequence is enzymatically removed. Hydroxylationof proline and lysine residues and glycosylation ofhydroxylysines in the procollagen molecule also takeplace at this site. The procollagen molecule containsTable 48–3. Order and location of processing ofthe fibrillar collagen precursor.Intracellular1. Cleavage of signal peptide2. Hydroxylation of prolyl residues and some lysylresidues; glycosylation of some hydroxylysyl residues3. Formation of intrachain and interchain S–S bonds in extensionpeptides4. Formation of triple helixExtracellular1. Cleavage of amino and carboxyl terminal propeptides2. Assembly of collagen fibers in quarter-staggered alignment3. Oxidative deamination of ε-amino groups of lysyl andhydroxylysyl residues to aldehydes4. Formation of intra- and interchain cross-links via Schiffbases and aldol condensation productspolypeptide extensions (extension peptides) of 20–35kDa at both its amino and carboxyl terminal ends, neitherof which is present in mature collagen. Both extensionpeptides contain cysteine residues. While theamino terminal propeptide forms only intrachain disulfidebonds, the carboxyl terminal propeptides formboth intrachain and interchain disulfide bonds. Formationof these disulfide bonds assists in the registration ofthe three collagen molecules to form the triple helix,winding from the carboxyl terminal end. After formationof the triple helix, no further hydroxylation of prolineor lysine or glycosylation of hydroxylysines cantake place. Self-assembly is a cardinal principle in thebiosynthesis of collagen.Following secretion from the cell by way of theGolgi apparatus, extracellular enzymes called procollagenaminoproteinase and procollagen carboxyproteinaseremove the extension peptides at the amino andcarboxyl terminal ends, respectively. Cleavage of thesepropeptides may occur within crypts or folds in the cellmembrane. Once the propeptides are removed, thetriple helical collagen molecules, containing approximately1000 amino acids per chain, spontaneously assembleinto collagen fibers. These are further stabilizedby the formation of inter- and intrachain cross-linksthrough the action of lysyl oxidase, as described previously.The same cells that secrete collagen also secrete fibronectin,a large glycoprotein present on cell surfaces,in the extracellular matrix, and in blood (see below). Fibronectinbinds to aggregating precollagen fibers andalters the kinetics of fiber formation in the pericellularmatrix. Associated with fibronectin and procollagen in

538 / CHAPTER 48this matrix are the proteoglycans heparan sulfate andchondroitin sulfate (see below). In fact, type IX collagen,a minor collagen type from cartilage, contains attachedproteoglycan chains. Such interactions mayserve to regulate the formation of collagen fibers and todetermine their orientation in tissues.Once formed, collagen is relatively metabolically stable.However, its breakdown is increased during starvationand various inflammatory states. Excessive productionof collagen occurs in a number of conditions, eg,hepatic cirrhosis.A Number of Genetic Diseases Result FromAbnormalities in the Synthesis of CollagenAbout 30 genes encode collagen, and its pathway ofbiosynthesis is complex, involving at least eight enzyme-catalyzedposttranslational steps. Thus, it is notsurprising that a number of diseases (Table 48–4) aredue to mutations in collagen genes or in genes encodingsome of the enzymes involved in these posttranslationalmodifications. The diseases affecting bone(eg, osteogenesis imperfecta) and cartilage (eg, thechondrodysplasias) will be discussed later in this chapter.Ehlers-Danlos syndrome comprises a group of inheriteddisorders whose principal clinical features arehyperextensibility of the skin, abnormal tissue fragility,and increased joint mobility. The clinical picture isvariable, reflecting underlying extensive genetic heterogeneity.At least 10 types have been recognized, mostbut not all of which reflect a variety of lesions in thesynthesis of collagen. Type IV is the most serious becauseof its tendency for spontaneous rupture of arteriesor the bowel, reflecting abnormalities in type III collagen.Patients with type VI, due to a deficiency of lysylhydroxylase, exhibit marked joint hypermobility and atendency to ocular rupture. A deficiency of procollagenN-proteinase, causing formation of abnormal thin, irregularcollagen fibrils, results in type VIIC, manifestedby marked joint hypermobility and soft skin.Alport syndrome is the designation applied to anumber of genetic disorders (both X-linked and autosomal)affecting the structure of type IV collagen fibers,the major collagen found in the basement membranesof the renal glomeruli (see discussion of laminin,below). Mutations in several genes encoding type IVcollagen fibers have been demonstrated. The presentingsign is hematuria, and patients may eventually developend-stage renal disease. Electron microscopy revealscharacteristic abnormalities of the structure of the basementmembrane and lamina densa.In epidermolysis bullosa, the skin breaks and blistersas a result of minor trauma. The dystrophic form isTable 48–4. Diseases caused by mutations incollagen genes or by deficiencies in the activitiesof posttranslational enzymes involved in thebiosynthesis of collagen. 1Gene or Enzyme Disease 2COL1A1, COL1A2 Osteogenesis imperfecta, type 1 3 (MIM1566200)Osteoporosis 4 (MIM 166710)Ehlers-Danlos syndrome type VII autosomaldominant (130060)COL2A1Severe chondrodysplasiasOsteoarthritis 4 (MIM 120140)COL3A1Ehlers-Danlos syndrome type IV (MIM130050)COL4A3–COL4A6 Alport syndrome (including both autosomaland X-linked forms) (MIM 104200)COL7A1Epidermolysis bullosa, dystrophic (MIM131750)COL10A1Schmid metaphysial chondrodysplasia(MIM 156500)Lysyl hydroxylase Ehlers-Danlos syndrome type VI (MIM225400)Procollagen Ehlers-Danlos syndrome type VII auto-N-proteinase somal recessive (MIM 225410)Lysyl hydroxylase Menkes disease 5 (MIM 309400)1 Adapted from Prockop DJ, Kivirrikko KI: Collagens: molecular biology,diseases, and potentials for therapy. Annu Rev Biochem1995;64:403.2 Genetic linkage to collagen genes has been shown for a fewother conditions not listed here.3 At least four types of osteogenesis imperfecta are recognized;the great majority of mutations in all types are in the COL1A1 andCOL1A2 genes.4 At present applies to only a relatively small number of suchpatients.5 Secondary to a deficiency of copper (Chapter 50).due to mutations in COL7A1, affecting the structure oftype VII collagen. This collagen forms delicate fibrilsthat anchor the basal lamina to collagen fibrils in thedermis. These anchoring fibrils have been shown to bemarkedly reduced in this form of the disease, probablyresulting in the blistering. Epidermolysis bullosa simplex,another variant, is due to mutations in keratin 5(Chapter 49).Scurvy affects the structure of collagen. However, itis due to a deficiency of ascorbic acid (Chapter 45) andis not a genetic disease. Its major signs are bleeding

THE EXTRACELLULAR MATRIX / 539gums, subcutaneous hemorrhages, and poor woundhealing. These signs reflect impaired synthesis of collagendue to deficiencies of prolyl and lysyl hydroxylases,both of which require ascorbic acid as a cofactor.ELASTIN CONFERS EXTENSIBILITY& RECOIL ON LUNG, BLOODVESSELS, & LIGAMENTSElastin is a connective tissue protein that is responsiblefor properties of extensibility and elastic recoil in tissues.Although not as widespread as collagen, elastin ispresent in large amounts, particularly in tissues that requirethese physical properties, eg, lung, large arterialblood vessels, and some elastic ligaments. Smaller quantitiesof elastin are also found in skin, ear cartilage, andseveral other tissues. In contrast to collagen, there appearsto be only one genetic type of elastin, althoughvariants arise by alternative splicing (Chapter 37) of thehnRNA for elastin. Elastin is synthesized as a solublemonomer of 70 kDa called tropoelastin. Some of theprolines of tropoelastin are hydroxylated to hydroxyprolineby prolyl hydroxylase, though hydroxylysineand glycosylated hydroxylysine are not present. Unlikecollagen, tropoelastin is not synthesized in a pro- formwith extension peptides. Furthermore, elastin does notcontain repeat Gly-X-Y sequences, triple helical structure,or carbohydrate moieties.After secretion from the cell, certain lysyl residues oftropoelastin are oxidatively deaminated to aldehydes bylysyl oxidase, the same enzyme involved in this processin collagen. However, the major cross-links formed inelastin are the desmosines, which result from the condensationof three of these lysine-derived aldehydes withan unmodified lysine to form a tetrafunctional crosslinkunique to elastin. Once cross-linked in its mature,extracellular form, elastin is highly insoluble and extremelystable and has a very low turnover rate. Elastinexhibits a variety of random coil conformations that permitthe protein to stretch and subsequently recoil duringthe performance of its physiologic functions.Table 48–5 summarizes the main differences betweencollagen and elastin.Deletions in the elastin gene (located at 7q11.23)have been found in approximately 90% of subjects withWilliams syndrome, a developmental disorder affectingconnective tissue and the central nervous system.The mutations, by affecting synthesis of elastin, probablyplay a causative role in the supravalvular aorticstenosis often found in this condition. A number ofskin diseases (eg, scleroderma) are associated with accumulationof elastin. Fragmentation or, alternatively, adecrease of elastin is found in conditions such as pulmonaryemphysema, cutis laxa, and aging of the skin.Table 48–5. Major differences between collagenand elastin.CollagenElastin1. Many different genetic One genetic typetypes2. Triple helix No triple helix; random coilconformations permittingstretching3. (Gly-X-Y) n repeating No (Gly-X-Y) n repeatingstructurestructure4. Presence of hydroxylysine No hydroxylysine5. Carbohydrate-containing No carbohydrate6. Intramolecular aldol Intramolecular desmosinecross-linkscross-links7. Presence of extension No extension peptides presentpeptides during bio- during biosynthesissynthesisMARFAN SYNDROME IS DUE TOMUTATIONS IN THE GENE FOR FIBRILLIN,A PROTEIN PRESENT IN MICROFIBRILSMarfan syndrome is a relatively prevalent inherited diseaseaffecting connective tissue; it is inherited as an autosomaldominant trait. It affects the eyes (eg, causingdislocation of the lens, known as ectopia lentis), theskeletal system (most patients are tall and exhibit longdigits [arachnodactyly] and hyperextensibility of thejoints), and the cardiovascular system (eg, causingweakness of the aortic media, leading to dilation of theascending aorta). Abraham Lincoln may have had thiscondition. Most cases are caused by mutations in thegene (on chromosome 15) for fibrillin; missense mutationshave been detected in several patients with Marfansyndrome.Fibrillin is a large glycoprotein (about 350 kDa)that is a structural component of microfibrils, 10- to12-nm fibers found in many tissues. Fibrillin is secreted(subsequent to a proteolytic cleavage) into the extracellularmatrix by fibroblasts and becomes incorporatedinto the insoluble microfibrils, which appear to providea scaffold for deposition of elastin. Of special relevanceto Marfan syndrome, fibrillin is found in the zonularfibers of the lens, in the periosteum, and associatedwith elastin fibers in the aorta (and elsewhere); these locationsrespectively explain the ectopia lentis, arachnodactyly,and cardiovascular problems found in thesyndrome. Other proteins (eg, emelin and two microfibril-associatedproteins) are also present in thesemicrofibrils, and it appears likely that abnormalities ofthem may cause other connective tissue disorders. An-

540 / CHAPTER 48other gene for fibrillin exists on chromosome 5; mutationsin this gene are linked to causation of congenitalcontractural arachnodactyly but not to Marfan syndrome.The probable sequence of events leading toMarfan syndrome is summarized in Figure 48–2.FIBRONECTIN IS AN IMPORTANTGLYCOPROTEIN INVOLVED IN CELLADHESION & MIGRATIONFibronectin is a major glycoprotein of the extracellularmatrix, also found in a soluble form in plasma. It consistsof two identical subunits, each of about 230 kDa,joined by two disulfide bridges near their carboxyl terminals.The gene encoding fibronectin is very large,containing some 50 exons; the RNA produced by itstranscription is subject to considerable alternative splicing,and as many as 20 different mRNAs have been detectedin various tissues. Fibronectin contains threetypes of repeating motifs (I, II, and III), which are organizedinto functional domains (at least seven); functionsof these domains include binding heparin (seebelow) and fibrin, collagen, DNA, and cell surfaces(Figure 48–3). The amino acid sequence of the fibronectinreceptor of fibroblasts has been derived, andthe protein is a member of the transmembrane integrinclass of proteins (Chapter 51). The integrins are heterodimers,containing various types of α and βpolypeptide chains. Fibronectin contains an Arg-Gly-Asp (RGD) sequence that binds to the receptor. TheRGD sequence is shared by a number of other proteinspresent in the ECM that bind to integrins present incell surfaces. Synthetic peptides containing the RGDsequence inhibit the binding of fibronectin to cell surfaces.Figure 48–4 illustrates the interaction of collagen,fibronectin, and laminin, all major proteins of theMutations in gene (on chromosome 15)for fibrillin, a large glycoprotein present inelastin-associated microfibrilsAbnormalities of the structure of fibrillinStructures of the suspensory ligament of the eye,the periosteum, and the media of the aorta affectedEctopia lentis, arachnodactyly,and dilation of the ascending aortaFigure 48–2. Probable sequence of events in thecausation of the major signs exhibited by patients withMarfan syndrome (MIM 154700).ECM, with a typical cell (eg, fibroblast) present in thematrix.The fibronectin receptor interacts indirectly withactin microfilaments (Chapter 49) present in the cytosol(Figure 48–5). A number of proteins, collectivelyknown as attachment proteins, are involved; these includetalin, vinculin, an actin-filament capping protein,and α-actinin. Talin interacts with the receptor andvinculin, whereas the latter two interact with actin. Theinteraction of fibronectin with its receptor provides oneroute whereby the exterior of the cell can communicatewith the interior and thus affect cell behavior. Via theinteraction with its cell receptor, fibronectin plays animportant role in the adhesion of cells to the ECM. It isalso involved in cell migration by providing a bindingsite for cells and thus helping them to steer their waythrough the ECM. The amount of fibronectin aroundmany transformed cells is sharply reduced, partly explainingtheir faulty interaction with the ECM.LAMININ IS A MAJOR PROTEINCOMPONENT OF RENAL GLOMERULAR& OTHER BASAL LAMINASBasal laminas are specialized areas of the ECM that surroundepithelial and some other cells (eg, muscle cells);here we discuss only the laminas found in the renalglomerulus. In that structure, the basal lamina is contributedby two separate sheets of cells (one endothelialand one epithelial), each disposed on opposite sides ofthe lamina; these three layers make up the glomerularmembrane. The primary components of the basal laminaare three proteins—laminin, entactin, and type IVcollagen—and the GAG heparin or heparan sulfate.These components are synthesized by the underlyingcells.Laminin (about 850 kDa, 70 nm long) consists ofthree distinct elongated polypeptide chains (A, B 1 , andB 2 ) linked together to form an elongated cruciformshape. It has binding sites for type IV collagen, heparin,and integrins on cell surfaces. The collagen interactswith laminin (rather than directly with the cell surface),which in turn interacts with integrins or other lamininreceptor proteins, thus anchoring the lamina to thecells. Entactin, also known as “nidogen,” is a glycoproteincontaining an RGD sequence; it binds to lamininand is a major cell attachment factor. The relativelythick basal lamina of the renal glomerulus has an importantrole in glomerular filtration, regulating thepassage of large molecules (most plasma proteins) acrossthe glomerulus into the renal tubule. The glomerularmembrane allows small molecules, such as inulin (5.2kDa), to pass through as easily as water. On the otherhand, only a small amount of the protein albumin (69

THE EXTRACELLULAR MATRIX / 541RGDHeparin AFibrin ACollagen DNA Cell A Heparin B Cell B Fibrin BFigure 48–3. Schematic representation of fibronectin. Seven functionaldomains of fibronectin are represented; two different types of domain forheparin, cell-binding, and fibrin are shown. The domains are composed ofvarious combinations of three structural motifs (I, II, and III), not depictedin the figure. Also not shown is the fact that fibronectin is a dimer joinedby disulfide bridges near the carboxyl terminals of the monomers. The approximatelocation of the RGD sequence of fibronectin, which interactswith a variety of fibronectin integrin receptors on cell surfaces, is indicatedby the arrow. (Redrawn after Yamada KM: Adhesive recognition sequences.J Biol Chem 1991;266:12809.)kDa), the major plasma protein, passes through thenormal glomerulus. This is explained by two sets offacts: (1) The pores in the glomerular membrane arelarge enough to allow molecules up to about 8 nm topass through. (2) Albumin is smaller than this pore size,but it is prevented from passing through easily by thenegative charges of heparan sulfate and of certain sialicacid-containing glycoproteins present in the lamina.These negative charges repel albumin and most plasmaproteins, which are negatively charged at the pH ofblood. The normal structure of the glomerulus may beseverely damaged in certain types of glomerulonephritis(eg, caused by antibodies directed against variouscomponents of the glomerular membrane). This altersthe pores and the amounts and dispositions of the negativelycharged macromolecules referred to above, andrelatively massive amounts of albumin (and of certainHeparinS-SS-Sα βIntegrinreceptorCollagenFibronectinOUTSIDEPlasma membraneCollagenFibronectinα βα βTalinVinculinCapping proteinα-ActinINSIDEα βLamininFigure 48–4. Schematic representation of a cell interactingthrough various integrin receptors with collagen,fibronectin, and laminin present in the ECM. (Specificsubunits are not indicated.) (Redrawn after YamadaKM: Adhesive recognition sequences. J Biol Chem1991;266:12809.)ActinFigure 48–5. Schematic representation of fibronectininteracting with an integrin fibronectin receptorsituated in the exterior of the plasma membrane of acell of the ECM and of various attachment proteins interactingindirectly or directly with an actin microfilamentin the cytosol. For simplicity, the attachment proteinsare represented as a complex.

542 / CHAPTER 48other plasma proteins) can pass through into the urine,resulting in severe albuminuria.PROTEOGLYCANS& GLYCOSAMINOGLYCANSThe Glycosaminoglycans Foundin Proteoglycans Are Built Upof Repeating DisaccharidesProteoglycans are proteins that contain covalentlylinked glycosaminoglycans. A number of them havebeen characterized and given names such as syndecan,betaglycan, serglycin, perlecan, aggrecan, versican,decorin, biglycan, and fibromodulin. They vary in tissuedistribution, nature of the core protein, attachedglycosaminoglycans, and function. The proteins boundcovalently to glycosaminoglycans are called “core proteins”;they have proved difficult to isolate and characterize,but the use of recombinant DNA technology isbeginning to yield important information about theirstructures. The amount of carbohydrate in a proteoglycanis usually much greater than is found in a glycoproteinand may comprise up to 95% of its weight. Figures48–6 and 48–7 show the general structure of one particularproteoglycan, aggrecan, the major type found incartilage. It is very large (about 2 × 10 3 kDa), with itsoverall structure resembling that of a bottle brush. Itcontains a long strand of hyaluronic acid (one type ofGAG) to which link proteins are attached noncovalently.In turn, these latter interact noncovalently withcore protein molecules from which chains of otherGAGs (keratan sulfate and chondroitin sulfate in thiscase) project. More details on this macromolecule aregiven when cartilage is discussed below.There are at least seven glycosaminoglycans(GAGs): hyaluronic acid, chondroitin sulfate, keratansulfates I and II, heparin, heparan sulfate, and dermatansulfate. A GAG is an unbranched polysaccharidemade up of repeating disaccharides, one component ofwhich is always an amino sugar (hence the nameGAG), either D-glucosamine or D-galactosamine. Theother component of the repeating disaccharide (exceptin the case of keratan sulfate) is a uronic acid, eitherL-glucuronic acid (GlcUA) or its 5′-epimer, L-iduronicacid (IdUA). With the exception of hyaluronic acid, allthe GAGs contain sulfate groups, either as O-esters oras N-sulfate (in heparin and heparan sulfate).Hyaluronic acid affords another exception becausethere is no clear evidence that it is attached covalentlyto protein, as the definition of a proteoglycan givenabove specifies. Both GAGs and proteoglycans haveproved difficult to work with, partly because of theircomplexity. However, they are major components ofthe ground substance; they have a number of importantFigure 48–6. Dark field electron micrograph of aproteoglycan aggregate in which the proteoglycansubunits and filamentous backbone are particularlywell extended. (Reproduced, with permission, fromRosenberg L, Hellman W, Kleinschmidt AK: Electron microscopicstudies of proteoglycan aggregates from bovinearticular cartilage. J Biol Chem 1975;250:1877.)biologic roles; and they are involved in a number of diseaseprocesses—so that interest in them is increasingrapidly.Biosynthesis of GlycosaminoglycansInvolves Attachment to Core Proteins,Chain Elongation, & Chain TerminationA. ATTACHMENT TO CORE PROTEINSThe linkage between GAGs and their core proteins isgenerally one of three types.1. An O-glycosidic bond between xylose (Xyl) andSer, a bond that is unique to proteoglycans. This linkageis formed by transfer of a Xyl residue to Ser fromUDP-xylose. Two residues of Gal are then added to the

THE EXTRACELLULAR MATRIX / 543Hyaluronic acidLink proteinKeratan sulfateChondroitin sulfateCore proteinSubunitsFigure 48–7. Schematic representation of the proteoglycanaggrecan. (Reproduced, with permission, fromLennarz WJ:The Biochemistry of Glycoproteins and Proteoglycans.Plenum Press, 1980.)Xyl residue, forming a link trisaccharide, Gal-Gal-Xyl-Ser. Further chain growth of the GAG occurs on theterminal Gal.2. An O-glycosidic bond forms between GalNAc(N-acetylgalactosamine) and Ser (Thr) (Figure 47–1[a]), present in keratan sulfate II. This bond is formedby donation to Ser (or Thr) of a GalNAc residue, employingUDP-GalNAc as its donor.3. An N-glycosylamine bond between GlcNAc(N-acetylglucosamine) and the amide nitrogen of Asn,as found in N-linked glycoproteins (Figure 47–1[b]).Its synthesis is believed to involve dolichol-P-Poligosaccharide.The synthesis of the core proteins occurs in the endoplasmicreticulum, and formation of at least someof the above linkages also occurs there. Most of the latersteps in the biosynthesis of GAG chains and their subsequentmodifications occur in the Golgi apparatus.B. CHAIN ELONGATIONAppropriate nucleotide sugars and highly specificGolgi-located glycosyltransferases are employed to synthesizethe oligosaccharide chains of GAGs. The “oneenzyme, one linkage” relationship appears to hold here,as in the case of certain types of linkages found in glycoproteins.The enzyme systems involved in chain elongationare capable of high-fidelity reproduction of complexGAGs.C. CHAIN TERMINATIONThis appears to result from (1) sulfation, particularly atcertain positions of the sugars, and (2) the progressionof the growing GAG chain away from the membranesite where catalysis occurs.D. FURTHER MODIFICATIONSAfter formation of the GAG chain, numerous chemicalmodifications occur, such as the introduction of sulfategroups onto GalNAc and other moieties and theepimerization of GlcUA to IdUA residues. The enzymescatalyzing sulfation are designated sulfotransferases anduse 3′-phosphoadenosine-5′-phosphosulfate (PAPS; activesulfate) as the sulfate donor. These Golgi-locatedenzymes are highly specific, and distinct enzymes catalyzesulfation at different positions (eg, carbons 2, 3, 4,and 6) on the acceptor sugars. An epimerase catalyzesconversions of glucuronyl to iduronyl residues.The Various Glycosaminoglycans ExhibitDifferences in Structure & HaveCharacteristic DistributionsThe seven GAGs named above differ from each other ina number of the following properties: amino sugar composition,uronic acid composition, linkages betweenthese components, chain length of the disaccharides, thepresence or absence of sulfate groups and their positionsof attachment to the constituent sugars, the nature ofthe core proteins to which they are attached, the natureof the linkage to core protein, their tissue and subcellulardistribution, and their biologic functions.The structures (Figure 48–8) and the distributionsof each of the GAGs will now be briefly discussed. Themajor features of the seven GAGs are summarized inTable 48–6.A. HYALURONIC ACIDHyaluronic acid consists of an unbranched chain of repeatingdisaccharide units containing GlcUA and Glc-NAc. Hyaluronic acid is present in bacteria and iswidely distributed among various animals and tissues,including synovial fluid, the vitreous body of the eye,cartilage, and loose connective tissues.B. CHONDROITIN SULFATES (CHONDROITIN4-SULFATE & CHONDROITIN 6-SULFATE)Proteoglycans linked to chondroitin sulfate by the Xyl-Ser O-glycosidic bond are prominent components ofcartilage (see below). The repeating disaccharide issimilar to that found in hyaluronic acid, containing

544 / CHAPTER 48β1,4 β1,3Hyaluronic acid: GlcUA GlcNAcβ1,4GlcUAβ1,3GlcNAc β1,4β1,4 β1,3Chondroitin sulfates: GlcUA GalNAcβ1,4 β1,3GlcUAβ1,3Galβ1,4GalXylβSer4- or 6-SulfateKeratan sulfatesI and II:β1,4 β1,3GlcNAcβ1,4 β1,3Gal GlcNAcGal(GlcNAc, Man)1,6βGlcNAc Asn (keratan sulfate I)6-Sulfate6-SulfateGalNAcαThr (Ser) (keratan sulfate II)Gal-NeuAcHeparin andheparan sulfate:α1,4 α1,4IdUA6-Sulfateα1,4 β1,4GlcN GlcUAα1,4 β1,3GlcNAc GlcUAβ1,3Galβ1,4GalXylβSer2-SulfateSO 3 – or AcDermatan sulfate:β1,4 α1,3IdUAβ1,4 β1,3GalNAc GlcUAβ1,4 β1,3GalNAc GlcUAβ1,3Galβ1,4GalXylβSer2-Sulfate4-SulfateFigure 48–8. Summary of structures of glycosaminoglycans and their attachments to core proteins. (GlcUA,D-glucuronic acid; IdUA, L-iduronic acid; GlcN, D-glucosamine; GalN, D-galactosamine; Ac, acetyl; Gal, D-galactose;Xyl, D-xylose; Ser, L-serine; Thr, L-threonine; Asn, L-asparagine; Man, D-mannose; NeuAc, N-acetylneuraminicacid.) The summary structures are qualitative representations only and do not reflect, for example, theuronic acid composition of hybrid glycosaminoglycans such as heparin and dermatan sulfate, which containboth L-iduronic and D-glucuronic acid. Neither should it be assumed that the indicated substituents are alwayspresent, eg, whereas most iduronic acid residues in heparin carry a 2′-sulfate group, a much smaller proportionof these residues are sulfated in dermatan sulfate. The presence of link trisaccharides (Gal-Gal-Xyl) in the chondroitinsulfates, heparin, and heparan and dermatan sulfates is shown. (Slightly modified and reproduced, withpermission, from Lennarz WJ: The Biochemistry of Glycoproteins and Proteoglycans. Plenum Press, 1980.)Table 48–6. Major properties of the glycosaminoglycans.GAG Sugars Sulfate 1 Linkage of Protein LocationHA GIcNAc, GlcUA Nil No firm evidence Synovial fluid, vitreous humor, loose connective tissueCS GaINAc, GlcUA GalNAc Xyl-Ser; associated withHA via link proteins Cartilage, bone, corneaKS I GlcNAc, Gal GlcNAc GlcNAc-Asn CorneaKS II GlcNAc, Gal Same as KS I GalNAc-Thr Loose connective tissueHeparin GlcN, IdUA GlcN Ser Mast cellsGlcNIdUAHeparan sulfate GlcN, GlcUA GlcN Xyl-Ser Skin fibroblasts, aortic wallDermatan GalNAc, IdUA, GaINAc Xyl-Ser Wide distributionsulfate (GlcUA) IdUa1 The sulfate is attached to various positions of the sugars indicated (see Figure 48–7).

THE EXTRACELLULAR MATRIX / 545GlcUA but with GalNAc replacing GlcNAc. TheGalNAc is substituted with sulfate at either its 4′ or its6′ position, with approximately one sulfate being presentper disaccharide unit.C. KERATAN SULFATES I & IIAs shown in Figure 48–8, the keratan sulfates consist ofrepeating Gal-GlcNAc disaccharide units containingsulfate attached to the 6′ position of GlcNAc or occasionallyof Gal. Type I is abundant in cornea, and typeII is found along with chondroitin sulfate attached tohyaluronic acid in loose connective tissue. Types I andII have different attachments to protein (Figure 48–8).D. HEPARINThe repeating disaccharide contains glucosamine(GlcN) and either of the two uronic acids (Figure48–9). Most of the amino groups of the GlcN residuesare N-sulfated, but a few are acetylated. The GlcN alsocarries a C 6 sulfate ester.Approximately 90% of the uronic acid residues areIdUA. Initially, all of the uronic acids are GlcUA, but a5′-epimerase converts approximately 90% of theGlcUA residues to IdUA after the polysaccharide chainis formed. The protein molecule of the heparin proteoglycanis unique, consisting exclusively of serine andglycine residues. Approximately two-thirds of the serineresidues contain GAG chains, usually of 5–15 kDa butoccasionally much larger. Heparin is found in the granulesof mast cells and also in liver, lung, and skin.E. HEPARAN SULFATEThis molecule is present on many cell surfaces as aproteoglycan and is extracellular. It contains GlcN withfewer N-sulfates than heparin, and, unlike heparin, itspredominant uronic acid is GlcUA.F. DERMATAN SULFATEThis substance is widely distributed in animal tissues.Its structure is similar to that of chondroitin sulfate, exceptthat in place of a GlcUA in β-1,3 linkage toGalNAc it contains an IdUA in an α-1,3 linkage toGalNAc. Formation of the IdUA occurs, as in heparinand heparan sulfate, by 5′-epimerization of GlcUA. Becausethis is regulated by the degree of sulfation and becausesulfation is incomplete, dermatan sulfate containsboth IdUA-GalNAc and GlcUA-GalNAc disaccharides.Deficiencies of Enzymes That DegradeGlycosaminoglycans Result inMucopolysaccharidosesBoth exo- and endoglycosidases degrade GAGs. Likemost other biomolecules, GAGs are subject toturnover, being both synthesized and degraded. Inadult tissues, GAGs generally exhibit relatively slowturnover, their half-lives being days to weeks.Understanding of the degradative pathways forGAGs, as in the case of glycoproteins (Chapter 47) andglycosphingolipids (Chapter 24), has been greatly aidedby elucidation of the specific enzyme deficiencies thatoccur in certain inborn errors of metabolism. WhenGAGs are involved, these inborn errors are called mucopolysaccharidoses(Table 48–7).Degradation of GAGs is carried out by a battery oflysosomal hydrolases. These include certain endoglycosidases,various exoglycosidases, and sulfatases, generallyacting in sequence to degrade the various GAGs. Anumber of them are indicated in Table 48–7.The mucopolysaccharidoses share a commonmechanism of causation, as illustrated in Figure 48–10.They are inherited in an autosomal recessive manner,with Hurler and Hunter syndromes being perhaps themost widely studied. None are common. In some cases,a family history of a mucopolysaccharidosis is obtained.Specific laboratory investigations of help in their diagnosisare urine testing for the presence of increasedOCH 2 OSO 3–OHOGlcNHNSO 3–OO–CO 2OHIdUAOSO 3–OCH 2 OSO 3–OHOGlcNHNSO 3–OO–CO 2OHIdUAOHOCH 2 OSO 3–OHOGlcNHNSO 3–OCO 2–OHOOHGlcUAOCH 2 OSO 3–OHOHNAcGlcNAcFigure 48–9. Structure of heparin. The polymer section illustrates structural features typical of heparin;however, the sequence of variously substituted repeating disaccharide units has been arbitrarily selected. Inaddition, non-O-sulfated or 3-O-sulfated glucosamine residues may also occur. (Modified, redrawn, and reproduced,with permission, from Lindahl U et al: Structure and biosynthesis of heparin-like polysaccharides. Fed Proc1977;36:19.)O

546 / CHAPTER 48Table 48–7. Biochemical defects and diagnostic tests in mucopolysaccharidoses (MPS) andmucolipidoses (ML). 1 Alternative UrinaryName Designation 2,3 Enzymatic Defect MetabolitesMucopolysaccharidosesHurler, Scheie, MPS I α-L-Iduronidase Dermatan sulfate, heparan sulfateHurler-Scheie(MIM 252800)Hunter (MIM 309900) MPS II Iduronate sulfatase Dermatan sulfate, heparan sulfateSanfilippo A MPS IIIA Heparan sulfate N-sulfatase Heparan sulfate(MIM 252900)(sulfamidase)Sanfilippo B MPS IIIB α-N-Acetylglucosaminidase Heparan sulfate(MIM 252920)Sanfilippo C MPS IIIC Acetyltransferase Heparan sulfate(MIM 252930)Sanfilippo D MPS IIID N-Acetylglucosamine Heparan sulfate(MIM 252940)6-sulfataseMorquio A MPS IVA Galactosamine 6-sulfatase Keratan sulfate, chondroitin 6-sulfate(MIM 253000)Morquio B MPS IVB β-Galactosidase Keratan sulfate(MIM 253010)Maroteaux-Lamy MPS VI N-Acetylgalactosamine 4- Dermatan sulfate(MIM 253200) sulfatase (arylsulfatase B)Sly (MIM 253220) MPS VII β-Glucuronidase Dermatan sulfate, heparan sulfate, chondroitin4-sulfate, chondroitin 6-sulfateMucolipidosesSialidosis M LI Sialidase (neuraminidase) Glycoprotein fragments(MIM 256550)I-cell disease ML II UDP-N-acetylglucosamine: Glycoprotein fragments(MIM 252500)glycoprotein N-acetylglucosamininylphosphotransferase.(Acid hydrolasesthus lack phosphomannosylresidues.)Pseudo-Hurler ML III As for ML II but deficiency Glycoprotein fragmentspolydystrophyis incomplete(MIM 252600)1 Modified and reproduced, with permission, from DiNatale P, Neufeld EF: The biochemical diagnosis of mucopolysaccharidoses,mucolipidoses and related disorders. In: Perspectives in Inherited Metabolic Diseases, vol 2. Barr B et al (editors). Editiones Ermes(Milan), 1979.2 Fibroblasts, leukocytes, tissues, amniotic fluid cells, or serum can be used for the assay of many of the above enzymes. Patientswith these disorders exhibit a variety of clinical findings that may include cloudy corneas, mental retardation, stiff joints, cardiac abnormalities,hepatosplenomegaly, and short stature, depending on the specific disease and its severity.3 The term MPS V is no longer used. The existence of MPS VIII (suspected glucosamine 6-sulfatase deficiency: MIM 253230) has notbeen confirmed. At least one case of hyaluronidase deficiency (MPS IX; MIM 601492) has been reported.amounts of GAGs and assays of suspected enzymes inwhite cells, fibroblasts, or sometimes in serum. In certaincases, a tissue biopsy is performed and the GAGthat has accumulated can be determined by electrophoresis.DNA tests are increasingly available. Prenataldiagnosis can be made using amniotic cells orchorionic villus biopsy.The term “mucolipidosis” was introduced to denotediseases that combined features common to bothmucopolysaccharidoses and sphingolipidoses (Chapter24). Three mucolipidoses are listed in Table 48–7. Insialidosis (mucolipidosis I, ML-I), various oligosaccharidesderived from glycoproteins and certain gangliosidescan accumulate in tissues. I-cell disease (ML-II)

THE EXTRACELLULAR MATRIX / 547Mutation(s) in a gene encoding a lysosomal hydrolaseinvolved in the degradation of one or more GAGsDefective lysosomal hydrolaseAccumulation of substrate in various tissues, includingliver, spleen, bone, skin, and central nervous systemFigure 48–10. Simplified scheme of causation of amucopolysaccharidosis, such as Hurler syndrome (MIM252800), in which the affected enzyme is α-L-iduronidase.Marked accumulation of the GAGs in the tissuesmentioned in the figure could cause hepatomegaly,splenomegaly, disturbances of growth, coarse facies,and mental retardation, respectively.and pseudo-Hurler polydystrophy (ML-III) are describedin Chapter 47. The term “mucolipidosis” is retainedbecause it is still in relatively widespread clinicalusage, but it is not appropriate for these two latter diseasessince the mechanism of their causation involvesmislocation of certain lysosomal enzymes. Genetic defectsof the catabolism of the oligosaccharide chains ofglycoproteins (eg, mannosidosis, fucosidosis) are alsodescribed in Chapter 47. Most of these defects are characterizedby increased excretion of various fragments ofglycoproteins in the urine, which accumulate becauseof the metabolic block, as in the case of the mucolipidoses.Hyaluronidase is one important enzyme involvedin the catabolism of both hyaluronic acid and chondroitinsulfate. It is a widely distributed endoglycosidasethat cleaves hexosaminidic linkages. From hyaluronicacid, the enzyme will generate a tetrasaccharide withthe structure (GlcUA-β-1,3-GlcNAc-β-1,4) 2 , whichcan be degraded further by a β-glucuronidase and β-Nacetylhexosaminidase.Surprisingly, only one case of anapparent genetic deficiency of this enzyme appears tohave been reported.Proteoglycans Have Numerous FunctionsAs indicated above, proteoglycans are remarkably complexmolecules and are found in every tissue of thebody, mainly in the ECM or “ground substance.”There they are associated with each other and also withthe other major structural components of the matrix,collagen and elastin, in quite specific manners. Someproteoglycans bind to collagen and others to elastin.These interactions are important in determining thestructural organization of the matrix. Some proteoglycans(eg, decorin) can also bind growth factors such asTGF-β, modulating their effects on cells. In addition,some of them interact with certain adhesive proteinssuch as fibronectin and laminin (see above), also foundin the matrix. The GAGs present in the proteoglycansare polyanions and hence bind polycations and cationssuch as Na + and K + . This latter ability attracts water byosmotic pressure into the extracellular matrix and contributesto its turgor. GAGs also gel at relatively lowconcentrations. Because of the long extended nature ofthe polysaccharide chains of GAGs and their ability togel, the proteoglycans can act as sieves, restricting thepassage of large macromolecules into the ECM but allowingrelatively free diffusion of small molecules.Again, because of their extended structures and thehuge macromolecular aggregates they often form, theyoccupy a large volume of the matrix relative to proteins.A. SOME FUNCTIONS OF SPECIFICGAGS & PROTEOGLYCANSHyaluronic acid is especially high in concentration inembryonic tissues and is thought to play an importantrole in permitting cell migration during morphogenesisand wound repair. Its ability to attract water into theextracellular matrix and thereby “loosen it up” may beimportant in this regard. The high concentrations ofhyaluronic acid and chondroitin sulfates present in cartilagecontribute to its compressibility (see below).Chondroitin sulfates are located at sites of calcificationin endochondral bone and are also found in cartilage.They are also located inside certain neurons andmay provide an endoskeletal structure, helping tomaintain their shape.Both keratan sulfate I and dermatan sulfate arepresent in the cornea. They lie between collagen fibrilsand play a critical role in corneal transparency. Changesin proteoglycan composition found in corneal scars disappearwhen the cornea heals. The presence of dermatansulfate in the sclera may also play a role in maintainingthe overall shape of the eye. Keratan sulfate I isalso present in cartilage.Heparin is an important anticoagulant. It bindswith factors IX and XI, but its most important interactionis with plasma antithrombin III (discussed inChapter 51). Heparin can also bind specifically tolipoprotein lipase present in capillary walls, causing arelease of this enzyme into the circulation.Certain proteoglycans (eg, heparan sulfate) are associatedwith the plasma membrane of cells, with theircore proteins actually spanning that membrane. In itthey may act as receptors and may also participate inthe mediation of cell growth and cell-cell communication.The attachment of cells to their substratum in cultureis mediated at least in part by heparan sulfate. Thisproteoglycan is also found in the basement membraneof the kidney along with type IV collagen and laminin

548 / CHAPTER 48(see above), where it plays a major role in determiningthe charge selectiveness of glomerular filtration.Proteoglycans are also found in intracellular locationssuch as the nucleus; their function in this organellehas not been elucidated. They are present insome storage or secretory granules, such as the chromaffingranules of the adrenal medulla. It has been postulatedthat they play a role in release of the contents ofsuch granules. The various functions of GAGs are summarizedin Table 48–8.B. ASSOCIATIONS WITH MAJOR DISEASES& WITH AGINGHyaluronic acid may be important in permittingtumor cells to migrate through the ECM. Tumor cellscan induce fibroblasts to synthesize greatly increasedamounts of this GAG, thereby perhaps facilitating theirown spread. Some tumor cells have less heparan sulfateat their surfaces, and this may play a role in the lack ofadhesiveness that these cells display.The intima of the arterial wall contains hyaluronicacid and chondroitin sulfate, dermatan sulfate, and heparansulfate proteoglycans. Of these proteoglycans,dermatan sulfate binds plasma low-density lipoproteins.In addition, dermatan sulfate appears to be themajor GAG synthesized by arterial smooth musclecells. Because it is these cells that proliferate in atheroscleroticlesions in arteries, dermatan sulfate may playan important role in development of the atheroscleroticplaque.Table 48–8. Some functions ofglycosaminoglycans and proteoglycans. 1• Act as structural components of the ECM• Have specific interactions with collagen, elastin, fibronectin,laminin, and other proteins such as growth factors• As polyanions, bind polycations and cations• Contribute to the characteristic turgor of various tissues• Act as sieves in the ECM• Facilitate cell migration (HA)• Have role in compressibility of cartilage in weight-bearing(HA, CS)• Play role in corneal transparency (KS I and DS)• Have structural role in sclera (DS)• Act as anticoagulant (heparin)• Are components of plasma membranes, where they mayact as receptors and participate in cell adhesion and cell-cellinteractions (eg, HS)• Determine charge-selectiveness of renal glomerulus (HS)• Are components of synaptic and other vesicles (eg, HS)1 ECM, extracellular matrix; HA, hyaluronic acid; CS, chondroitinsulfate; KS I, keratan sulfate I; DS, dermatan sulfate; HS, heparansulfate.In various types of arthritis, proteoglycans may actas autoantigens, thus contributing to the pathologicfeatures of these conditions. The amount of chondroitinsulfate in cartilage diminishes with age, whereasthe amounts of keratan sulfate and hyaluronic acid increase.These changes may contribute to the developmentof osteoarthritis. Changes in the amounts of cer-Table 48–9. The principal proteins foundin bone. 1ProteinsCollagensCollagen type ICollagen type VNoncollagen proteinsPlasma proteinsProteoglycans 2CS-PG I (biglycan)CS-PG II (decorin)CS-PG IIIBone SPARC 3 protein(osteonectin)Osteocalcin (bone Glaprotein)OsteopontinBone sialoproteinBone morphogeneticproteins (BMPs)CommentsApproximately 90% of total boneprotein. Composed of two α1(I)and one α2(I) chains.Minor component.Mixture of various plasma proteins.Contains two GAG chains; found inother tissues.Contains one GAG chain; found inother tissues.Bone-specific.Not bone-specific.Contains γ-carboxyglutamateresidues that bind to hydroxyapatite.Bone-specific.Not bone-specific. Glycosylatedand phosphorylated.Bone-specific. Heavily glycosylated,and sulfated on tyrosine.A family (eight or more) of secretedproteins with a variety of actionson bone; many induce ectopicbone growth.1 Various functions have been ascribed to the noncollagenproteins, including roles in mineralization; however, most ofthem are still speculative. It is considered unlikely that thenoncollagen proteins that are not bone-specific play a keyrole in mineralization. A number of other proteins are alsopresent in bone, including a tyrosine-rich acidic matrix protein(TRAMP), some growth factors (eg, TGFβ), and enzymesinvolved in collagen synthesis (eg, lysyl oxidase).2 CS-PG, chondroitin sulfate–proteoglycan; these are similar tothe dermatan sulfate PGs (DS-PGs) of cartilage (Table 48–11).3 SPARC, secreted protein acidic and rich in cysteine.

THE EXTRACELLULAR MATRIX / 549Osteoclast Mesenchyme Newly formed matrix (osteoid)Osteoblast Osteocyte Bone matrixFigure 48–11. Schematic illustration of the major cells present in membranousbone. Osteoblasts (lighter color) are synthesizing type I collagen, which forms a matrixthat traps cells. As this occurs, osteoblasts gradually differentiate to become osteocytes.(Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text &Atlas, 10th ed. McGraw-Hill, 2003.)tain GAGs in the skin are also observed with aging andhelp to account for the characteristic changes noted inthis organ in the elderly.An exciting new phase in proteoglycan research isopening up with the findings that mutations that affectindividual proteoglycans or the enzymes needed fortheir synthesis alter the regulation of specific signalingpathways in drosophila and Caenorhabditis elegans, thusaffecting development; it already seems likely that similareffects exist in mice and humans.BONE IS A MINERALIZEDCONNECTIVE TISSUEBone contains both organic and inorganic material.The organic matter is mainly protein. The principalproteins of bone are listed in Table 48–9; type I collagenis the major protein, comprising 90–95% of theorganic material. Type V collagen is also present insmall amounts, as are a number of noncollagen proteins,some of which are relatively specific to bone.The inorganic or mineral component is mainly crystallinehydroxyapatite—Ca 10 (PO 4 ) 6 (OH) 2 —alongwith sodium, magnesium, carbonate, and fluoride; approximately99% of the body’s calcium is contained inbone (Chapter 45). Hydroxyapatite confers on bonethe strength and resilience required by its physiologicroles.Bone is a dynamic structure that undergoes continuingcycles of remodeling, consisting of resorption followedby deposition of new bone tissue. This remodelingpermits bone to adapt to both physical (eg,increases in weight-bearing) and hormonal signals.The major cell types involved in bone resorptionand deposition are osteoclasts and osteoblasts (Figure48–11). The former are associated with resorption andthe latter with deposition of bone. Osteocytes are descendedfrom osteoblasts; they also appear to be involvedin maintenance of bone matrix but will not bediscussed further here.Osteoclasts are multinucleated cells derived frompluripotent hematopoietic stem cells. Osteoclasts possessan apical membrane domain, exhibiting a ruffledborder that plays a key role in bone resorption (Figure48–12). A proton-translocating ATPase expels protonsacross the ruffled border into the resorption area, whichis the microenvironment of low pH shown in the figure.This lowers the local pH to 4.0 or less, thus increasingthe solubility of hydroxyapatite and allowingdemineralization to occur. Lysosomal acid proteases arereleased that digest the now accessible matrix proteins.

550 / CHAPTER 48Blood capillaryNucleusOsteoclastGolgiNucleusRuffledborderLysosomesCO 2 + H 2 O H + + HCO 3–Section ofcircumferentialclear zoneBone matrixMicroenvironment of low pHand lysosomal enzymesFigure 48–12. Schematic illustration of some aspects of the role of the osteoclast inbone resorption. Lysosomal enzymes and hydrogen ions are released into the confinedmicroenvironment created by the attachment between bone matrix and the peripheralclear zone of the osteoclast. The acidification of this confined space facilitates the dissolutionof calcium phosphate from bone and is the optimal pH for the activity of lysosomalhydrolases. Bone matrix is thus removed, and the products of bone resorptionare taken up into the cytoplasm of the osteoclast, probably digested further, and transferredinto capillaries. The chemical equation shown in the figure refers to the action ofcarbonic anhydrase II, described in the text. (Reproduced, with permission, from JunqueiraLC, Carneiro J: Basic Histology: Text & Atlas, 10th ed. McGraw-Hill, 2003.)Osteoblasts—mononuclear cells derived from pluripotentmesenchymal precursors—synthesize most of theproteins found in bone (Table 48–9) as well as variousgrowth factors and cytokines. They are responsible forthe deposition of new bone matrix (osteoid) and itssubsequent mineralization. Osteoblasts control mineralizationby regulating the passage of calcium and phosphateions across their surface membranes. The lattercontain alkaline phosphatase, which is used to generatephosphate ions from organic phosphates. The mechanismsinvolved in mineralization are not fully understood,but several factors have been implicated. Alkalinephosphatase contributes to mineralization but in itselfis not sufficient. Small vesicles (matrix vesicles) containingcalcium and phosphate have been described at sitesof mineralization, but their role is not clear. Type I collagenappears to be necessary, with mineralization beingfirst evident in the gaps between successive molecules.Recent interest has focused on acidic phosphoproteins,such as bone sialoprotein, acting as sites of nucleation.These proteins contain motifs (eg, poly-Asp and poly-Glu stretches) that bind calcium and may provide aninitial scaffold for mineralization. Some macromolecules,such as certain proteoglycans and glycoproteins,can also act as inhibitors of nucleation.It is estimated that approximately 4% of compactbone is renewed annually in the typical healthy adult,whereas approximately 20% of trabecular bone is replaced.Many factors are involved in the regulation of bonemetabolism, only a few of which will be mentionedhere. Some stimulate osteoblasts (eg, parathyroid hormoneand 1,25-dihydroxycholecalciferol) and othersinhibit them (eg, corticosteroids). Parathyroid hormoneand 1,25-dihydroxycholecalciferol also stimulate osteoclasts,whereas calcitonin and estrogens inhibit them.

THE EXTRACELLULAR MATRIX / 551Table 48–10. Some metabolic and geneticdiseases affecting bone and cartilage.DiseaseCommentsDwarfismOften due to a deficiency of growthhormone, but has many other causes.RicketsDue to a deficiency of vitamin Dduring childhood.Osteomalacia Due to a deficiency of vitamin Dduring adulthood.Hyperparathyroidism Excess parathormone causes boneresorption.Osteogenesis Due to a variety of mutations in theimperfecta (eg, COL1A1 and COL1A2 genes affectingMIM 166200) the synthesis and structure of type Icollagen.Osteoporosis Commonly postmenopausal or inother cases is more gradual and relatedto age; a small number of casesare due to mutations in the COL1A1and COL1A2 genes and possibly in thevitamin D receptor gene (MIM 166710)Osteoarthritis A small number of cases are due tomutations in the COL1A genes.Several chondro- Due to mutations in COL2A1 genes.dysplasiasPfeiffer syndrome 1 Mutations in the gene encoding fi-(MIM 100600) broblast growth receptor 1 (FGFR1).Jackson-Weiss Mutations in the gene encoding(MIM 123150) FGFR2.and Crouzon(MIM 123500)syndromes 1Achondroplasia Mutations in the gene encoding(MIM 100800) FGFR3.and thanatophoricdysplasia 2(MIM 187600)1 The Pfeiffer, Jackson-Weiss, and Crouzon syndromes are craniosynostosissyndromes; craniosynostosis is a term signifying prematurefusion of sutures in the skull.2 Thanatophoric (Gk thanatos “death” + phoros “bearing”) dysplasiais the most common neonatal lethal skeletal dysplasia, displayingfeatures similar to those of homozygous achondroplasia.MANY METABOLIC & GENETICDISORDERS INVOLVE BONEA number of the more important examples of metabolicand genetic disorders that affect bone are listed inTable 48–10.Osteogenesis imperfecta (brittle bones) is characterizedby abnormal fragility of bones. The scleras areoften abnormally thin and translucent and may appearblue owing to a deficiency of connective tissue. Fourtypes of this condition (mild, extensive, severe, andvariable) have been recognized, of which the extensivetype occurring in the newborn is the most ominous.Affected infants may be born with multiple fracturesand not survive. Over 90% of patients with osteogenesisimperfecta have mutations in the COL1A1 andCOL1A2 genes, encoding proα1(I) and proα2(I)chains, respectively. Over 100 mutations in these twogenes have been documented and include partial genedeletions and duplications. Other mutations affectRNA splicing, and the most frequent type results in thereplacement of glycine by another bulkier amino acid,affecting formation of the triple helix. In general, thesemutations result in decreased expression of collagen orTable 48–11. The principal proteins foundin cartilage.ProteinsCollagen proteinsCollagen type IIComments90–98% of total articular cartilagecollagen. Composed of threeα1(II) chains.Collagens V, VI, IX, Type IX cross-links to type II colla-X, XI gen. Type XI may help control diameterof type II fibrils.Noncollagen proteinsProteoglycansAggrecanLarge nonaggregatingproteoglycanDS-PG I (biglycan) 1DS-PG II (decorin)ChondronectinAnchorin C IIThe major proteoglycan of cartilage.Found in some types of cartilage.Similar to CS-PG I of bone.Similar to CS-PG II of bone.May play role in binding type II collagento surface of cartilage.May bind type II collagen to surfaceof chondrocyte.1 The core proteins of DS-PG I and DS-PG II are homologous tothose of CS-PG I and CS-PG II found in bone (Table 48–9). A possibleexplanation is that osteoblasts lack the epimerase required toconvert glucuronic acid to iduronic acid, the latter of which isfound in dermatan sulfate.

552 / CHAPTER 48in structurally abnormal proα chains that assemble intoabnormal fibrils, weakening the overall structure ofbone. When one abnormal chain is present, it may interactwith two normal chains, but folding may be prevented,resulting in enzymatic degradation of all of thechains. This is called “procollagen suicide” and is an exampleof a dominant negative mutation, a result oftenseen when a protein consists of multiple different subunits.Osteopetrosis (marble bone disease), characterizedby increased bone density, is due to inability to resorbbone. One form occurs along with renal tubular acidosisand cerebral calcification. It is due to mutations inthe gene (located on chromosome 8q22) encoding carbonicanhydrase II (CA II), one of four isozymes of carbonicanhydrase present in human tissues. The reactioncatalyzed by carbonic anhydrase is shown below:Fibril67 nmReaction II is spontaneous. In osteoclasts involved inbone resorption, CA II apparently provides protons toneutralize the OH − ions left inside the cell when H +ions are pumped across their ruffled borders (seeabove). Thus, if CA II is deficient in activity in osteoclasts,normal bone resorption does not occur, and osteopetrosisresults. The mechanism of the cerebral calcificationis not clear, whereas the renal tubular acidosisreflects deficient activity of CA II in the renal tubules.Osteoporosis is a generalized progressive reductionin bone tissue mass per unit volume causing skeletalweakness. The ratio of mineral to organic elements isunchanged in the remaining normal bone. Fractures ofvarious bones, such as the head of the femur, occur veryeasily and represent a huge burden to both the affectedpatients and to the health care budget of society.Among other factors, estrogens and interleukins-1 and-6 appear to be intimately involved in the causation ofosteoporosis.THE MAJOR COMPONENTS OFCARTILAGE ARE TYPE II COLLAGEN& CERTAIN PROTEOGLYCANSThe principal proteins of hyaline cartilage (the majortype of cartilage) are listed in Table 48–11. Type II collagenis the principal protein (Figure 48–13), and a numberof other minor types of collagen are also present. InHyaluronic acidType II collagen fibrilHyaluronic acidLink proteinChondroitin sulfateProteoglycanCollagen (type II)Core proteinFigure 48–13. Schematic representation of the molecular organization in cartilagematrix. Link proteins noncovalently bind the core protein (lighter color) of proteoglycansto the linear hyaluronic acid molecules (darker color). The chondroitin sulfate sidechains of the proteoglycan electrostatically bind to the collagen fibrils, forming across-linked matrix. The oval outlines the area enlarged in the lower part of the figure.(Reproduced, with permission, from Junqueira LC, Carneiro J: Basic Histology: Text & Atlas,10th ed. McGraw-Hill, 2003.)

THE EXTRACELLULAR MATRIX / 553addition to these components, elastic cartilage containselastin and fibroelastic cartilage contains type I collagen.Cartilage contains a number of proteoglycans, whichplay an important role in its compressibility. Aggrecan(about 2 × 10 3 kDa) is the major proteoglycan. As shownin Figure 48–14, it has a very complex structure, containingseveral GAGs (hyaluronic acid, chondroitin sulfate,and keratan sulfate) and both link and core proteins.The core protein contains three domains: A, B, and C.The hyaluronic acid binds noncovalently to domain A ofthe core protein as well as to the link protein, which stabilizesthe hyaluronate–core protein interactions. Thekeratan sulfate chains are located in domain B, whereasthe chondroitin sulfate chains are located in domain C;both of these types of GAGs are bound covalently to thecore protein. The core protein also contains both O- andN-linked oligosaccharide chains.The other proteoglycans found in cartilage havesimpler structures than aggrecan.Chondronectin is involved in the attachment oftype II collagen to chondrocytes.Cartilage is an avascular tissue and obtains most ofits nutrients from synovial fluid. It exhibits slow butcontinuous turnover. Various proteases (eg, collagenasesand stromalysin) synthesized by chondrocytes candegrade collagen and the other proteins found in cartilage.Interleukin-1 (IL-1) and tumor necrosis factor α(TNFα) appear to stimulate the production of suchproteases, whereas transforming growth factor β(TGFβ) and insulin-like growth factor 1 (IGF-I) generallyexert an anabolic influence on cartilage.THE MOLECULAR BASES OF THECHONDRODYSPLASIAS INCLUDEMUTATIONS IN GENES ENCODINGTYPE II COLLAGEN & FIBROBLASTGROWTH FACTOR RECEPTORSChondrodysplasias are a mixed group of hereditary disordersaffecting cartilage. They are manifested by shortlimbeddwarfism and numerous skeletal deformities. Anumber of them are due to a variety of mutations in theCOL2A1 gene, leading to abnormal forms of type IIcollagen. One example is Stickler syndrome, manifestedby degeneration of joint cartilage and of the vitreousbody of the eye.The best-known of the chondrodysplasias is achondroplasia,the commonest cause of short-limbeddwarfism. Affected individuals have short limbs, nor-Domain A Domain B Domain CHyaluronatebindingregionLinkproteinCoreproteinN-linkedoligosaccharideHyaluronic acidKeratansulfateChondroitinsulfateO-linkedoligosaccharideFigure 48–14. Schematic diagram of the aggrecan from bovine nasal cartilage. Astrand of hyaluronic acid is shown on the left. The core protein (about 210 kDa) hasthree major domains. Domain A, at its amino terminal end, interacts with approximatelyfive repeating disaccharides in hyaluronate. The link protein interacts withboth hyaluronate and domain A, stabilizing their interactions. Approximately 30 keratansulfate chains are attached, via GalNAc-Ser linkages, to domain B. Domain Ccontains about 100 chondroitin sulfate chains attached via Gal-Gal-Xyl-Ser linkagesand about 40 O-linked oligosaccharide chains. One or more N-linked glycan chainsare also found near the carboxyl terminal of the core protein. (Reproduced, with permission,from Moran LA et al: Biochemistry, 2nd ed. Neil Patterson Publishers, 1994.)

554 / CHAPTER 48mal trunk size, macrocephaly, and a variety of otherskeletal abnormalities. The condition is often inheritedas an autosomal dominant trait, but many cases are dueto new mutations. The molecular basis of achondroplasiais outlined in Figure 48–15. Achondroplasia is not acollagen disorder but is due to mutations in the geneencoding fibroblast growth factor receptor 3(FGFR3). Fibroblast growth factors are a family of atleast nine proteins that affect the growth and differentiationof cells of mesenchymal and neuroectodermal origin.Their receptors are transmembrane proteins andform a subgroup of the family of receptor tyrosine kinases.FGFR3 is one member of this subgroup and mediatesthe actions of FGF3 on cartilage. In almost allcases of achondroplasia that have been investigated, themutations were found to involve nucleotide 1138 andresulted in substitution of arginine for glycine (residuenumber 380) in the transmembrane domain of the protein,rendering it inactive. No such mutation was foundin unaffected individuals. As indicated in Table 48–10,other skeletal dysplasias (including certain craniosynostosissyndromes) are also due to mutations in genes encodingFGF receptors. Another type of skeletal dysplasia(diastrophic dysplasia) has been found to be due tomutation in a sulfate transporter. Thus, thanks to recombinantDNA technology, a new era in understandingof skeletal dysplasias has begun.Mutations of nucleotide 1138 in the geneencoding FGFR3 on chromosome 4Replacement in FGFR3 of Gly (codon 380) by ArgDefective function of FGFR3Abnormal development and growth of cartilageleading to short-limbed dwarfism and other featuresFigure 48–15. Simplified scheme of the causation ofachondroplasia (MIM 100800). In most cases studied sofar, the mutation has been a G to A transition at nucleotide1138. In a few cases, the mutation was a G to Ctransversion at the same nucleotide. This particular nucleotideis a real “hot spot” for mutation. Both mutationsresult in replacement of a Gly residue by an Argresidue in the transmembrane segment of the receptor.A few cases involving replacement of Gly by Cys atcodon 375 have also been reported.SUMMARY• The major components of the ECM are the structuralproteins collagen, elastin, and fibrillin; a numberof specialized proteins (eg, fibronectin andlaminin); and various proteoglycans.• Collagen is the most abundant protein in the animalkingdom; approximately 19 types have been isolated.All collagens contain greater or lesser stretches oftriple helix and the repeating structure (Gly-X-Y) n .• The biosynthesis of collagen is complex, featuringmany posttranslational events, including hydroxylationof proline and lysine.• Diseases associated with impaired synthesis of collageninclude scurvy, osteogenesis imperfecta, Ehlers-Danlos syndrome (many types), and Menkes disease.• Elastin confers extensibility and elastic recoil on tissues.Elastin lacks hydroxylysine, Gly-X-Y sequences,triple helical structure, and sugars but containsdesmosine and isodesmosine cross-links not found incollagen.• Fibrillin is located in microfibrils. Mutations in thegene for fibrillin cause Marfan syndrome.• The glycosaminoglycans (GAGs) are made up of repeatingdisaccharides containing a uronic acid (glucuronicor iduronic) or hexose (galactose) and a hexosamine(galactosamine or glucosamine). Sulfate isalso frequently present.• The major GAGs are hyaluronic acid, chondroitin4- and 6-sulfates, keratan sulfates I and II, heparin,heparan sulfate, and dermatan sulfate.• The GAGs are synthesized by the sequential actionsof a battery of specific enzymes (glycosyltransferases,epimerases, sulfotransferases, etc) and are degradedby the sequential action of lysosomal hydrolases. Geneticdeficiencies of the latter result in mucopolysaccharidoses(eg, Hurler syndrome).• GAGs occur in tissues bound to various proteins(linker proteins and core proteins), constituting proteoglycans.These structures are often of very highmolecular weight and serve many functions in tissues.• Many components of the ECM bind to proteins ofthe cell surface named integrins; this constitutes onepathway by which the exteriors of cells can communicatewith their interiors.• Bone and cartilage are specialized forms of the ECM.Collagen I and hydroxyapatite are the major constituentsof bone. Collagen II and certain proteoglycansare major constituents of cartilage.

THE EXTRACELLULAR MATRIX / 555• The molecular causes of a number of heritable diseasesof bone (eg, osteogenesis imperfecta) and of cartilage(eg, the chondrodystrophies) are being revealedby the application of recombinant DNA technology.REFERENCESBandtlow CE, Zimmermann DR: Proteoglycans in the developingbrain: new conceptual insights for old proteins. Physiol Rev2000;80:1267.Bikle DD: Biochemical markers in the assessment of bone diseases.Am J Med 1997;103:427.Burke D et al: Fibroblast growth factor receptors: lessons from thegenes. Trends Biochem Sci 1998;23:59.Compston JE: Sex steroids and bone. Physiol Rev 2001;81:419.Fuller GM, Shields D: Molecular Basis of Medical Cell Biology. Appleton& Lange, 1998.Herman T, Horvitz HR: Three proteins involved in Caenorhabditiselegans vulval invagination are similar to components of a glycosylationpathway. Proc Natl Acad Sci U S A 1999;96:974.Prockop DJ, Kivirikko KI: Collagens: molecular biology, diseases,and potential therapy. Annu Rev Biochem 1995;64:403.Pyeritz RE: Ehlers-Danlos syndrome. N Engl J Med 2000;342:730.Sage E: Regulation of interactions between cells and extracellularmatrix: a command performance on several stages. J Clin Invest2001;107:781. (This article introduces a series of six articleson cell-matrix interaction. The topics covered are celladhesion and de-adhesion, thrombospondins, syndecans,SPARC, osteopontin, and Ehlers-Danlos syndrome. All ofthe articles can be accessed at www.jci.org.)Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001 (This comprehensivefour-volume text contains chapters on disorders ofcollagen biosynthesis and structure, Marfan syndrome, themucopolysaccharidoses, achondroplasia, Alport syndrome,and craniosynostosis syndromes.)Selleck SB: Genetic dissection of proteoglycan function inDrosophila and C. elegans. Semin Cell Dev Biol 2001;12:127.

Muscle & the Cytoskeleton 49Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEProteins play an important role in movement at boththe organ (eg, skeletal muscle, heart, and gut) and cellularlevels. In this chapter, the roles of specific proteinsand certain other key molecules (eg, Ca 2+ ) in muscularcontraction are described. A brief coverage of cytoskeletalproteins is also presented.Knowledge of the molecular bases of a number ofconditions that affect muscle has advanced greatly in recentyears. Understanding of the molecular basis ofDuchenne-type muscular dystrophy was greatly enhancedwhen it was found that it was due to mutationsin the gene encoding dystrophin. Significant progresshas also been made in understanding the molecularbasis of malignant hyperthermia, a serious complicationfor some patients undergoing certain types of anesthesia.Heart failure is a very common medical condition,with a variety of causes; its rational therapyrequires understanding of the biochemistry of heartmuscle. One group of conditions that cause heart failureare the cardiomyopathies, some of which are geneticallydetermined. Nitric oxide (NO) has beenfound to be a major regulator of smooth muscle tone.Many widely used vasodilators—such as nitroglycerin,used in the treatment of angina pectoris—act by increasingthe formation of NO. Muscle, partly becauseof its mass, plays major roles in the overall metabolismof the body.MUSCLE TRANSDUCES CHEMICALENERGY INTO MECHANICAL ENERGYMuscle is the major biochemical transducer (machine)that converts potential (chemical) energy into kinetic(mechanical) energy. Muscle, the largest single tissue inthe human body, makes up somewhat less than 25% ofbody mass at birth, more than 40% in the young adult,and somewhat less than 30% in the aged adult. We556shall discuss aspects of the three types of muscle foundin vertebrates: skeletal, cardiac, and smooth. Bothskeletal and cardiac muscle appear striated upon microscopicobservation; smooth muscle is nonstriated. Althoughskeletal muscle is under voluntary nervous control,the control of both cardiac and smooth muscle isinvoluntary.The Sarcoplasm of Muscle CellsContains ATP, Phosphocreatine,& Glycolytic EnzymesStriated muscle is composed of multinucleated musclefiber cells surrounded by an electrically excitable plasmamembrane, the sarcolemma. An individual musclefiber cell, which may extend the entire length of themuscle, contains a bundle of many myofibrils arrangedin parallel, embedded in intracellular fluid termed sarcoplasm.Within this fluid is contained glycogen, thehigh-energy compounds ATP and phosphocreatine,and the enzymes of glycolysis.The Sarcomere Is the FunctionalUnit of MuscleAn overall view of voluntary muscle at several levels oforganization is presented in Figure 49–1.When the myofibril is examined by electron microscopy,alternating dark and light bands (anisotropicbands, meaning birefringent in polarized light; andisotropic bands, meaning not altered by polarized light)can be observed. These bands are thus referred to as Aand I bands, respectively. The central region of the Aband (the H band) appears less dense than the rest ofthe band. The I band is bisected by a very dense andnarrow Z line (Figure 49–2).The sarcomere is defined as the region between twoZ lines (Figures 49–1 and 49–2) and is repeated alongthe axis of a fibril at distances of 1500–2300 nm dependingupon the state of contraction.

MUSCLE & THE CYTOSKELETON / 557AMuscleBMuscle fasciculusC20–100 µmMuscle fiberHbandZlineAbandIbandD1–2 µmZ – Sarcomere – ZMyofibrilFigure 49–1. The structure of voluntary muscle. The sarcomere is the region between theZ lines. (Drawing by Sylvia Colard Keene. Reproduced, with permission, from Bloom W, FawcettDW: A Textbook of Histology, 10th ed. Saunders, 1975.)The striated appearance of voluntary and cardiacmuscle in light microscopic studies results from theirhigh degree of organization, in which most muscle fibercells are aligned so that their sarcomeres are in parallelregister (Figure 49–1).Thick Filaments Contain Myosin;Thin Filaments Contain Actin,Tropomyosin, & TroponinWhen myofibrils are examined by electron microscopy,it appears that each one is constructed of two types oflongitudinal filaments. One type, the thick filament,confined to the A band, contains chiefly the proteinmyosin. These filaments are about 16 nm in diameterand arranged in cross-section as a hexagonal array (Figure49–2, center; right-hand cross-section).The thin filament (about 7 nm in diameter) lies inthe I band and extends into the A band but not into itsH zone (Figure 49–2). Thin filaments contain the proteinsactin, tropomyosin, and troponin (Figure 49–3).In the A band, the thin filaments are arranged aroundthe thick (myosin) filament as a secondary hexagonalarray. Each thin filament lies symmetrically betweenthree thick filaments (Figure 49–2, center; mid crosssection),and each thick filament is surrounded symmetricallyby six thin filaments.The thick and thin filaments interact via crossbridgesthat emerge at intervals of 14 nm along thethick filaments. As depicted in Figure 49–2, the crossbridges(drawn as arrowheads at each end of the myosinfilaments, but not shown extending fully across to thethin filaments) have opposite polarities at the two endsof the thick filaments. The two poles of the thick filamentsare separated by a 150-nm segment (the M band,not labeled in the figure) that is free of projections.The Sliding Filament Cross-BridgeModel Is the Foundation on WhichCurrent Thinking About MuscleContraction Is BuiltThis model was proposed independently in the 1950sby Henry Huxley and Andrew Huxley and their colleagues.It was largely based on careful morphologic observationson resting, extended, and contracting muscle.Basically, when muscle contracts, there is no changein the lengths of the thick and thin filaments, but theH zones and the I bands shorten (see legend to Fig-

558 / CHAPTER 49H bandA. ExtendedI bandA bandZ line2300 nmActin filaments6-nm diameterα-ActininMyosin filaments16-nm diameterCross section:B. ContractedThinfilament6-nm diameterThickfilament1500 nm16-nm diameterFigure 49–2. Arrangement of filaments in striated muscle. A: Extended. The positions of theI, A, and H bands in the extended state are shown. The thin filaments partly overlap the ends of thethick filaments, and the thin filaments are shown anchored in the Z lines (often called Z disks). Inthe lower part of Figure 49–2A, “arrowheads,” pointing in opposite directions, are shown emanatingfrom the myosin (thick) filaments. Four actin (thin) filaments are shown attached to two Z linesvia α-actinin. The central region of the three myosin filaments, free of arrowheads, is called theM band (not labeled). Cross-sections through the M bands, through an area where myosin andactin filaments overlap and through an area in which solely actin filaments are present, are shown.B: Contracted. The actin filaments are seen to have slipped along the sides of the myosin fibers towardeach other. The lengths of the thick filaments (indicated by the A bands) and the thin filaments(distance between Z lines and the adjacent edges of the H bands) have not changed. However,the lengths of the sarcomeres have been reduced (from 2300 nm to 1500 nm), and thelengths of the H and I bands are also reduced because of the overlap between the thick and thinfilaments. These morphologic observations provided part of the basis for the sliding filamentmodel of muscle contraction.

MUSCLE & THE CYTOSKELETON / 559G-actinF-actin6–7 nmTropomyosinTroponin38.5 nmTpCTpITpT35.5 nmThe assembled thin filamentFigure 49–3. Schematic representation of the thin filament, showing the spatial configuration of its threemajor protein components: actin, myosin, and tropomyosin. The upper panel shows individual molecules ofG-actin. The middle panel shows actin monomers assembled into F-actin. Individual molecules of tropomyosin(two strands wound around one another) and of troponin (made up of its three subunits) are also shown. Thelower panel shows the assembled thin filament, consisting of F-actin, tropomyosin, and the three subunits oftroponin (TpC, TpI, and TpT).ure 49–2). Thus, the arrays of interdigitating filamentsmust slide past one another during contraction. Crossbridgesthat link thick and thin filaments at certainstages in the contraction cycle generate and sustain thetension. The tension developed during muscle contractionis proportionate to the filament overlap and to thenumber of cross-bridges. Each cross-bridge head is connectedto the thick filament via a flexible fibrous segmentthat can bend outward from the thick filament.This flexible segment facilitates contact of the headwith the thin filament when necessary but is also sufficientlypliant to be accommodated in the interfilamentspacing.ACTIN & MYOSIN ARE THE MAJORPROTEINS OF MUSCLEThe mass of a muscle is made up of 75% water andmore than 20% protein. The two major proteins areactin and myosin.Monomeric G-actin (43 kDa; G, globular) makesup 25% of muscle protein by weight. At physiologicionic strength and in the presence of Mg 2+ , G-actinpolymerizes noncovalently to form an insoluble doublehelical filament called F-actin (Figure 49–3). TheF-actin fiber is 6–7 nm thick and has a pitch or repeatingstructure every 35.5 nm.

560 / CHAPTER 49Myosins constitute a family of proteins, with atleast 15 members having been identified. The myosindiscussed in this chapter is myosin-II, and when myosinis referred to in this text, it is this species that is meantunless otherwise indicated. Myosin-I is a monomericspecies that binds to cell membranes. It may serve as alinkage between microfilaments and the cell membranein certain locations.Myosin contributes 55% of muscle protein byweight and forms the thick filaments. It is an asymmetrichexamer with a molecular mass of approximately460 kDa. Myosin has a fibrous tail consisting of two intertwinedhelices. Each helix has a globular head portionattached at one end (Figure 49–4). The hexamerconsists of one pair of heavy (H) chains each of approximately200 kDA molecular mass, and two pairs oflight (L) chains each with a molecular mass of approximately20 kDa. The L chains differ, one being calledthe essential light chain and the other the regulatorylight chain. Skeletal muscle myosin binds actin to formactomyosin (actin-myosin), and its intrinsic ATPase activityis markedly enhanced in this complex. Isoformsof myosin exist whose amounts can vary in differentanatomic, physiologic, and pathologic situations.The structures of actin and of the head of myosinhave been determined by x-ray crystallography; thesestudies have confirmed a number of earlier findingsconcerning their structures and have also given rise tomuch new information.Limited Digestion of Myosin WithProteases Has Helped to ElucidateIts Structure & FunctionWhen myosin is digested with trypsin, two myosinfragments (meromyosins) are generated. Light meromyosin(LMM) consists of aggregated, insoluble α-helicalfibers from the tail of myosin (Figure 49–4). LMMLLLLLL9 nmLGGLLG G G G HMM S-1LLLPAPAINTRYPSINHMM134 nmLMMHMM S-285 nmFigure 49–4. Diagram of a myosin molecule showing the two intertwined α-helices (fibrous portion), theglobular region or head (G), the light chains (L), and the effects of proteolytic cleavage by trypsin and papain.The globular region (myosin head) contains an actin-binding site and an L chain-binding site and also attachesto the remainder of the myosin molecule.

MUSCLE & THE CYTOSKELETON / 561exhibits no ATPase activity and does not bind toF-actin.Heavy meromyosin (HMM; molecular mass about340 kDa) is a soluble protein that has both a fibrousportion and a globular portion (Figure 49–4). It exhibitsATPase activity and binds to F-actin. Digestionof HMM with papain generates two subfragments, S-1and S-2. The S-2 fragment is fibrous in character, hasno ATPase activity, and does not bind to F-actin.S-1 (molecular mass approximately 115 kDa) doesexhibit ATPase activity, binds L chains, and in the absenceof ATP will bind to and decorate actin with “arrowheads”(Figure 49–5). Both S-1 and HMM exhibitATPase activity, which is accelerated 100- to 200-fold bycomplexing with F-actin. As discussed below, F-actingreatly enhances the rate at which myosin ATPase releasesits products, ADP and P i . Thus, although F-actindoes not affect the hydrolysis step per se, its ability topromote release of the products produced by the ATPaseactivity greatly accelerates the overall rate of catalysis.CHANGES IN THE CONFORMATIONOF THE HEAD OF MYOSIN DRIVEMUSCLE CONTRACTIONHow can hydrolysis of ATP produce macroscopicmovement? Muscle contraction essentially consists ofthe cyclic attachment and detachment of the S-1 head ofmyosin to the F-actin filaments. This process can also bereferred to as the making and breaking of cross-bridges.The attachment of actin to myosin is followed by conformationalchanges which are of particular importancein the S-1 head and are dependent upon which nucleotideis present (ADP or ATP). These changes resultin the power stroke, which drives movement of actinfilaments past myosin filaments. The energy for thepower stroke is ultimately supplied by ATP, which ishydrolyzed to ADP and P i . However, the power strokeitself occurs as a result of conformational changes inthe myosin head when ADP leaves it.The major biochemical events occurring during onecycle of muscle contraction and relaxation can be representedin the five steps shown in Figure 49–6:(1) In the relaxation phase of muscle contraction,the S-1 head of myosin hydrolyzes ATP to ADP and P i ,but these products remain bound. The resultant ADP-P i -myosin complex has been energized and is in a socalledhigh-energy conformation.(2) When contraction of muscle is stimulated (viaevents involving Ca 2+ , troponin, tropomyosin, andactin, which are described below), actin becomes accessibleand the S-1 head of myosin finds it, binds it, andforms the actin-myosin-ADP-P i complex indicated.(3) Formation of this complex promotes the releaseof P i , which initiates the power stroke. This is followedby release of ADP and is accompanied by a largeconformational change in the head of myosin in relationto its tail (Figure 49–7), pulling actin about 10 nmtoward the center of the sarcomere. This is the powerstroke. The myosin is now in a so-called low-energystate, indicated as actin-myosin.(4) Another molecule of ATP binds to the S-1 head,forming an actin-myosin-ATP complex.(5) Myosin-ATP has a low affinity for actin, andactin is thus released. This last step is a key componentof relaxation and is dependent upon the bindingof ATP to the actin-myosin complex.ActinATP-MyosinH 2 OActin-MyosinATP51ATP4ADP-P i -MyosinActin-Myosin2ActinADP+P i3Actin-MyosinADP-P iFigure 49–5. The decoration of actin filaments withthe S-1 fragments of myosin to form “arrowheads.”(Courtesy of JA Spudich.)Figure 49–6. The hydrolysis of ATP drives the cyclicassociation and dissociation of actin and myosin in fivereactions described in the text. (Modified from Stryer L:Biochemistry, 2nd ed. Freeman, 1981.)

562 / CHAPTER 49123Thick filamentLMMS-2 S-1Thin filamentFigure 49–7. Representation of the active crossbridgesbetween thick and thin filaments. This diagramwas adapted by AF Huxley from HE Huxley: Themechanism of muscular contraction. Science1969;164:1356. The latter proposed that the force involvedin muscular contraction originates in a tendencyfor the myosin head (S-1) to rotate relative to the thinfilament and is transmitted to the thick filament by theS-2 portion of the myosin molecule acting as an inextensiblelink. Flexible points at each end of S-2 permitS-1 to rotate and allow for variations in the separationbetween filaments. The present figure is based on HEHuxley’s proposal but also incorporates elastic (the coilsin the S-2 portion) and stepwise-shortening elements(depicted here as four sites of interaction between theS-1 portion and the thin filament). (See Huxley AF, SimmonsRM: Proposed mechanism of force generation instriated muscle. Nature [Lond] 1971;233:533.) Thestrengths of binding of the attached sites are higher inposition 2 than in position 1 and higher in position 3than position 2. The myosin head can be detached fromposition 3 with the utilization of a molecule of ATP; thisis the predominant process during shortening. Themyosin head is seen to vary in its position from about90° to about 45°, as indicated in the text. (S-1, myosinhead; S-2, portion of the myosin molecule; LMM, lightmeromyosin) (see legend to Figure 49–4). (Reproducedfrom Huxley AF: Muscular contraction. J Physiol 1974;243:1. By kind permission of the author and the Journal ofPhysiology.)Another cycle then commences with the hydrolysisof ATP (step 1 of Figure 49–6), re-forming the highenergyconformation.Thus, hydrolysis of ATP is used to drive the cycle,with the actual power stroke being the conformationalchange in the S-1 head that occurs upon the release ofADP. The hinge regions of myosin (referred to as flexiblepoints at each end of S-2 in the legend to Figure49–7) permit the large range of movement of S-1 andalso allow S-1 to find actin filaments.If intracellular levels of ATP drop (eg, after death),ATP is not available to bind the S-1 head (step 4above), actin does not dissociate, and relaxation (step 5)does not occur. This is the explanation for rigor mortis,the stiffening of the body that occurs after death.Calculations have indicated that the efficiency ofcontraction is about 50%; that of the internal combustionengine is less than 20%.Tropomyosin & the Troponin ComplexPresent in Thin Filaments Perform KeyFunctions in Striated MuscleIn striated muscle, there are two other proteins that areminor in terms of their mass but important in terms oftheir function. Tropomyosin is a fibrous molecule thatconsists of two chains, alpha and beta, that attach toF-actin in the groove between its filaments (Figure 49–3).Tropomyosin is present in all muscular and muscle-likestructures. The troponin complex is unique to striatedmuscle and consists of three polypeptides. Troponin T(TpT) binds to tropomyosin as well as to the other twotroponin components. Troponin I (TpI) inhibits theF-actin-myosin interaction and also binds to the othercomponents of troponin. Troponin C (TpC) is a calcium-bindingpolypeptide that is structurally and functionallyanalogous to calmodulin, an important calcium-bindingprotein widely distributed in nature.Four molecules of calcium ion are bound per moleculeof troponin C or calmodulin, and both molecules havea molecular mass of 17 kDa.Ca 2+ Plays a Central Role in Regulationof Muscle ContractionThe contraction of muscles from all sources occurs bythe general mechanism described above. Muscles fromdifferent organisms and from different cells and tissueswithin the same organism may have different molecularmechanisms responsible for the regulation of their contractionand relaxation. In all systems, Ca 2+ plays a keyregulatory role. There are two general mechanisms ofregulation of muscle contraction: actin-based andmyosin-based. The former operates in skeletal and cardiacmuscle, the latter in smooth muscle.Actin-Based Regulation Occursin Striated MuscleActin-based regulation of muscle occurs in vertebrateskeletal and cardiac muscles, both striated. In the gen-

MUSCLE & THE CYTOSKELETON / 563eral mechanism described above (Figure 49–6), theonly potentially limiting factor in the cycle of musclecontraction might be ATP. The skeletal muscle systemis inhibited at rest; this inhibition is relieved to activatecontraction. The inhibitor of striated muscle is the troponinsystem, which is bound to tropomyosin andF-actin in the thin filament (Figure 49–3). In striatedmuscle, there is no control of contraction unless thetropomyosin-troponin systems are present along withthe actin and myosin filaments. As described above,tropomyosin lies along the groove of F-actin, andthe three components of troponin—TpT, TpI, andTpC—are bound to the F-actin–tropomyosin complex.TpI prevents binding of the myosin head to its F-actinattachment site either by altering the conformation ofF-actin via the tropomyosin molecules or by simplyrolling tropomyosin into a position that directly blocksthe sites on F-actin to which the myosin heads attach.Either way prevents activation of the myosin ATPasethat is mediated by binding of the myosin head toF-actin. Hence, the TpI system blocks the contractioncycle at step 2 of Figure 49–6. This accounts for the inhibitedstate of relaxed striated muscle.The Sarcoplasmic ReticulumRegulates Intracellular Levelsof Ca 2+ in Skeletal MuscleIn the sarcoplasm of resting muscle, the concentrationof Ca 2+ is 10 −8 to 10 −7 mol/L. The resting state isachieved because Ca 2+ is pumped into the sarcoplasmicreticulum through the action of an active transport system,called the Ca 2+ ATPase (Figure 49–8), initiatingrelaxation. The sarcoplasmic reticulum is a network offine membranous sacs. Inside the sarcoplasmic reticulum,Ca 2+ is bound to a specific Ca 2+ -binding proteindesignated calsequestrin. The sarcomere is surroundedby an excitable membrane (the T tubule system) composedof transverse (T) channels closely associated withthe sarcoplasmic reticulum.When the sarcolemma is excited by a nerve impulse,the signal is transmitted into the T tubule system and aCa 2+ release channel in the nearby sarcoplasmic reticulumopens, releasing Ca 2+ from the sarcoplasmic reticuluminto the sarcoplasm. The concentration of Ca 2+ inthe sarcoplasm rises rapidly to 10 −5 mol/L. The Ca 2+ -binding sites on TpC in the thin filament are quicklyoccupied by Ca 2+ . The TpC-4Ca 2+ interacts with TpIand TpT to alter their interaction with tropomyosin.Accordingly, tropomyosin moves out of the way or altersthe conformation of F-actin so that the myosinhead-ADP-P i (Figure 49–6) can interact with F-actin tostart the contraction cycle.The Ca 2+ release channel is also known as the ryanodinereceptor (RYR). There are two isoforms of thisSarcolemmaDihydropyridinereceptorCa 2+ releasechannelCa 2+CisternaCa 2+CalsequestrinCa 2+SarcomereT tubuleCa 2+Ca 2+ Ca 2+CalsequestrinCisternaCa 2+ ATPaseFigure 49–8. Diagram of the relationships amongthe sarcolemma (plasma membrane), a T tubule, andtwo cisternae of the sarcoplasmic reticulum of skeletalmuscle (not to scale). The T tubule extends inward fromthe sarcolemma. A wave of depolarization, initiated bya nerve impulse, is transmitted from the sarcolemmadown the T tubule. It is then conveyed to the Ca 2+ releasechannel (ryanodine receptor), perhaps by interactionbetween it and the dihydropyridine receptor (slowCa 2+ voltage channel), which are shown in close proximity.Release of Ca 2+ from the Ca 2+ release channel intothe cytosol initiates contraction. Subsequently, Ca 2+ ispumped back into the cisternae of the sarcoplasmicreticulum by the Ca 2+ ATPase (Ca 2+ pump) and storedthere, in part bound to calsequestrin.receptor, RYR1 and RYR2, the former being present inskeletal muscle and the latter in heart muscle and brain.Ryanodine is a plant alkaloid that binds to RYR1 andRYR2 specifically and modulates their activities. TheCa 2+ release channel is a homotetramer made up of foursubunits of kDa 565. It has transmembrane sequencesat its carboxyl terminal, and these probably form theCa 2+ channel. The remainder of the protein protrudesinto the cytosol, bridging the gap between the sarcoplasmicreticulum and the transverse tubular membrane.The channel is ligand-gated, Ca 2+ and ATPworking synergistically in vitro, although how it operatesin vivo is not clear. A possible sequence of eventsleading to opening of the channel is shown in Figure49–9. The channel lies very close to the dihydropyridinereceptor (DHPR; a voltage-gated slow K type

564 / CHAPTER 49Depolarization of nerveDepolarization of skeletal muscleDepolarization of the transverse tubular membraneCharge movement of the slow Ca 2+ voltagechannel (DHPR) of the transverse tubular membraneOpening of the Ca 2+ release channel (RYR1)Figure 49–9. Possible chain of events leading toopening of the Ca 2+ release channel. As indicated in thetext, the Ca 2+ voltage channel and the Ca 2+ releasechannel have been shown to interact with each other invitro via specific regions in their polypeptide chains.(DHPR, dihydropyridine receptor; RYR1, ryanodine receptor1.)Ca 2+ channel) of the transverse tubule system (Figure49–8). Experiments in vitro employing an affinity columnchromatography approach have indicated that a37-amino-acid stretch in RYR1 interacts with one specificloop of DHPR.Relaxation occurs when sarcoplasmic Ca 2+ fallsbelow 10 −7 mol/L owing to its resequestration into thesarcoplasmic reticulum by Ca 2+ ATPase. TpC.4Ca 2+thus loses its Ca 2+ . Consequently, troponin, via interactionwith tropomyosin, inhibits further myosin headand F-actin interaction, and in the presence of ATP themyosin head detaches from the F-actin.Thus, Ca 2+ controls skeletal muscle contraction andrelaxation by an allosteric mechanism mediated byTpC, TpI, TpT, tropomyosin, and F-actin.A decrease in the concentration of ATP in the sarcoplasm(eg, by excessive usage during the cycle of contraction-relaxationor by diminished formation, such asmight occur in ischemia) has two major effects: (1) TheCa 2+ ATPase (Ca 2+ pump) in the sarcoplasmic reticulumceases to maintain the low concentration of Ca 2+in the sarcoplasm. Thus, the interaction of the myosinheads with F-actin is promoted. (2) The ATP-dependentdetachment of myosin heads from F-actin cannotoccur, and rigidity (contracture) sets in. The conditionof rigor mortis, following death, is an extension ofthese events.Muscle contraction is a delicate dynamic balance ofthe attachment and detachment of myosin heads toF-actin, subject to fine regulation via the nervoussystem.Table 49–1 summarizes the overall events in contractionand relaxation of skeletal muscle.Mutations in the Gene Encoding the Ca 2 +Release Channel Are One Cause of HumanMalignant HyperthermiaSome genetically predisposed patients experience a severereaction, designated malignant hyperthermia, onexposure to certain anesthetics (eg, halothane) and depolarizingskeletal muscle relaxants (eg, succinylcholine).The reaction consists primarily of rigidity ofskeletal muscles, hypermetabolism, and high fever. Ahigh cytosolic concentration of Ca 2+ in skeletal muscleis a major factor in its causation. Unless malignanthyperthermia is recognized and treated immediately,patients may die acutely of ventricular fibrillation orsurvive to succumb subsequently from other seriouscomplications. Appropriate treatment is to stop theanesthetic and administer the drug dantrolene intravenously.Dantrolene is a skeletal muscle relaxant thatacts to inhibit release of Ca 2+ from the sarcoplasmicreticulum into the cytosol, thus preventing the increaseof cytosolic Ca 2+ found in malignant hyperthermia.Table 49–1. Sequence of events in contractionand relaxation of skeletal muscle. 1Steps in contraction(1) Discharge of motor neuron(2) Release of transmitter (acetylcholine) at motor endplate(3) Binding of acetylcholine to nicotinic acetylcholine receptors(4) Increased Na + and K + conductance in endplate membrane(5) Generation of endplate potential(6) Generation of action potential in muscle fibers(7) Inward spread of depolarization along T tubules(8) Release of Ca 2+ from terminal cisterns of sarcoplasmicreticulum and diffusion to thick and thin filaments(9) Binding of Ca 2+ to troponin C, uncovering myosinbinding sites of actin(10) Formation of cross-linkages between actin andmyosin and sliding of thin on thick filaments, producingshorteningSteps in relaxation(1) Ca 2+ pumped back into sarcoplasmic reticulum(2) Release of Ca 2+ from troponin(3) Cessation of interaction between actin and myosin1 Reproduced, with permission, from Ganong WF: Review of MedicalPhysiology, 21st ed. McGraw-Hill, 2003.

MUSCLE & THE CYTOSKELETON / 565Malignant hyperthermia also occurs in swine. Susceptibleanimals homozygous for malignant hyperthermiarespond to stress with a fatal reaction (porcinestress syndrome) similar to that exhibited by humans.If the reaction occurs prior to slaughter, it affects thequality of the pork adversely, resulting in an inferiorproduct. Both events can result in considerable economiclosses for the swine industry.The finding of a high level of cytosolic Ca 2+ in musclein malignant hyperthermia suggested that the conditionmight be caused by abnormalities of the Ca 2+ATPase or of the Ca 2+ release channel. No abnormalitieswere detected in the former, but sequencing ofcDNAs for the latter protein proved insightful, particularlyin swine. All cDNAs from swine with malignanthyperthermia so far examined have shown a substitutionof T for C1843, resulting in the substitution ofCys for Arg 615 in the Ca 2+ release channel. The mutationaffects the function of the channel in that it opensmore easily and remains open longer; the net result ismassive release of Ca 2+ into the cytosol, ultimately causingsustained muscle contraction.The picture is more complex in humans, since malignanthyperthermia exhibits genetic heterogeneity.Members of a number of families who suffer from malignanthyperthermia have not shown genetic linkageto the RYR1 gene. Some humans susceptible to malignanthyperthermia have been found to exhibit thesame mutation found in swine, and others have a varietyof point mutations at different loci in the RYR1gene. Certain families with malignant hypertensionhave been found to have mutations affecting theDHPR. Figure 49–10 summarizes the probable chainof events in malignant hyperthermia. The majorpromise of these findings is that, once additional mutationsare detected, it will be possible to screen, usingsuitable DNA probes, for individuals at risk of developingmalignant hyperthermia during anesthesia. Currentscreening tests (eg, the in vitro caffeine-halothanetest) are relatively unreliable. Affected individualscould then be given alternative anesthetics, whichwould not endanger their lives. It should also be possible,if desired, to eliminate malignant hyperthermiafrom swine populations using suitable breeding practices.Another condition due to mutations in the RYR1gene is central core disease. This is a rare myopathypresenting in infancy with hypotonia and proximalmuscle weakness. Electron microscopy reveals an absenceof mitochondria in the center of many type I (seebelow) muscle fibers. Damage to mitochondria inducedby high intracellular levels of Ca 2+ secondary to abnormalfunctioning of RYR1 appears to be responsible forthe morphologic findings.Mutations in the RYR1 geneAltered Ca 2+ release channel protein (RYR1)(eg, substitution of Cys for Arg 615 )Mutated channel opens more easily and stays openlonger, thus flooding the cytosol with Ca 2+High intracellular levels of Ca 2+ stimulate sustainedmuscle contraction (rigidity); high Ca 2+ also stimulatesbreakdown of glycogen, glycolysis, and aerobicmetabolism (resulting in excessive production of heat)Figure 49–10. Simplified scheme of the causation ofmalignant hyperthermia (MIM 145600). At least 17 differentpoint mutations have been detected in the RYR1gene, some of which are associated with central coredisease (MIM 117000). It is estimated that at least 50%of families with members who have malignant hyperthermiaare linked to the RYR1 gene. Some individualswith mutations in the gene encoding DHPR have alsobeen detected; it is possible that mutations in othergenes for proteins involved in certain aspects of musclemetabolism will also be found.MUTATIONS IN THE GENE ENCODINGDYSTROPHIN CAUSE DUCHENNEMUSCULAR DYSTROPHYA number of additional proteins play various roles inthe structure and function of muscle. They include titin(the largest protein known), nebulin, α-actinin, desmin,dystrophin, and calcineurin. Some properties of theseproteins are summarized in Table 49–2.Dystrophin is of special interest. Mutations in thegene encoding this protein have been shown to be thecause of Duchenne muscular dystrophy and the milderBecker muscular dystrophy (see Figure 49–11). Theyare also implicated in some cases of dilated cardiomyopathy(see below). The gene encoding dystrophin isthe largest gene known (≈ 2300 kb) and is situated onthe X chromosome, accounting for the maternal inheritancepattern of Duchenne and Becker muscular dystrophies.As shown in Figure 49–12, dystrophin formspart of a large complex of proteins that attach to or interactwith the plasmalemma. Dystrophin links theactin cytoskeleton to the ECM and appears to beneeded for assembly of the synaptic junction. Impairmentof these processes by formation of defective dystrophinis presumably critical in the causation of

566 / CHAPTER 49Table 49–2. Some other important proteinsof muscle.Protein Location Comment or FunctionTitin Reaches from the Z Largest protein in body.line to the M line Role in relaxation ofmuscle.Nebulin From Z line along May regulate assemblylength of actin and length of actinfilamentsfilaments.α-Actinin Anchors actin to Z Stabilizes actinlinesfilaments.Desmin Lies alongside actin Attaches to plasmafilamentsmembrane (plasmalemma).Dystrophin Attached to plasma- Deficient in Duchennelemmamuscular dystrophy.Mutations of its genecan also cause dilatedcardiomyopathy.Calcineurin Cytosol A calmodulin-regulatedprotein phosphatase.May play importantroles in cardiac hypertrophyand in regulatingamounts of slow andfast twitch muscles.Myosin- Arranged trans- Binds myosin and titin.binding versely in sarcomere Plays a role in mainproteinC A-bandstaining the structuralintegrity of the sarcomere.Deletion of part of the structural gene for dystrophin,located on the X chromosomeDiminished synthesis of the mRNA for dystrophinLow levels or absence of dystrophinMuscle contraction/relaxation affected;precise mechanisms not elucidatedProgressive, usually fatal muscular weaknessFigure 49–11. Summary of the causation ofDuchenne muscular dystrophy (MIM 310200).Duchenne muscular dystrophy. Mutations in the genesencoding some of the components of the sarcoglycancomplex shown in Figure 49–12 are responsible forlimb-girdle and certain other congenital forms of musculardystrophy.CARDIAC MUSCLE RESEMBLES SKELETALMUSCLE IN MANY RESPECTSThe general picture of muscle contraction in the heartresembles that of skeletal muscle. Cardiac muscle, likeskeletal muscle, is striated and uses the actin-myosintropomyosin-troponinsystem described above. Unlikeskeletal muscle, cardiac muscle exhibits intrinsic rhythmicity,and individual myocytes communicate witheach other because of its syncytial nature. The T tubularsystem is more developed in cardiac muscle,whereas the sarcoplasmic reticulum is less extensiveand consequently the intracellular supply of Ca 2+ forcontraction is less. Cardiac muscle thus relies on extracellularCa 2+ for contraction; if isolated cardiac muscleis deprived of Ca 2+ , it ceases to beat within approximately1 minute, whereas skeletal muscle can continueto contract without an extracellular source of Ca 2+ .Cyclic AMP plays a more prominent role in cardiacthan in skeletal muscle. It modulates intracellular levelsof Ca 2+ through the activation of protein kinases; theseenzymes phosphorylate various transport proteins inthe sarcolemma and sarcoplasmic reticulum and also inthe troponin-tropomyosin regulatory complex, affectingintracellular levels of Ca 2+ or responses to it. Thereis a rough correlation between the phosphorylation ofTpI and the increased contraction of cardiac muscle inducedby catecholamines. This may account for the inotropiceffects (increased contractility) of β-adrenergiccompounds on the heart. Some differences amongskeletal, cardiac, and smooth muscle are summarized inTable 49–3.Ca 2+ Enters Myocytes via Ca 2+ Channels& Leaves via the Na + -Ca 2+ Exchanger& the Ca 2+ ATPaseAs stated above, extracellular Ca 2+ plays an importantrole in contraction of cardiac muscle but not in skeletalmuscle. This means that Ca 2+ both enters and leavesmyocytes in a regulated manner. We shall briefly considerthree transmembrane proteins that play roles inthis process.A. Ca 2+ CHANNELSCa 2+ enters myocytes via these channels, which allowentry only of Ca 2+ ions. The major portal of entry is the

MUSCLE & THE CYTOSKELETON / 567Figure 49–12. Organization of dystrophin and other proteins in relation to the plasma membrane of muscle cells.Dystrophin is part of a large oligomeric complex associated with several other protein complexes. The dystroglycancomplex consists of α-dystroglycan, which associates with the basal lamina protein merosin, and β-dystroglycan,which binds α-dystroglycan and dystrophin. Syntrophin binds to the carboxyl terminal of dystrophin. The sarcoglycancomplex consists of four transmembrane proteins: α-, β-, γ-, and δ-sarcoglycan. The function of the sarcoglycancomplex and the nature of the interactions within the complex and between it and the other complexes are notclear. The sarcoglycan complex is formed only in striated muscle, and its subunits preferentially associate with eachother, suggesting that the complex may function as a single unit. Mutations in the gene encoding dystrophin causeDuchenne and Becker muscular dystrophy; mutations in the genes encoding the various sarcoglycans have beenshown to be responsible for limb-girdle dystrophies (eg, MIM 601173). (Reproduced, with permission, from Duggan DJet al: Mutations in the sarcoglycan genes in patients with myopathy. N Engl J Med 1997;336:618.)L-type (long-duration current, large conductance) orslow Ca 2+ channel, which is voltage-gated, openingduring depolarization induced by spread of the cardiacaction potential and closing when the action potentialdeclines. These channels are equivalent to the dihydropyridinereceptors of skeletal muscle (Figure 49–8).Slow Ca 2+ channels are regulated by cAMP-dependentprotein kinases (stimulatory) and cGMP-protein kinases(inhibitory) and are blocked by so-called calciumchannel blockers (eg, verapamil). Fast (or T, transient)Ca 2+ channels are also present in the plasmalemma,though in much lower numbers; they probably contributeto the early phase of increase of myoplasmicCa 2+ .The resultant increase of Ca 2+ in the myoplasm actson the Ca 2+ release channel of the sarcoplasmic reticulumto open it. This is called Ca 2+ -induced Ca 2+ release(CICR). It is estimated that approximately 10% of theCa 2+ involved in contraction enters the cytosol fromthe extracellular fluid and 90% from the sarcoplasmicreticulum. However, the former 10% is important, asthe rate of increase of Ca 2+ in the myoplasm is important,and entry via the Ca 2+ channels contributes appreciablyto this.B. Ca 2+ -Na + EXCHANGERThis is the principal route of exit of Ca 2+ from myocytes.In resting myocytes, it helps to maintain a lowlevel of free intracellular Ca 2+ by exchanging one Ca 2+for three Na + . The energy for the uphill movement ofCa 2+ out of the cell comes from the downhill movementof Na + into the cell from the plasma. This exchangecontributes to relaxation but may run in the re-

568 / CHAPTER 49Table 49–3. Some differences between skeletal, cardiac, and smooth muscle.Skeletal Muscle Cardiac Muscle Smooth Muscle1. Striated. 1. Striated. 1. Nonstriated.2. No syncytium. 2. Syncytial. 2. Syncytial.3. Small T tubules. 3. Large T tubules. 3. Generally rudimentary T tubules.4. Sarcoplasmic reticulum well- 4. Sarcoplasmic reticulum present and 4. Sarcoplasmic reticulum often rudimendevelopedand Ca 2+ pump acts Ca 2+ pump acts relatively rapidly. tary and Ca 2+ pump acts slowly.rapidly.5. Plasmalemma lacks many hormone 5. Plasmalemma contains a variety of 5. Plasmalemma contains a variety ofreceptors. receptors (eg, α- and β-adrenergic). receptors (eg, α- and β-adrenergic).6. Nerve impulse initiates contraction. 6. Has intrinsic rhythmicity. 6. Contraction initiated by nerve impulses,hormones, etc.7. Extracellular fluid Ca 2+ not important 7. Extracellular fluid Ca 2+ important 7. Extracellular fluid Ca 2+ important forfor contraction. for contraction. contraction.8. Troponin system present. 8. Troponin system present. 8. Lacks troponin system; uses regulatoryhead of myosin.9. Caldesmon not involved. 9. Caldesmon not involved. 9. Caldesmon is important regulatoryprotein.10. Very rapid cycling of the 10. Relatively rapid cycling of the cross- 10. Slow cycling of the cross-bridges percross-bridges.bridges. mits slow prolonged contractionand less utilization of ATP.verse direction during excitation. Because of the Ca 2+ -Na + exchanger, anything that causes intracellular Na +(Na + i) to rise will secondarily cause Ca 2+ i to rise, causingmore forceful contraction. This is referred to as apositive inotropic effect. One example is when the drugdigitalis is used to treat heart failure. Digitalis inhibitsthe sarcolemmal Na + -K + ATPase, diminishing exit ofNa + and thus increasing Na + i. This in turn causes Ca 2+to increase, via the Ca 2+ -Na + exchanger. The increasedCa 2+ i results in increased force of cardiac contraction, ofbenefit in heart failure.important in skeletal muscle. Mutations in genes encodingion channels have been shown to be responsiblefor a number of relatively rare conditions affecting muscle.These and other diseases due to mutations of ionchannels have been termed channelopathies; some arelisted in Table 49–5.Table 49–4. Major types of ion channels foundin cells.C. Ca 2+ ATPASEThis Ca 2+ pump, situated in the sarcolemma, also contributesto Ca 2+ exit but is believed to play a relativelyminor role as compared with the Ca 2+ -Na + exchanger.It should be noted that there are a variety of ionchannels (Chapter 41) in most cells, for Na + , K + , Ca 2+ ,etc. Many of them have been cloned in recent years andtheir dispositions in their respective membranes workedout (number of times each one crosses its membrane,location of the actual ion transport site in the protein,etc). They can be classified as indicated in Table 49–4.Cardiac muscle is rich in ion channels, and they are alsoTypeExternalligand-gatedCommentOpen in response to a specific extracellularmolecule, eg, acetylcholine.Open or close in response to a specific intra-cellular molecule, eg, a cyclic nucleotide.Open in response to a change in membranepotential, eg, Na + , K + , and Ca 2+ channels inheart.Open in response to change in mechanicalpressure.Internalligand-gatedVoltage-gatedMechanicallygated

MUSCLE & THE CYTOSKELETON / 569Table 49–5. Some disorders (channelopathies)due to mutations in genes encoding polypeptideconstituents of ion channels. 1Ion Channel and MajorDisorder 2Organs InvolvedCentral core disease Ca 2+ release channel (RYR1)(MIM 117000)Skeletal muscleCystic fibrosisCFTR (Cl − channel)(MIM 219700)Lungs, pancreasHyperkalemic periodic Sodium channelparalysis (MIM 170500) Skeletal muscleHypokalemic periodic Slow Ca 2+ voltage channel (DHPR)paralysis (MIM 114208) Skeletal muscleMalignant hyperthermia Ca 2+ release channel (RYR1)(MIM 180901)Skeletal muscleMyotonia congenita Chloride channel(MIM 160800)Skeletal muscle1 Data in part from Ackerman NJ, Clapham DE: Ion channels—basic science and clinical disease. N Engl J Med 1997;336:1575.2 Other channelopathies include the long QT syndrome (MIM192500); pseudoaldosteronism (Liddle syndrome, MIM 177200);persistent hyperinsulinemic hypoglycemia of infancy (MIM601820); hereditary X-linked recessive type II nephrolithiasis of infancy(Dent syndrome, MIM 300009); and generalized myotonia,recessive (Becker disease, MIM 255700). The term “myotonia” signifiesany condition in which muscles do not relax after contraction.Inherited Cardiomyopathies Are Dueto Disorders of Cardiac EnergyMetabolism or to AbnormalMyocardial ProteinsAn inherited cardiomyopathy is any structural or functionalabnormality of the ventricular myocardium dueto an inherited cause. There are nonheritable types ofcardiomyopathy, but these will not be described here.As shown in Table 49–6, the causes of inherited cardiomyopathiesfall into two broad classes: (1) disordersof cardiac energy metabolism, mainly reflecting mutationsin genes encoding enzymes or proteins involved infatty acid oxidation (a major source of energy for themyocardium) and oxidative phosphorylation; and(2) mutations in genes encoding proteins involved in oraffecting myocardial contraction, such as myosin,tropomyosin, the troponins, and cardiac myosinbindingprotein C. Mutations in the genes encodingthese latter proteins cause familial hypertrophic cardiomyopathy,which will now be discussed.Table 49–6. Biochemical causes of inheritedcardiomyopathies. 1,2Proteins or ProcessCauseAffectedInborn errors of fatty acid Carnitine entry into cells andoxidationmitochondriaCertain enzymes of fatty acidoxidationDisorders of mitochondrial Proteins encoded by mitooxidativephosphorylation chondrial genesProteins encoded by nucleargenesAbnormalities of myocardial β-Myosin heavy chains, tropocontractileand structural nin, tropomyosin, dysproteinstrophin1 Based on Kelly DP, Strauss AW: Inherited cardiomyopathies.N Engl J Med 1994;330:913.2 Mutations (eg, point mutations, or in some cases deletions) inthe genes (nuclear or mitochondrial) encoding various proteins,enzymes, or tRNA molecules are the fundamental causes of theinherited cardiomyopathies. Some conditions are mild, whereasothers are severe and may be part of a syndrome affecting othertissues.Mutations in the Cardiac -Myosin HeavyChain Gene Are One Cause of FamilialHypertrophic CardiomyopathyFamilial hypertrophic cardiomyopathy is one of themost frequent hereditary cardiac diseases. Patients exhibithypertrophy—often massive—of one or both ventricles,starting early in life, and not related to any extrinsiccause such as hypertension. Most cases aretransmitted in an autosomal dominant manner; the restare sporadic. Until recently, its cause was obscure. However,this situation changed when studies of one affectedfamily showed that a missense mutation (ie, substitutionof one amino acid by another) in the β-myosinheavy chain gene was responsible for the condition.Subsequent studies have shown a number of missensemutations in this gene, all coding for highly conservedresidues. Some individuals have shown other mutations,such as formation of an α/β-myosin heavy chain hybridgene. Patients with familial hypertrophic cardiomyopathycan show great variation in clinical picture. This inpart reflects genetic heterogeneity; ie, mutation in anumber of other genes (eg, those encoding cardiacactin, tropomyosin, cardiac troponins I and T, essentialand regulatory myosin light chains, and cardiac myosinbindingprotein C) may also cause familial hypertrophic

570 / CHAPTER 49cardiomyopathy. In addition, mutations at differentsites in the gene for β-myosin heavy chain may affect thefunction of the protein to a greater or lesser extent. Themissense mutations are clustered in the head and headrodregions of myosin heavy chain. One hypothesis isthat the mutant polypeptides (“poison polypeptides”)cause formation of abnormal myofibrils, eventually resultingin compensatory hypertrophy. Some mutationsalter the charge of the amino acid (eg, substitution ofarginine for glutamine), presumably affecting the conformationof the protein more markedly and thus affectingits function. Patients with these mutations have asignificantly shorter life expectancy than patients inwhom the mutation produced no alteration in charge.Thus, definition of the precise mutations involved in thegenesis of FHC may prove to be of important prognosticvalue; it can be accomplished by appropriate use ofthe polymerase chain reaction on genomic DNA obtainedfrom one sample of blood lymphocytes. Figure49–13 is a simplified scheme of the events causing familialhypertrophic cardiomyopathy.Another type of cardiomyopathy is termed dilatedcardiomyopathy. Mutations in the genes encoding dystrophin,muscle LIM protein (so called because it wasfound to contain a cysteine-rich domain originally detectedin three proteins: Lin-II, Isl-1, and Mec-3), andthe cyclic response-element binding protein (CREB)have been implicated in the causation of this condition.The first two proteins help organize the contractile apparatusof cardiac muscle cells, and CREB is involvedPredominantly missense mutations in the β-myosinheavy chain gene on chromosome 14Mutant polypeptide chains (“poison polypeptides”)that lead to formation of defective myofibrilsCompensatory hypertrophy of oneor both cardiac ventriclesCardiomegaly and various cardiac signs andsymptoms, including sudden deathFigure 49–13. Simplified scheme of the causation offamilial hypertrophic cardiomyopathy (MIM 192600)due to mutations in the gene encoding β-myosin heavychain. Mutations in genes encoding other proteins,such as the troponins, tropomyosin, and cardiacmyosin-binding protein C can also cause this condition.Mutations in genes encoding yet other proteins (eg,dystrophin) are involved in the causation of dilatedcardiomyopathy.in the regulation of a number of genes in these cells.Current research is not only elucidating the molecularcauses of the cardiomyopathies but is also disclosingmutations that cause cardiac developmental disorders(eg, septal defects) and arrhythmias (eg, due to mutationsaffecting ion channels).Ca 2+ Also Regulates Contractionof Smooth MuscleWhile all muscles contain actin, myosin, and tropomyosin,only vertebrate striated muscles contain thetroponin system. Thus, the mechanisms that regulatecontraction must differ in various contractile systems.Smooth muscles have molecular structures similar tothose in striated muscle, but the sarcomeres are notaligned so as to generate the striated appearance.Smooth muscles contain α-actinin and tropomyosinmolecules, as do skeletal muscles. They do not have thetroponin system, and the light chains of smooth musclemyosin molecules differ from those of striated musclemyosin. Regulation of smooth muscle contraction ismyosin-based, unlike striated muscle, which is actinbased.However, like striated muscle, smooth musclecontraction is regulated by Ca 2+ .Phosphorylation of Myosin Light ChainsInitiates Contraction of Smooth MuscleWhen smooth muscle myosin is bound to F-actin in theabsence of other muscle proteins such as tropomyosin,there is no detectable ATPase activity. This absence ofactivity is quite unlike the situation described for striatedmuscle myosin and F-actin, which has abundantATPase activity. Smooth muscle myosin contains lightchains that prevent the binding of the myosin head toF-actin; they must be phosphorylated before they allowF-actin to activate myosin ATPase. The ATPase activitythen attained hydrolyzes ATP about tenfold moreslowly than the corresponding activity in skeletal muscle.The phosphate on the myosin light chains may forma chelate with the Ca 2+ bound to the tropomyosin-TpCactincomplex, leading to an increased rate of formationof cross-bridges between the myosin heads and actin.The phosphorylation of light chains initiates the attachment-detachmentcontraction cycle of smooth muscle.Myosin Light Chain Kinase Is Activatedby Calmodulin-4Ca 2+ & ThenPhosphorylates the Light ChainsSmooth muscle sarcoplasm contains a myosin lightchain kinase that is calcium-dependent. The Ca 2+ activationof myosin light chain kinase requires binding ofcalmodulin-4Ca 2+ to its kinase subunit (Figure 49–14).

MUSCLE & THE CYTOSKELETON / 571Calmodulin10 –5 mol/L Ca 2+ 10 –7 mol/L Ca 2+Ca 2+ • calmodulinATPL-myosin (inhibitsmyosin-actin interaction)H 2 PO 4–PHOSPHATASEMyosin kinase(inactive)Ca 2+ • CALMODULIN–MYOSINKINASE (ACTIVE)The calmodulin-4Ca 2+ -activated light chain kinasephosphorylates the light chains, which then ceases to inhibitthe myosin–F-actin interaction. The contractioncycle then begins.Smooth Muscle Relaxes Whenthe Concentration of Ca 2+ FallsBelow 10 −7 MolarADPpL-myosin (does notinhibit myosin-actin interaction)Figure 49–14. Regulation of smooth muscle contractionby Ca 2+ . pL-myosin is the phosphorylated lightchain of myosin; L-myosin is the dephosphorylatedlight chain. (Adapted from Adelstein RS, Eisenberg R: Regulationand kinetics of actin-myosin ATP interaction. AnnuRev Biochem 1980;49:921.)Relaxation of smooth muscle occurs when sarcoplasmicCa 2+ falls below 10 −7 mol/L. The Ca 2+ dissociates fromcalmodulin, which in turn dissociates from the myosinlight chain kinase, inactivating the kinase. No newphosphates are attached to the p-light chain, and lightchain protein phosphatase, which is continually activeand calcium-independent, removes the existing phosphatesfrom the light chains. Dephosphorylated myosinp-light chain then inhibits the binding of myosin headsto F-actin and the ATPase activity. The myosin headdetaches from the F-actin in the presence of ATP, butit cannot reattach because of the presence of dephosphorylatedp-light chain; hence, relaxation occurs.Table 49–7 summarizes and compares the regulationof actin-myosin interactions (activation of myosinATPase) in striated and smooth muscles.The myosin light chain kinase is not directly affectedor activated by cAMP. However, cAMP-activatedprotein kinase can phosphorylate the myosinlight chain kinase (not the light chains themselves). Thephosphorylated myosin light chain kinase exhibits a significantlylower affinity for calmodulin-Ca 2+ and thus isless sensitive to activation. Accordingly, an increase incAMP dampens the contraction response of smoothmuscle to a given elevation of sarcoplasmic Ca 2+ . Thismolecular mechanism can explain the relaxing effect ofβ-adrenergic stimulation on smooth muscle.Another protein that appears to play a Ca 2+ -dependentrole in the regulation of smooth muscle contractionis caldesmon (87 kDa). This protein is ubiquitousin smooth muscle and is also found in nonmuscle tissue.At low concentrations of Ca 2+ , it binds to tropomyosinand actin. This prevents interaction of actinwith myosin, keeping muscle in a relaxed state. Athigher concentrations of Ca 2+ , Ca 2+ -calmodulin bindscaldesmon, releasing it from actin. The latter is thenfree to bind to myosin, and contraction can occur.Caldesmon is also subject to phosphorylation-dephosphorylation;when phosphorylated, it cannot bindactin, again freeing the latter to interact with myosin.Caldesmon may also participate in organizing the structureof the contractile apparatus in smooth muscle.Many of its effects have been demonstrated in vitro,and its physiologic significance is still under investigation.As noted in Table 49–3, slow cycling of the crossbridgespermits slow prolonged contraction of smoothmuscle (eg, in viscera and blood vessels) with less utilizationof ATP compared with striated muscle. Theability of smooth muscle to maintain force at reducedvelocities of contraction is referred to as the latch state;this is an important feature of smooth muscle, and itsprecise molecular bases are under study.Nitric Oxide Relaxes the Smooth Muscleof Blood Vessels & Also Has Many OtherImportant Biologic FunctionsAcetylcholine is a vasodilator that acts by causing relaxationof the smooth muscle of blood vessels. However,it does not act directly on smooth muscle. A key observationwas that if endothelial cells were stripped awayfrom underlying smooth muscle cells, acetylcholine nolonger exerted its vasodilator effect. This finding indicatedthat vasodilators such as acetylcholine initially interactwith the endothelial cells of small blood vesselsvia receptors. The receptors are coupled to the phosphoinositidecycle, leading to the intracellular release of

572 / CHAPTER 49Table 49–7. Actin-myosin interactions in striated and smooth muscle.Smooth MuscleStriated Muscle(and Nonmuscle Cells)Proteins of muscle filaments Actin ActinMyosin Myosin 1TropomyosinTropomyosinTroponin (Tpl, TpT, TpC)Spontaneous interaction of F-actin and Yes Nomyosin alone (spontaneous activationof myosin ATPase by F-actinInhibitor of F-actin–myosin interaction (in- Troponin system (Tpl) Unphosphorylated myosin light chainhibitor of F-actin–dependent activationof ATPase)Contraction activated by Ca 2+ Ca 2+Direct effect of Ca 2+ 4Ca 2+ bind to TpC 4Ca 2+ bind to calmodulinEffect of protein-bound Ca 2+ TpC ⋅ 4Ca 2+ antagonizes Tpl inhibition Calmodulin ⋅ 4Ca 2+ activates myosin lightof F-actin–myosin interaction (allows chain kinase that phosphorylates myosinF-actin activation of ATPase)p-light chain. The phosphorylated p-lightchain no longer inhibits F-actin–myosininteraction (allows F-actin activation ofATPase).1 Light chains of myosin are different in striated and smooth muscles.Ca 2+ through the action of inositol trisphosphate. Inturn, the elevation of Ca 2+ leads to the liberation of endothelium-derivedrelaxing factor (EDRF), whichdiffuses into the adjacent smooth muscle. There, it reactswith the heme moiety of a soluble guanylyl cyclase,resulting in activation of the latter, with a consequentelevation of intracellular levels of cGMP (Figure49–15). This in turn stimulates the activities of certaincGMP-dependent protein kinases, which probablyphosphorylate specific muscle proteins, causing relaxation;however, the details are still being clarified. Theimportant coronary artery vasodilator nitroglycerin,widely used to relieve angina pectoris, acts to increaseintracellular release of EDRF and thus of cGMP.Quite unexpectedly, EDRF was found to be the gasnitric oxide (NO). NO is formed by the action of theenzyme NO synthase, which is cytosolic. The endothelialand neuronal forms of NO synthase are activated byCa 2+ (Table 49–8). The substrate is arginine, and theproducts are citrulline and NO:ArginineNO SYNTHASECitrulline + NONO synthase catalyzes a five-electron oxidation ofan amidine nitrogen of arginine. L-Hydroxyarginine isan intermediate that remains tightly bound to the en-zyme. NO synthase is a very complex enzyme, employingfive redox cofactors: NADPH, FAD, FMN, heme,and tetrahydrobiopterin. NO can also be formed fromnitrite, derived from vasodilators such as glyceryl trinitrateduring their metabolism. NO has a very shorthalf-life (approximately 3–4 seconds) in tissues becauseit reacts with oxygen and superoxide. The product ofthe reaction with superoxide is peroxynitrite (ONOO − ),which decomposes to form the highly reactive OH •radical. NO is inhibited by hemoglobin and otherheme proteins, which bind it tightly. Chemical inhibitorsof NO synthase are now available that canmarkedly decrease formation of NO. Administration ofsuch inhibitors to animals and humans leads to vasoconstrictionand a marked elevation of blood pressure,indicating that NO is of major importance in the maintenanceof blood pressure in vivo. Another importantcardiovascular effect is that by increasing synthesis ofcGMP, it acts as an inhibitor of platelet aggregation(Chapter 51).Since the discovery of the role of NO as a vasodilator,there has been intense experimental interest in thissubstance. It has turned out to have a variety of physiologicroles, involving virtually every tissue of the body(Table 49–9). Three major isoforms of NO synthasehave been identified, each of which has been cloned,and the chromosomal locations of their genes in hu-

MUSCLE & THE CYTOSKELETON / 573Glyceryltrinitrate↑Ca 2+AcetylcholineArginine+RNO + CitrullineENDOTHELIALCELLNO synthasephosphate, and (4) from two molecules of ADP in a reactioncatalyzed by adenylyl kinase (Figure 49–16). Theamount of ATP in skeletal muscle is only sufficient toprovide energy for contraction for a few seconds, sothat ATP must be constantly renewed from one ormore of the above sources, depending upon metabolicconditions. As discussed below, there are at least twodistinct types of fibers in skeletal muscle, one predominantlyactive in aerobic conditions and the other inanaerobic conditions; not unexpectedly, they use eachof the above sources of energy to different extents.Nitrate Nitrite NOcGMPproteinkinases+RelaxationGTP+GuanylylcyclasecGMPSMOOTH MUSCLE CELLFigure 49–15. Diagram showing formation in an endothelialcell of nitric oxide (NO) from arginine in a reactioncatalyzed by NO synthase. Interaction of an agonist(eg, acetylcholine) with a receptor (R) probablyleads to intracellular release of Ca 2+ via inositol trisphosphategenerated by the phosphoinositide pathway, resultingin activation of NO synthase. The NO subsequentlydiffuses into adjacent smooth muscle, where itleads to activation of guanylyl cyclase, formation ofcGMP, stimulation of cGMP-protein kinases, and subsequentrelaxation. The vasodilator nitroglycerin is shownentering the smooth muscle cell, where its metabolismalso leads to formation of NO.mans have been determined. Gene knockout experimentshave been performed on each of the three isoformsand have helped establish some of the postulatedfunctions of NO.To summarize, research in the past decade hasshown that NO plays an important role in many physiologicand pathologic processes.SEVERAL MECHANISMS REPLENISHSTORES OF ATP IN MUSCLEThe ATP required as the constant energy source for thecontraction-relaxation cycle of muscle can be generated(1) by glycolysis, using blood glucose or muscle glycogen,(2) by oxidative phosphorylation, (3) from creatineSkeletal Muscle Contains LargeSupplies of GlycogenThe sarcoplasm of skeletal muscle contains large storesof glycogen, located in granules close to the I bands.The release of glucose from glycogen is dependent on aspecific muscle glycogen phosphorylase (Chapter 18),which can be activated by Ca 2+ , epinephrine, and AMP.To generate glucose 6-phosphate for glycolysis in skeletalmuscle, glycogen phosphorylase b must be activatedto phosphorylase a via phosphorylation by phosphorylaseb kinase (Chapter 18). Ca 2+ promotes the activationof phosphorylase b kinase, also by phosphorylation.Thus, Ca 2+ both initiates muscle contraction andactivates a pathway to provide necessary energy. Thehormone epinephrine also activates glycogenolysis inmuscle. AMP, produced by breakdown of ADP duringmuscular exercise, can also activate phosphorylase bwithout causing phosphorylation. Muscle glycogenphosphorylase b is inactive in McArdle disease, one ofthe glycogen storage diseases (Chapter 18).Under Aerobic Conditions, MuscleGenerates ATP Mainly by OxidativePhosphorylationSynthesis of ATP via oxidative phosphorylation requiresa supply of oxygen. Muscles that have a high demandfor oxygen as a result of sustained contraction(eg, to maintain posture) store it attached to the hememoiety of myoglobin. Because of the heme moiety,muscles containing myoglobin are red, whereas muscleswith little or no myoglobin are white. Glucose, derivedfrom the blood glucose or from endogenous glycogen,and fatty acids derived from the triacylglycerols of adiposetissue are the principal substrates used for aerobicmetabolism in muscle.Creatine Phosphate Constitutes aMajor Energy Reserve in MuscleCreatine phosphate prevents the rapid depletion ofATP by providing a readily available high-energy phosphatethat can be used to regenerate ATP from ADP.

574 / CHAPTER 49Table 49–8. Summary of the nomenclature of the NO synthases and of the effects of knockout of theirgenes in mice. 1Result of GeneSubtype Name 2 Comments Knockout in Mice 31 nNOS Activity depends on elevated Ca 2+ . First Pyloric stenosis, resistant to vascular stroke, aggressiveidentified in neurons. Calmodulin-activated. sexual behavior (males).2 iNOS 4 Independent of elevated Ca 2+ . More susceptible to certain types of infection.Prominent in macrophages.3 eNOS Activity depends on elevated Ca 2+ . Elevated mean blood pressure.First identified in endothelial cells.1 Adapted from Snyder SH: No endothelial NO. Nature 1995;377:196.2 n, neuronal; i, inducible; e, endothelial.3 Gene knockouts were performed by homologous recombination in mice. The enzymes are characterized as neuronal, inducible(macrophage), and endothelial because these were the sites in which they were first identified. However, all three enzymes have beenfound in other sites, and the neuronal enzyme is also inducible. Each gene has been cloned, and its chromosomal location in humans hasbeen determined.4 iNOS is Ca 2+ -independent but binds calmodulin very tightly.Creatine phosphate is formed from ATP and creatine(Figure 49–16) at times when the muscle is relaxed anddemands for ATP are not so great. The enzyme catalyzingthe phosphorylation of creatine is creatine kinase(CK), a muscle-specific enzyme with clinical utility inthe detection of acute or chronic diseases of muscle.Table 49–9. Some physiologic functions andpathologic involvements of nitric oxide (NO).• Vasodilator, important in regulation of blood pressure• Involved in penile erection; sildenafil citrate (Viagra) affectsthis process by inhibiting a cGMP phosphodiesterase• Neurotransmitter in the brain and peripheral autonomicnervous system• Role in long-term potentiation• Role in neurotoxicity• Low level of NO involved in causation of pylorospasm in infantilehypertrophic pyloric stenosis• May have role in relaxation of skeletal muscle• May constitute part of a primitive immune system• Inhibits adhesion, activation, and aggregation of plateletsSKELETAL MUSCLE CONTAINS SLOW(RED) & FAST (WHITE) TWITCH FIBERSDifferent types of fibers have been detected in skeletalmuscle. One classification subdivides them into type I(slow twitch), type IIA (fast twitch-oxidative), and typeIIB (fast twitch-glycolytic). For the sake of simplicity,we shall consider only two types: type I (slow twitch, oxidative)and type II (fast twitch, glycolytic) (Table49–10). The type I fibers are red because they containmyoglobin and mitochondria; their metabolism is aerobic,and they maintain relatively sustained contractions.The type II fibers, lacking myoglobin and containingfew mitochondria, are white: they derive their energyfrom anaerobic glycolysis and exhibit relatively short durationsof contraction. The proportion of these twotypes of fibers varies among the muscles of the body, dependingon function (eg, whether or not a muscle is involvedin sustained contraction, such as maintainingposture). The proportion also varies with training; forexample, the number of type I fibers in certain leg musclesincreases in athletes training for marathons, whereasthe number of type II fibers increases in sprinters.A Sprinter Uses Creatine Phosphate& Anaerobic Glycolysis to Make ATP,Whereas a Marathon Runner UsesOxidative PhosphorylationIn view of the two types of fibers in skeletal muscle andof the various energy sources described above, it is ofinterest to compare their involvement in a sprint (eg,100 meters) and in the marathon (42.2 km; just over26 miles) (Table 49–11).The major sources of energy in the 100-m sprintare creatine phosphate (first 4–5 seconds) and thenanaerobic glycolysis, using muscle glycogen as thesource of glucose. The two main sites of metabolic controlare at glycogen phosphorylase and at PFK-1. Theformer is activated by Ca 2+ (released from the sarcoplasmicreticulum during contraction), epinephrine, and

MUSCLE & THE CYTOSKELETON / 575Creatine phosphateMuscle glycogenMUSCLEPHOSPHORYLASEGlucose 6-PCREATINEPHOSPHOKINASECreatineGLYCOLYSISADPOXIDATIVEPHOSPHORYLATIONATPMYOSINATPaseMusclecontractionADP + P iAMPADENYLYLKINASEADPFigure 49–16.The multiple sources of ATP in muscle.AMP. PFK-1 is activated by AMP, P i , and NH 3 . Attestingto the efficiency of these processes, the flux throughglycolysis can increase as much as 1000-fold during asprint.In contrast, in the marathon, aerobic metabolism isthe principal source of ATP. The major fuel sources areblood glucose and free fatty acids, largely derived fromthe breakdown of triacylglycerols in adipose tissue,stimulated by epinephrine. Hepatic glycogen is degradedto maintain the level of blood glucose. Muscleglycogen is also a fuel source, but it is degraded muchmore gradually than in a sprint. It has been calculatedthat the amounts of glucose in the blood, of glycogen inthe liver, of glycogen in muscle, and of triacylglycerol inadipose tissue are sufficient to supply muscle with energyduring a marathon for 4 minutes, 18 minutes, 70minutes, and approximately 4000 minutes, respectively.However, the rate of oxidation of fatty acids bymuscle is slower than that of glucose, so that oxidationof glucose and of fatty acids are both major sources ofenergy in the marathon.A number of procedures have been used by athletesto counteract muscle fatigue and inadequate strength.These include carbohydrate loading, soda (sodium bi-Table 49–11. Types of muscle fibers and majorfuel sources used by a sprinter and by a marathonrunner.Table 49–10. Characteristics of type I and type IIfibers of skeletal muscle.Type I Type IISlow Twitch Fast TwitchMyosin ATPase Low HighEnergy utilization Low HighMitochondria Many FewColor Red WhiteMyoglobin Yes NoContraction rate Slow FastDuration Prolonged ShortSprinter (100 m)Type II (glycolytic) fibers areused predominantly.Creatine phosphate is themajor energy source duringthe first 4–5 seconds.Glucose derived from muscleglycogen and metabolizedby anaerobic glycolysis isthe major fuel source.Muscle glycogen is rapidlydepleted.Marathon RunnerType I (oxidative) fibers areused predominantly.ATP is the major energysource throughout.Blood glucose and free fattyacids are the major fuelsources.Muscle glycogen is slowlydepleted.

576 / CHAPTER 49carbonate) loading, blood doping (administration ofred blood cells), and ingestion of creatine and androstenedione.Their rationales and efficacies will notbe discussed here.SKELETAL MUSCLE CONSTITUTESTHE MAJOR RESERVE OFPROTEIN IN THE BODYIn humans, skeletal muscle protein is the major nonfatsource of stored energy. This explains the very largelosses of muscle mass, particularly in adults, resultingfrom prolonged caloric undernutrition.The study of tissue protein breakdown in vivo is difficult,because amino acids released during intracellularbreakdown of proteins can be extensively reutilized forprotein synthesis within the cell, or the amino acidsmay be transported to other organs where they enteranabolic pathways. However, actin and myosin aremethylated by a posttranslational reaction, forming3-methylhistidine. During intracellular breakdown ofactin and myosin, 3-methylhistidine is released and excretedinto the urine. The urinary output of the methylatedamino acid provides a reliable index of the rate ofmyofibrillar protein breakdown in the musculature ofhuman subjects.Various features of muscle metabolism, most ofwhich are dealt with in other chapters of this text, aresummarized in Table 49–12.THE CYTOSKELETON PERFORMSMULTIPLE CELLULAR FUNCTIONSNonmuscle cells perform mechanical work, includingself-propulsion, morphogenesis, cleavage, endocytosis,exocytosis, intracellular transport, and changing cellshape. These cellular functions are carried out by an extensiveintracellular network of filamentous structuresconstituting the cytoskeleton. The cell cytoplasm isnot a sac of fluid, as once thought. Essentially all eukaryoticcells contain three types of filamentous structures:actin filaments (7–9.5 nm in diameter; alsoknown as microfilaments), microtubules (25 nm), andintermediate filaments (10–12 nm). Each type of filamentcan be distinguished biochemically and by theelectron microscope.Nonmuscle Cells Contain ActinThat Forms MicrofilamentsG-actin is present in most if not all cells of the body.With appropriate concentrations of magnesium andpotassium chloride, it spontaneously polymerizes toform double helical F-actin filaments like those seen inmuscle. There are at least two types of actin in nonmus-Table 49–12. Summary of major features ofthe biochemistry of skeletal muscle related toits metabolism. 1• Skeletal muscle functions under both aerobic (resting) andanaerobic (eg, sprinting) conditions, so both aerobic andanaerobic glycolysis operate, depending on conditions.• Skeletal muscle contains myoglobin as a reservoir of oxygen.• Skeletal muscle contains different types of fibers primarilysuited to anaerobic (fast twitch fibers) or aerobic (slowtwitch fibers) conditions.• Actin, myosin, tropomyosin, troponin complex (TpT, Tpl,and TpC), ATP, and Ca 2+ are key constituents in relation tocontraction.• The Ca 2+ ATPase, the Ca 2+ release channel, and calsequestrinare proteins involved in various aspects of Ca 2+ metabolismin muscle.• Insulin acts on skeletal muscle to increase uptake of glucose.• In the fed state, most glucose is used to synthesize glycogen,which acts as a store of glucose for use in exercise;“preloading” with glucose is used by some long-distanceathletes to build up stores of glycogen.• Epinephrine stimulates glycogenolysis in skeletal muscle,whereas glucagon does not because of absence of its receptors.• Skeletal muscle cannot contribute directly to blood glucosebecause it does not contain glucose-6-phosphatase.• Lactate produced by anaerobic metabolism in skeletal musclepasses to liver, which uses it to synthesize glucose,which can then return to muscle (the Cori cycle).• Skeletal muscle contains phosphocreatine, which acts as anenergy store for short-term (seconds) demands.• Free fatty acids in plasma are a major source of energy, particularlyunder marathon conditions and in prolonged starvation.• Skeletal muscle can utilize ketone bodies during starvation.• Skeletal muscle is the principal site of metabolism ofbranched-chain amino acids, which are used as an energysource.• Proteolysis of muscle during starvation supplies aminoacids for gluconeogenesis.• Major amino acids emanating from muscle are alanine (destinedmainly for gluconeogenesis in liver and forming partof the glucose-alanine cycle) and glutamine (destinedmainly for the gut and kidneys).1 This table brings together material from various chapters in thisbook.

MUSCLE & THE CYTOSKELETON / 577cle cells: β-actin and γ-actin. Both types can coexist inthe same cell and probably even copolymerize in thesame filament. In the cytoplasm, F-actin forms microfilamentsof 7–9.5 nm that frequently exist as bundlesof a tangled-appearing meshwork. These bundles areprominent just underlying the plasma membrane ofmany cells and are there referred to as stress fibers. Thestress fibers disappear as cell motility increases or uponmalignant transformation of cells by chemicals or oncogenicviruses.Although not organized as in muscle, actin filamentsin nonmuscle cells interact with myosin to cause cellularmovements.Microtubules Contain - & -TubulinsMicrotubules, an integral component of the cellular cytoskeleton,consist of cytoplasmic tubes 25 nm in diameterand often of extreme length. Microtubules are necessaryfor the formation and function of the mitoticspindle and thus are present in all eukaryotic cells.They are also involved in the intracellular movement ofendocytic and exocytic vesicles and form the majorstructural components of cilia and flagella. Microtubulesare a major component of axons and dendrites,in which they maintain structure and participate in theaxoplasmic flow of material along these neuronalprocesses.Microtubules are cylinders of 13 longitudinallyarranged protofilaments, each consisting of dimers ofα-tubulin and β-tubulin, closely related proteins of approximately50 kDa molecular mass. The tubulindimers assemble into protofilaments and subsequentlyinto sheets and then cylinders. A microtubule-organizingcenter, located around a pair of centrioles, nucleatesthe growth of new microtubules. A third species oftubulin, γ-tubulin, appears to play an important role inthis assembly. GTP is required for assembly. A varietyof proteins are associated with microtubules (microtubule-associatedproteins [MAPs], one of which is tau)and play important roles in microtubule assembly andstabilization. Microtubules are in a state of dynamicinstability, constantly assembling and disassembling.They exhibit polarity (plus and minus ends); this is importantin their growth from centrioles and in theirability to direct intracellular movement. For instance,in axonal transport, the protein kinesin, with amyosin-like ATPase activity, uses hydrolysis of ATP tomove vesicles down the axon toward the positive end ofthe microtubular formation. Flow of materials in theopposite direction, toward the negative end, is poweredby cytosolic dynein, another protein with ATPase activity.Similarly, axonemal dyneins power ciliary andflagellar movement. Another protein, dynamin, usesGTP and is involved in endocytosis. Kinesins, dyneins,dynamin, and myosins are referred to as molecularmotors.An absence of dynein in cilia and flagella results inimmotile cilia and flagella, leading to male sterility andchronic respiratory infection, a condition known asKartagener syndrome.Certain drugs bind to microtubules and thus interferewith their assembly or disassembly. These includecolchicine (used for treatment of acute gouty arthritis),vinblastine (a vinca alkaloid used for treating certaintypes of cancer), paclitaxel (Taxol) (effective againstovarian cancer), and griseofulvin (an antifungal agent).Intermediate Filaments Differ FromMicrofilaments & MicrotubulesAn intracellular fibrous system exists of filaments withan axial periodicity of 21 nm and a diameter of 8–10nm that is intermediate between that of microfilaments(6 nm) and microtubules (23 nm). Four classes of intermediatefilaments are found, as indicated in Table49–13. They are all elongated, fibrous molecules, witha central rod domain, an amino terminal head, and acarboxyl terminal tail. They form a structure like arope, and the mature filaments are composed oftetramers packed together in a helical manner. They areimportant structural components of cells, and most arerelatively stable components of the cytoskeleton, notundergoing rapid assembly and disassembly and notTable 49–13. Classes of intermediate filaments ofeukaryotic cells and their distributions.MolecularProteins Mass DistributionsKeratinsType I (acidic) 40–60 kDa Epithelial cells, hair,Type II (basic) 50–70 kDa nailsVimentin-likeVimentin 54 kDa Various mesenchymalcellsDesmin 53 kDa MuscleGlial fibrillary acid 50 kDa Glial cellsproteinPeripherin 66 kDa NeuronsNeurofilamentsLow (L), medium (M), 60–130 kDa Neuronsand high (H) 1LaminsA, B, and C 65–75 kDa Nuclear lamina1 Refers to their molecular masses.

578 / CHAPTER 49disappearing during mitosis, as do actin and many microtubularfilaments. An important exception to this isprovided by the lamins, which, subsequent to phosphorylation,disassemble at mitosis and reappear when it terminates.Keratins form a large family, with about 30 membersbeing distinguished. As indicated in Table 49–13,two major types of keratins are found; all individualkeratins are heterodimers made up of one member ofeach class.Vimentins are widely distributed in mesodermalcells, and desmin, glial fibrillary acidic protein, and peripherinare related to them. All members of the vimentin-likefamily can copolymerize with each other.Intermediate filaments are very prominent in nervecells; neurofilaments are classified as low, medium, andhigh on the basis of their molecular masses. Laminsform a meshwork in apposition to the inner nuclearmembrane. The distribution of intermediate filamentsin normal and abnormal (eg, cancer) cells can be studiedby the use of immunofluorescent techniques, usingantibodies of appropriate specificities. These antibodiesto specific intermediate filaments can also be of use topathologists in helping to decide the origin of certaindedifferentiated malignant tumors. These tumors maystill retain the type of intermediate filaments found intheir cell of origin.A number of skin diseases, mainly characterized byblistering, have been found to be due to mutations ingenes encoding various keratins. Three of these disordersare epidermolysis bullosa simplex, epidermolytichyperkeratosis, and epidermolytic palmoplantar keratoderma.The blistering probably reflects a diminished capacityof various layers of the skin to resist mechanicalstresses due to abnormalities in microfilament structure.SUMMARY• The myofibrils of skeletal muscle contain thick andthin filaments. The thick filaments contain myosin.The thin filaments contain actin, tropomyosin, andthe troponin complex (troponins T, I, and C).• The sliding filament cross-bridge model is the foundationof current thinking about muscle contraction.The basis of this model is that the interdigitating filamentsslide past one another during contraction andcross-bridges between myosin and actin generate andsustain the tension.• The hydrolysis of ATP is used to drive movement ofthe filaments. ATP binds to myosin heads and is hydrolyzedto ADP and P i by the ATPase activity of theactomyosin complex.• Ca 2+ plays a key role in the initiation of muscle contractionby binding to troponin C. In skeletal muscle,the sarcoplasmic reticulum regulates distributionof Ca 2+ to the sarcomeres, whereas inflow of Ca 2+ viaCa 2+ channels in the sarcolemma is of major importancein cardiac and smooth muscle.• Many cases of malignant hyperthermia in humansare due to mutations in the gene encoding the Ca 2+release channel.• A number of differences exist between skeletal andcardiac muscle; in particular, the latter contains a varietyof receptors on its surface.• Some cases of familial hypertrophic cardiomyopathyare due to missense mutations in the gene coding forβ-myosin heavy chain.• Smooth muscle, unlike skeletal and cardiac muscle,does not contain the troponin system; instead, phosphorylationof myosin light chains initiates contraction.• Nitric oxide is a regulator of vascular smooth muscle;blockage of its formation from arginine causes anacute elevation of blood pressure, indicating that regulationof blood pressure is one of its many functions.• Duchenne-type muscular dystrophy is due to mutationsin the gene, located on the X chromosome, encodingthe protein dystrophin.• Two major types of muscle fibers are found in humans:white (anaerobic) and red (aerobic). The formerare particularly used in sprints and the latter inprolonged aerobic exercise. During a sprint, muscleuses creatine phosphate and glycolysis as energysources; in the marathon, oxidation of fatty acids isof major importance during the later phases.• Nonmuscle cells perform various types of mechanicalwork carried out by the structures constituting thecytoskeleton. These structures include actin filaments(microfilaments), microtubules (composed primarilyof α- tubulin and β-tubulin), and intermediate filaments.The latter include keratins, vimentin-like proteins,neurofilaments, and lamins.REFERENCESAckerman MJ, Clapham DE: Ion channels—basic science and clinicaldisease. N Engl J Med 1997;336:1575.Andreoli TE: Ion transport disorders: introductory comments. AmJ Med 1998;104:85. (First of a series of articles on ion transportdisorders published between January and August, 1998.Topics covered were structure and function of ion channels,arrhythmias and antiarrhythmic drugs, Liddle syndrome,cholera, malignant hyperthermia, cystic fibrosis, the periodicparalyses and Bartter syndrome, and Gittelman syndrome.)Fuller GM, Shields D: Molecular Basis of Medical Cell Biology. Appleton& Lange, 1998.

MUSCLE & THE CYTOSKELETON / 579Geeves MA, Holmes KC: Structural mechanism of muscle contraction.Annu Rev Biochem 1999;68:728.Hille B: Ion Channels of Excitable Membranes. Sinauer, 2001.Howard J: Mechanics of Motor Proteins and the Cytoskeleton. Sinauer,2001.Lodish H et al (editors): Molecular Cell Biology, 4th ed. Freeman,2000. (Chapters 18 and 19 of this text contain comprehensivedescriptions of cell motility and cell shape.)Loke J, MacLennan DH: Malignant hyperthermia and central coredisease: disorders of Ca 2+ release channels. Am J Med1998;104:470.Mayer B, Hemmens B: Biosynthesis and action of nitric oxide inmammalian cells. Trends Biochem Sci 1998;22:477.Scriver CR et al (editors): The Metabolic and Molecular Bases of InheritedDisease, 8th ed. McGraw-Hill, 2001. (This comprehensivefour-volume text contains coverage of malignant hyperthermia[Chapter 9], channelopathies [Chapter 204],hypertrophic cardiomyopathy [Chapter 213], the musculardystrophies [Chapter 216], and disorders of intermediate filamentsand their associated proteins [Chapter 221].)

Plasma Proteins & Immunoglobulins 50Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEThe fundamental role of blood in the maintenance ofhomeostasis and the ease with which blood can be obtainedhave meant that the study of its constituents hasbeen of central importance in the development of biochemistryand clinical biochemistry. The basic propertiesof a number of plasma proteins, including theimmunoglobulins (antibodies), are described in thischapter. Changes in the amounts of various plasma proteinsand immunoglobulins occur in many diseases andcan be monitored by electrophoresis or other suitableprocedures. As indicated in an earlier chapter, alterationsof the activities of certain enzymes found in plasma areof diagnostic use in a number of pathologic conditions.THE BLOOD HAS MANY FUNCTIONSThe functions of blood—except for specific cellularones such as oxygen transport and cell-mediated immunologicdefense—are carried out by plasma and itsconstituents (Table 50–1).Plasma consists of water, electrolytes, metabolites,nutrients, proteins, and hormones. The water and electrolytecomposition of plasma is practically the same asthat of all extracellular fluids. Laboratory determinationsof levels of Na + , K + , Ca 2+ , Cl − , HCO − 3 , PaCO 2 ,and of blood pH are important in the management ofmany patients.PLASMA CONTAINS A COMPLEXMIXTURE OF PROTEINSThe concentration of total protein in human plasma isapproximately 7.0–7.5 g/dL and comprises the majorpart of the solids of the plasma. The proteins of theplasma are actually a complex mixture that includes notonly simple proteins but also conjugated proteins suchas glycoproteins and various types of lipoproteins.Thousands of antibodies are present in human plasma,though the amount of any one antibody is usually quitelow under normal circumstances. The relative dimensionsand molecular masses of some of the most importantplasma proteins are shown in Figure 50–1.The separation of individual proteins from a complexmixture is frequently accomplished by the use ofsolvents or electrolytes (or both) to remove differentprotein fractions in accordance with their solubilitycharacteristics. This is the basis of the so-called saltingoutmethods, which find some usage in the determinationof protein fractions in the clinical laboratory.Thus, one can separate the proteins of the plasma intothree major groups—fibrinogen, albumin, and globulins—bythe use of varying concentrations of sodiumor ammonium sulfate.The most common method of analyzing plasmaproteins is by electrophoresis. There are many types ofelectrophoresis, each using a different supportingmedium. In clinical laboratories, cellulose acetate iswidely used as a supporting medium. Its use permitsresolution, after staining, of plasma proteins into fivebands, designated albumin, α 1 , α 2 , β, and γ fractions,respectively (Figure 50–2). The stained strip of celluloseacetate (or other supporting medium) is called anelectrophoretogram. The amounts of these five bandscan be conveniently quantified by use of densitometricscanning machines. Characteristic changes in theamounts of one or more of these five bands are foundin many diseases.The Concentration of Protein in Plasma IsImportant in Determining the Distributionof Fluid Between Blood & TissuesIn arterioles, the hydrostatic pressure is about 37 mmHg, with an interstitial (tissue) pressure of 1 mm Hgopposing it. The osmotic pressure (oncotic pressure)exerted by the plasma proteins is approximately 25 mmHg. Thus, a net outward force of about 11 mm Hgdrives fluid out into the interstitial spaces. In venules,the hydrostatic pressure is about 17 mm Hg, with theoncotic and interstitial pressures as described above;thus, a net force of about 9 mm Hg attracts water backinto the circulation. The above pressures are often referredto as the Starling forces. If the concentration ofplasma proteins is markedly diminished (eg, due to severeprotein malnutrition), fluid is not attracted backinto the intravascular compartment and accumulates inthe extravascular tissue spaces, a condition known asedema. Edema has many causes; protein deficiency isone of them.580

PLASMA PROTEINS & IMMUNOGLOBULINS / 581Table 50–1. Major functions of blood.(1) Respiration—transport of oxygen from the lungs to thetissues and of CO 2 from the tissues to the lungs(2) Nutrition—transport of absorbed food materials(3) Excretion—transport of metabolic waste to the kidneys,lungs, skin, and intestines for removal(4) Maintenance of the normal acid-base balance in thebody(5) Regulation of water balance through the effects ofblood on the exchange of water between the circulatingfluid and the tissue fluid(6) Regulation of body temperature by the distribution ofbody heat(7) Defense against infection by the white blood cells andcirculating antibodies(8) Transport of hormones and regulation of metabolism(9) Transport of metabolites(10) CoagulationPlasma Proteins Have BeenStudied ExtensivelyBecause of the relative ease with which they can be obtained,plasma proteins have been studied extensively inboth humans and animals. Considerable information isavailable about the biosynthesis, turnover, structure,and functions of the major plasma proteins. Alterationsof their amounts and of their metabolism in many diseasestates have also been investigated. In recent years,many of the genes for plasma proteins have been clonedand their structures determined.The preparation of antibodies specific for the individualplasma proteins has greatly facilitated theirstudy, allowing the precipitation and isolation of pureproteins from the complex mixture present in tissues orplasma. In addition, the use of isotopes has made possiblethe determination of their pathways of biosynthesisand of their turnover rates in plasma.The following generalizations have emerged fromstudies of plasma proteins.Albumin69,000β 1 -Globulin90,000α 1 -Lipoprotein200,00010 nmScaleNa + CI –GlucoseHemoglobin64,500γ-Globulin156,000β 1 -Lipoprotein1,300,000A. MOST PLASMA PROTEINS ARESYNTHESIZED IN THE LIVERThis has been established by experiments at the wholeanimallevel (eg, hepatectomy) and by use of the isolatedperfused liver preparation, of liver slices, of liverhomogenates, and of in vitro translation systems usingpreparations of mRNA extracted from liver. However,the γ-globulins are synthesized in plasma cells and certainplasma proteins are synthesized in other sites, suchas endothelial cells.B. PLASMA PROTEINS ARE GENERALLY SYNTHESIZEDON MEMBRANE-BOUND POLYRIBOSOMESThey then traverse the major secretory route in the cell(rough endoplasmic membrane → smooth endoplasmicmembrane → Golgi apparatus → secretory vesicles) priorto entering the plasma. Thus, most plasma proteins aresynthesized as preproteins and initially contain aminoterminal signal peptides (Chapter 46). They are usuallysubjected to various posttranslational modifications (proteolysis,glycosylation, phosphorylation, etc) as they travelthrough the cell. Transit times through the hepatocytefrom the site of synthesis to the plasma vary from 30 minutesto several hours or more for individual proteins.Fibrinogen340,000Figure 50–1. Relative dimensions and approximatemolecular masses of protein molecules in the blood(Oncley).C. MOST PLASMA PROTEINS ARE GLYCOPROTEINSAccordingly, they generally contain either N- or O-linked oligosaccharide chains, or both (Chapter 47). Albuminis the major exception; it does not contain sugarresidues. The oligosaccharide chains have various functions(Table 47–2). Removal of terminal sialic acid

582 / CHAPTER 50AC+ –Albumin α 1 α 2 β γBD+ –Albumin α 1 α 2 β γFigure 50–2. Technique of cellulose acetate zone electrophoresis. A: A small amount of serum or otherfluid is applied to a cellulose acetate strip. B: Electrophoresis of sample in electrolyte buffer is performed.C: Separated protein bands are visualized in characteristic positions after being stained. D: Densitometerscanning from cellulose acetate strip converts bands to characteristic peaks of albumin, α 1 -globulin, α 2 -globulin,β-globulin, and γ-globulin. (Reproduced, with permission, from Parslow TG et al [editors]: Medical Immunology,10th ed. McGraw-Hill, 2001.)residues from certain plasma proteins (eg, ceruloplasmin)by exposure to neuraminidase can markedlyshorten their half-lives in plasma (Chapter 47).D. MANY PLASMA PROTEINS EXHIBIT POLYMORPHISMA polymorphism is a mendelian or monogenic trait thatexists in the population in at least two phenotypes, neitherof which is rare (ie, neither of which occurs withfrequency of less than 1–2%). The ABO blood groupsubstances (Chapter 52) are the best-known examplesof human polymorphisms. Human plasma proteinsthat exhibit polymorphism include α 1 -antitrypsin, haptoglobin,transferrin, ceruloplasmin, and immunoglobulins.The polymorphic forms of these proteins can bedistinguished by different procedures (eg, various typesof electrophoresis or isoelectric focusing), in which eachform may show a characteristic migration. Analyses ofthese human polymorphisms have proved to be of genetic,anthropologic, and clinical interest.E. EACH PLASMA PROTEIN HAS A CHARACTERISTICHALF-LIFE IN THE CIRCULATIONThe half-life of a plasma protein can be determined bylabeling the isolated pure protein with 131 I under mild,nondenaturing conditions. This isotope unites covalentlywith tyrosine residues in the protein. The labeled proteinis freed of unbound 131 I and its specific activity (disintegrationsper minute per milligram of protein) determined.A known amount of the radioactive protein isthen injected into a normal adult subject, and samples ofblood are taken at various time intervals for determinationsof radioactivity. The values for radioactivity areplotted against time, and the half-life of the protein (thetime for the radioactivity to decline from its peak valueto one-half of its peak value) can be calculated from theresulting graph, discounting the times for the injectedprotein to equilibrate (mix) in the blood and in the extravascularspaces. The half-lives obtained for albuminand haptoglobin in normal healthy adults are approximately20 and 5 days, respectively. In certain diseases,the half-life of a protein may be markedly altered. For instance,in some gastrointestinal diseases such as regionalileitis (Crohn disease), considerable amounts of plasmaproteins, including albumin, may be lost into the bowelthrough the inflamed intestinal mucosa. Patients withthis condition have a protein-losing gastroenteropathy,and the half-life of injected iodinated albumin in thesesubjects may be reduced to as little as 1 day.

PLASMA PROTEINS & IMMUNOGLOBULINS / 583F. THE LEVELS OF CERTAIN PROTEINS IN PLASMAINCREASE DURING ACUTE INFLAMMATORY STATES ORSECONDARY TO CERTAIN TYPES OF TISSUE DAMAGEThese proteins are called “acute phase proteins” (or reactants)and include C-reactive protein (CRP, so-namedbecause it reacts with the C polysaccharide of pneumococci),α 1 -antitrypsin, haptoglobin, α 1 -acid glycoprotein,and fibrinogen. The elevations of the levels of theseproteins vary from as little as 50% to as much as 1000-fold in the case of CRP. Their levels are also usually elevatedduring chronic inflammatory states and in patientswith cancer. These proteins are believed to play arole in the body’s response to inflammation. For example,C-reactive protein can stimulate the classic complementpathway, and α 1 -antitrypsin can neutralize certainproteases released during the acute inflammatory state.CRP is used as a marker of tissue injury, infection, andinflammation, and there is considerable interest in itsuse as a predictor of certain types of cardiovascular conditionssecondary to atherosclerosis. Interleukin-1(IL-1), a polypeptide released from mononuclear phagocyticcells, is the principal—but not the sole—stimulatorof the synthesis of the majority of acute phase reactantsby hepatocytes. Additional molecules such as IL-6are involved, and they as well as IL-1 appear to work atthe level of gene transcription.Table 50–2 summarizes the functions of many ofthe plasma proteins. The remainder of the material inthis chapter presents basic information regarding selectedplasma proteins: albumin, haptoglobin, transferrin,ceruloplasmin, α 1 -antitrypsin, α 2 -macroglobulin,the immunoglobulins, and the complement system.The lipoproteins are discussed in Chapter 25.Albumin Is the Major Proteinin Human PlasmaAlbumin (69 kDa) is the major protein of humanplasma (3.4–4.7 g/dL) and makes up approximately60% of the total plasma protein. About 40% of albuminis present in the plasma, and the other 60% is presentin the extracellular space. The liver produces about12 g of albumin per day, representing about 25% oftotal hepatic protein synthesis and half its secreted protein.Albumin is initially synthesized as a preproprotein.Its signal peptide is removed as it passes into thecisternae of the rough endoplasmic reticulum, and ahexapeptide at the resulting amino terminal is subsequentlycleaved off farther along the secretory pathway.The synthesis of albumin is depressed in a variety ofdiseases, particularly those of the liver. The plasma ofpatients with liver disease often shows a decrease in theratio of albumin to globulins (decreased albuminglobulinratio). The synthesis of albumin decreases rela-Table 50–2. Some functions of plasma proteins.FunctionPlasma ProteinsAntiproteases Antichymotrypsinα 1 -Antitrypsin (α 1 -antiproteinase)α 2 -MacroglobulinAntithrombinBlood clotting Various coagulation factors, fibrinogenEnzymes Function in blood, eg, coagulationfactors, cholinesteraseLeakage from cells or tissues, eg, aminotransferasesHormones Erythropoietin 1Immune defense Immunoglobulins, complement proteins,β 2 -microglobulinInvolvement in Acute phase response proteins (eg,inflammatory C-reactive protein, α 1 -acid glycoresponsesprotein [orosomucoid])Oncofetal α 1 -Fetoprotein (AFP)Transport or Albumin (various ligands, including bilibindingrubin, free fatty acids, ions [Ca 2+ ],proteinsmetals [eg, Cu 2+ , Zn 2+ ], metheme,steroids, other hormones, and a varietyof drugsCeruloplasmin (contains Cu 2+ ; albuminprobably more important in physiologictransport of Cu 2+ )Corticosteroid-binding globulin (transcortin)(binds cortisol)Haptoglobin (binds extracorpuscularhemoglobin)Lipoproteins (chylomicrons, VLDL, LDL,HDL)Hemopexin (binds heme)Retinol-binding protein (binds retinol)Sex hormone-binding globulin (bindstestosterone, estradiol)Thyroid-binding globulin (binds T 4 , T 3 )Transferrin (transport iron)Transthyretin (formerly prealbumin;binds T 4 and forms a complex withretinol-binding protein)1 Various other protein hormones circulate in the blood but arenot usually designated as plasma proteins. Similarly, ferritin is alsofound in plasma in small amounts, but it too is not usually characterizedas a plasma protein.

584 / CHAPTER 50tively early in conditions of protein malnutrition, suchas kwashiorkor.Mature human albumin consists of one polypeptidechain of 585 amino acids and contains 17 disulfidebonds. By the use of proteases, albumin can be subdividedinto three domains, which have different functions.Albumin has an ellipsoidal shape, which meansthat it does not increase the viscosity of the plasma asmuch as an elongated molecule such as fibrinogen does.Because of its relatively low molecular mass (about 69kDa) and high concentration, albumin is thought to beresponsible for 75–80% of the osmotic pressure ofhuman plasma. Electrophoretic studies have shown thatthe plasma of certain humans lacks albumin. Thesesubjects are said to exhibit analbuminemia. One causeof this condition is a mutation that affects splicing.Subjects with analbuminemia show only moderateedema, despite the fact that albumin is the major determinantof plasma osmotic pressure. It is thought thatthe amounts of the other plasma proteins increase andcompensate for the lack of albumin.Another important function of albumin is its abilityto bind various ligands. These include free fatty acids(FFA), calcium, certain steroid hormones, bilirubin,and some of the plasma tryptophan. In addition, albuminappears to play an important role in transport ofcopper in the human body (see below). A variety ofdrugs, including sulfonamides, penicillin G, dicumarol,and aspirin, are bound to albumin; this finding has importantpharmacologic implications.Preparations of human albumin have been widelyused in the treatment of hemorrhagic shock and ofburns. However, this treatment is under review becausesome recent studies have suggested that administration ofalbumin in these conditions may increase mortality rates.Haptoglobin Binds ExtracorpuscularHemoglobin, Preventing Free HemoglobinFrom Entering the KidneyHaptoglobin (Hp) is a plasma glycoprotein that bindsextracorpuscular hemoglobin (Hb) in a tight noncovalentcomplex (Hb-Hp). The amount of haptoglobin inhuman plasma ranges from 40 mg to 180 mg of hemoglobin-bindingcapacity per deciliter. Approximately10% of the hemoglobin that is degraded each day is releasedinto the circulation and is thus extracorpuscular.The other 90% is present in old, damaged red bloodcells, which are degraded by cells of the histiocytic system.The molecular mass of hemoglobin is approximately65 kDa, whereas the molecular mass of the simplestpolymorphic form of haptoglobin (Hp 1-1) foundin humans is approximately 90 kDa. Thus, the Hb-Hpcomplex has a molecular mass of approximately 155kDa. Free hemoglobin passes through the glomerulusof the kidney, enters the tubules, and tends to precipitatetherein (as can happen after a massive incompatibleblood transfusion, when the capacity of haptoglobin tobind hemoglobin is grossly exceeded) (Figure 50–3).However, the Hb-Hp complex is too large to passthrough the glomerulus. The function of Hp thus appearsto be to prevent loss of free hemoglobin into thekidney. This conserves the valuable iron present in hemoglobin,which would otherwise be lost to the body.Human haptoglobin exists in three polymorphicforms, known as Hp 1-1, Hp 2-1, and Hp 2-2. Hp 1-1migrates in starch gel electrophoresis as a single band,whereas Hp 2-1 and Hp 2-2 exhibit much more complexband patterns. Two genes, designated Hp 1 and Hp 2 ,direct these three phenotypes, with Hp 2-1 being theheterozygous phenotype. It has been suggested that thehaptoglobin polymorphism may be associated withthe prevalence of many inflammatory diseases.The levels of haptoglobin in human plasma vary andare of some diagnostic use. Low levels of haptoglobin arefound in patients with hemolytic anemias. This is explainedby the fact that whereas the half-life of haptoglobinis approximately 5 days, the half-life of the Hb-Hpcomplex is about 90 minutes, the complex being rapidlyremoved from plasma by hepatocytes. Thus, when haptoglobinis bound to hemoglobin, it is cleared from theplasma about 80 times faster than normally. Accordingly,the level of haptoglobin falls rapidly in situationswhere hemoglobin is constantly being released from redblood cells, such as occurs in hemolytic anemias. Haptoglobinis an acute phase protein, and its plasma level iselevated in a variety of inflammatory states.Certain other plasma proteins bind heme but nothemoglobin. Hemopexin is a β 1 -globulin that bindsfree heme. Albumin will bind some metheme (ferricheme) to form methemalbumin, which then transfersthe metheme to hemopexin.Absorption of Iron From the SmallIntestine Is Tightly RegulatedTransferrin (Tf) is a plasma protein that plays a centralrole in transporting iron around the body to sites whereA. Hb → Kidney → Excreted in urine or precipitates in tubules;(MW 65,000)iron is lost to bodyB. Hb + Hp → Hb : Hp complex → Kidney(MW 65,000) (MW 90,000)(MW 155,000)Catabolized by liver cells;iron is conserved and reusedFigure 50–3. Different fates of free hemoglobin andof the hemoglobin-haptoglobin complex.

PLASMA PROTEINS & IMMUNOGLOBULINS / 585Table 50–3. Distribution of iron in a 70-kgadult male. 1Transferrin3–4 mgHemoglobin in red blood cells2500 mgIn myoglobin and various enzymes300 mgIn stores (ferritin and hemosiderin)1000 mgAbsorption1 mg/dLosses1 mg/d1 In an adult female of similar weight, the amount in stores wouldgenerally be less (100–400 mg) and the losses would be greater(1.5–2 mg/d).it is needed. Before we discuss it further, certain aspectsof iron metabolism will be reviewed.Iron is important in the human body because of itsoccurrence in many hemoproteins such as hemoglobin,myoglobin, and the cytochromes. It is ingested in thediet either as heme or nonheme iron (Figure 50–4); asshown, these different forms involve separate pathways.Absorption of iron in the proximal duodenum is tightlyregulated, as there is no physiologic pathway for its excretionfrom the body. Under normal circumstances,the body guards its content of iron zealously, so that ahealthy adult male loses only about 1 mg/d, which is replacedby absorption. Adult females are more prone tostates of iron deficiency because some may lose excessiveblood during menstruation. The amounts of iron in variousbody compartments are shown in Table 50–3.Enterocytes in the proximal duodenum are responsiblefor absorption of iron. Incoming iron in the Fe 3+state is reduced to Fe 2+ by a ferrireductase present onthe surface of enterocytes. Vitamin C in food also favorsreduction of ferric iron to ferrous iron. The transfer ofiron from the apical surfaces of enterocytes into their interiorsis performed by a proton-coupled divalent metaltransporter (DMT1). This protein is not specific foriron, as it can transport a wide variety of divalent cations.Once inside an enterocyte, iron can either be storedas ferritin or transferred across the basolateral membraneinto the plasma, where it is carried by transferrin(see below). Passage across the basolateral membraneappears to be carried out by another protein, possiblyiron regulatory protein 1 (IREG1). This protein mayinteract with the copper-containing protein hephaestin,a protein similar to ceruloplasmin (see below). Hephaestinis thought to have a ferroxidase activity, which isimportant in the release of iron from cells. Thus, Fe 2+ isconverted back to Fe 3+ , the form in which it is transportedin the plasma by transferrin.Overall regulation of iron absorption is complexand not well understood mechanistically. It occurs atIntestinallumenBrushborderEnterocyteBloodHemeFe 3 +reductaseFe 2 +HTDMT1HemeHOFe 2 +Fe 2 +Fe 2 +Fe 3 + -ferritinHPFPFe 2 +Fe 3 +ShedFe 3 + −TFFigure 50–4. Absorption of iron. Fe 3+ is converted to Fe 2+ by ferric reductase,and Fe 2+ is transported into the enterocyte by the apical membrane iron transporterDMT1. Heme is transported into the enterocyte by a separate hemetransporter (HT), and heme oxidase (HO) releases Fe 2+ from the heme. Some ofthe intracellular Fe 2+ is converted to Fe 3+ and bound by ferritin. The remainderbinds to the basolateral Fe 2+ transporter (FP) and is transported into the bloodstream,aided by hephaestin (HP). In plasma, Fe 3+ is bound to the iron transportprotein transferrin (TF). (Reproduced, with permission, from Ganong WF: Review ofMedical Physiology, 21st ed. McGraw-Hill, 2003.)

586 / CHAPTER 50the level of the enterocyte, where further absorption ofiron is blocked if a sufficient amount has been taken up(so-called dietary regulation exerted by “mucosalblock”). It also appears to be responsive to the overallrequirement of erythropoiesis for iron (erythropoieticregulation). Absorption is excessive in hereditary hemochromatosis(see below).Transferrin Shuttles Iron to SitesWhere It Is NeededTransferrin (Tf ) is a β 1 -globulin with a molecular massof approximately 76 kDa. It is a glycoprotein and issynthesized in the liver. About 20 polymorphic formsof transferrin have been found. It plays a central role inthe body’s metabolism of iron because it transports iron(2 mol of Fe 3+ per mole of Tf) in the circulation to siteswhere iron is required, eg, from the gut to the bonemarrow and other organs. Approximately 200 billionred blood cells (about 20 mL) are catabolized per day,releasing about 25 mg of iron into the body—most ofwhich will be transported by transferrin.There are receptors (TfRs) on the surfaces of manycells for transferrin. It binds to these receptors and is internalizedby receptor-mediated endocytosis (comparethe fate of LDL; Chapter 25). The acid pH inside thelysosome causes the iron to dissociate from the protein.The dissociated iron leaves the endosome via DMT1 toenter the cytoplasm. Unlike the protein component ofLDL, apoTf is not degraded within the lysosome. Instead,it remains associated with its receptor, returns tothe plasma membrane, dissociates from its receptor,reenters the plasma, picks up more iron, and again deliversthe iron to needy cells.Abnormalities of the glycosylation of transferrinoccur in the congenital disorders of glycosylation(Chapter 47) and in chronic alcohol abuse. Their detectionby, for example, isoelectric focusing is used to helpdiagnose these conditions.Iron Deficiency AnemiaIs Extremely PrevalentAttention to iron metabolism is particularly importantin women for the reason mentioned above. Additionally,in pregnancy, allowances must be made forthe growing fetus. Older people with poor dietaryhabits (“tea and toasters”) may develop iron deficiency.Iron deficiency anemia due to inadequate intake, inadequateutilization, or excessive loss of iron is one of themost prevalent conditions seen in medical practice.The concentration of transferrin in plasma is approximately300 mg/dL. This amount of transferrin canbind 300 µg of iron per deciliter, so that this representsthe total iron-binding capacity of plasma. However,the protein is normally only one-third saturated withiron. In iron deficiency anemia, the protein is even lesssaturated with iron, whereas in conditions of storage ofexcess iron in the body (eg, hemochromatosis) the saturationwith iron is much greater than one-third.Ferritin Stores Iron in CellsFerritin is another protein that is important in the metabolismof iron. Under normal conditions, it storesiron that can be called upon for use as conditions require.In conditions of excess iron (eg, hemochromatosis),body stores of iron are greatly increased and muchmore ferritin is present in the tissues, such as the liverand spleen. Ferritin contains approximately 23% iron,and apoferritin (the protein moiety free of iron) has amolecular mass of approximately 440 kDa. Ferritin iscomposed of 24 subunits of 18.5 kDa, which surroundin a micellar form some 3000–4500 ferric atoms. Normally,there is a little ferritin in human plasma. However,in patients with excess iron, the amount of ferritinin plasma is markedly elevated. The amount of ferritinin plasma can be conveniently measured by a sensitiveand specific radioimmunoassay and serves as an indexof body iron stores.Synthesis of the transferrin receptor (TfR) and thatof ferritin are reciprocally linked to cellular iron content.Specific untranslated sequences of the mRNAs forboth proteins (named iron response elements) interactwith a cytosolic protein sensitive to variations in levelsof cellular iron (iron-responsive element-binding protein).When iron levels are high, cells use stored ferritinmRNA to synthesize ferritin, and the TfR mRNA is degraded.In contrast, when iron levels are low, the TfRmRNA is stabilized and increased synthesis of receptorsoccurs, while ferritin mRNA is apparently stored in aninactive form. This is an important example of controlof expression of proteins at the translational level.Hemosiderin is a somewhat ill-defined molecule; itappears to be a partly degraded form of ferritin but stillcontaining iron. It can be detected by histologic stains(eg, Prussian blue) for iron, and its presence is determinedhistologically when excessive storage of ironoccurs.Hereditary Hemochromatosis Is Dueto Mutations in the HFE GeneHereditary (primary) hemochromatosis is a very prevalentautosomal recessive disorder in certain parts of theworld (eg, Scotland, Ireland, and North America). It ischaracterized by excessive storage of iron in tissues, leadingto tissue damage. Total body iron ranges between2.5 g and 3.5 g in normal adults; in primary hemochromatosisit usually exceeds 15 g. The accumulated iron

PLASMA PROTEINS & IMMUNOGLOBULINS / 587damages organs and tissues such as the liver, pancreaticislets, and heart, perhaps in part due to effects on freeradical production (Chapter 52). Melanin and variousamounts of iron accumulate in the skin, accounting forthe slate-gray color often seen. The precise cause ofmelanin accumulation is not clear. The frequent coexistenceof diabetes mellitus (due to islet damage) and theskin pigmentation led to use of the term bronze diabetesfor this condition. In 1995, Feder and colleaguesisolated a gene, now known as HFE, located on chromosome6 close to the major histocompatibility complexgenes. The encoded protein (HFE) was found to be relatedto MHC class 1 antigens. Initially, two differentmissense mutations were found in HFE in individualswith hereditary hemochromatosis. The more frequentmutation was one that changed cysteinyl residue 282 toa tyrosyl residue (CY282Y), disrupting the structure ofthe protein. The other mutation changed histidyl residue63 to an aspartyl residue (H63D). Some patientswith hereditary hemochromatosis have neither mutation,perhaps due to other mutations in HFE or becauseone or more other genes may be involved in its causation.Genetic screening for this condition has been evaluatedbut is not presently recommended. However, testingfor HFE mutations in individuals with elevatedserum iron concentrations may be useful.HFE has been shown to be located in cells in thecrypts of the small intestine, the site of iron absorption.There is evidence that it associates with β 2 -microglobulin,an association that may be necessary for its stability,intracellular processing, and cell surface expression. Thecomplex interacts with the transferrin receptor (TfR);how this leads to excessive storage of iron when HFE isaltered by mutation is under close study. The mousehomolog of HFE has been knocked out, resulting in apotentially useful animal model of hemochromatosis.A scheme of the likely main events in the causation ofhereditary hemochromatosis is set forth in Figure 50–5.Secondary hemochromatosis can occur after repeatedtransfusions (eg, for treatment of sickle cell anemia),excessive oral intake of iron (eg, by African Bantupeoples who consume alcoholic beverages fermented incontainers made of iron), or a number of other conditions.Table 50–4 summarizes laboratory tests useful in theassessment of patients with abnormalities of iron metabolism.Ceruloplasmin Binds Copper, & Low Levelsof This Plasma Protein Are AssociatedWith Wilson DiseaseCeruloplasmin (about 160 kDa) is an α 2 -globulin. Ithas a blue color because of its high copper content andMutations in HFE, located on chromosome 6p21.3,leading to abnormalities in the structureof its protein productLoss of regulation of absorption of ironin the small intestineAccumulation of iron in various tissues, but particularlyliver, pancreatic islets, skin, and heart muscleIron directly or indirectly causes damage to theabove tissues, resulting in hepatic cirrhosis, diabetesmellitus, skin pigmentation, and cardiac problemsFigure 50–5. Tentative scheme of the main eventsin causation of primary hemochromatosis (MIM235200). The two principal mutations are CY282Y andH63D (see text). Mutations in genes other than HFE arealso involved in some cases.carries 90% of the copper present in plasma. Each moleculeof ceruloplasmin binds six atoms of copper verytightly, so that the copper is not readily exchangeable.Albumin carries the other 10% of the plasma copperbut binds the metal less tightly than does ceruloplasmin.Albumin thus donates its copper to tissues morereadily than ceruloplasmin and appears to be more importantthan ceruloplasmin in copper transport in thehuman body. Ceruloplasmin exhibits a copper-dependentoxidase activity, but its physiologic significancehas not been clarified. The amount of ceruloplasmin inplasma is decreased in liver disease. In particular, lowlevels of ceruloplasmin are found in Wilson disease(hepatolenticular degeneration), a disease due to abnormalmetabolism of copper. In order to clarify the descriptionof Wilson disease, we shall first consider themetabolism of copper in the human body and thenMenkes disease, another condition involving abnormalcopper metabolism.Table 50–4. Laboratory tests for assessingpatients with disorders of iron metabolism.• Red blood cell count and estimation of hemoglobin• Determinations of plasma iron, total iron-binding capacity(TIBC), and % transferrin saturation• Determination of ferritin in plasma by radioimmunoassay• Prussian blue stain of tissue sections• Determination of amount of iron (µg/g) in a tissue biopsy

588 / CHAPTER 50Copper Is a Cofactor for Certain EnzymesCopper is an essential trace element. It is required inthe diet because it is the metal cofactor for a variety ofenzymes (see Table 50–5). Copper accepts and donateselectrons and is involved in reactions involving dismutation,hydroxylation, and oxygenation. However, excesscopper can cause problems because it can oxidizeproteins and lipids, bind to nucleic acids, and enhancethe production of free radicals. It is thus important tohave mechanisms that will maintain the amount ofcopper in the body within normal limits. The body ofthe normal adult contains about 100 mg of copper, locatedmostly in bone, liver, kidney, and muscle. Thedaily intake of copper is about 2–4 mg, with about50% being absorbed in the stomach and upper smallintestine and the remainder excreted in the feces. Copperis carried to the liver bound to albumin, taken upby liver cells, and part of it is excreted in the bile. Copperalso leaves the liver attached to ceruloplasmin,which is synthesized in that organ.The Tissue Levels of Copper & of CertainOther Metals Are Regulated inPart by MetallothioneinsMetallothioneins are a group of small proteins (about6.5 kDa), found in the cytosol of cells, particularly ofliver, kidney, and intestine. They have a high content ofcysteine and can bind copper, zinc, cadmium, and mercury.The SH groups of cysteine are involved in bindingthe metals. Acute intake (eg, by injection) of copper andof certain other metals increases the amount (induction)of these proteins in tissues, as does administration ofcertain hormones or cytokines. These proteins mayfunction to store the above metals in a nontoxic formand are involved in their overall metabolism in thebody. Sequestration of copper also diminishes theamount of this metal available to generate free radicals.Menkes Disease Is Due to Mutationsin the Gene Encoding a Copper-Binding P-Type ATPaseMenkes disease (“kinky” or “steely” hair disease) is adisorder of copper metabolism. It is X-linked, affectsTable 50–5. Some important enzymes thatcontain copper.• Amine oxidase• Copper-dependent superoxide dismutase• Cytochrome oxidase• Tyrosinaseonly male infants, involves the nervous system, connectivetissue, and vasculature, and is usually fatal in infancy.In 1993, it was reported that the basis of Menkesdisease was mutations in the gene for a copper-bindingP-type ATPase. Interestingly, the enzyme showed structuralsimilarity to certain metal-binding proteins in microorganisms.This ATPase is thought to be responsiblefor directing the efflux of copper from cells. When alteredby mutation, copper is not mobilized normallyfrom the intestine, in which it accumulates, as it does ina variety of other cells and tissues, from which it cannotexit. Despite the accumulation of copper, the activitiesof many copper-dependent enzymes are decreased, perhapsbecause of a defect of its incorporation into theapoenzymes. Normal liver expresses very little of theATPase, which explains the absence of hepatic involvementin Menkes disease. This work led to the suggestionthat liver might contain a different copper-bindingATPase, which could be involved in the causation ofWilson disease. As described below, this turned out tobe the case.Wilson Disease Is Also Due to Mutationsin a Gene Encoding a Copper-BindingP-Type ATPaseWilson disease is a genetic disease in which copper failsto be excreted in the bile and accumulates in liver,brain, kidney, and red blood cells. It can be regarded asan inability to maintain a near-zero copper balance, resultingin copper toxicosis. The increase of copper inliver cells appears to inhibit the coupling of copper toapoceruloplasmin and leads to low levels of ceruloplasminin plasma. As the amount of copper accumulates,patients may develop a hemolytic anemia, chronic liverdisease (cirrhosis, hepatitis), and a neurologic syndromeowing to accumulation of copper in the basal gangliaand other centers. A frequent clinical finding is theKayser-Fleischer ring. This is a green or golden pigmentring around the cornea due to deposition of copperin Descemet’s membrane. The major laboratorytests of copper metabolism are listed in Table 50–6. IfWilson disease is suspected, a liver biopsy should beperformed; a value for liver copper of over 250 µg pergram dry weight along with a plasma level of ceruloplasminof under 20 mg/dL is diagnostic.The cause of Wilson disease was also revealed in1993, when it was reported that a variety of mutationsin a gene encoding a copper-binding P-type ATPasewere responsible. The gene is estimated to encode aprotein of 1411 amino acids, which is highly homologousto the product of the gene affected in Menkes disease.In a manner not yet fully explained, a nonfunctionalATPase causes defective excretion of copper intothe bile, a reduction of incorporation of copper into

PLASMA PROTEINS & IMMUNOGLOBULINS / 589Table 50–6. Major laboratory tests used in theinvestigation of diseases of copper metabolism. 1Serum copperCeruloplasminUrinary copperLiver copperTestNormal AdultRange10–22 µmol/L200–600 mg/L< 1 µmol/24 h20–50 µg/g dry weight1 Based on Gaw A et al: Clinical Biochemistry. Churchill Livingstone,1995.apoceruloplasmin, and the accumulation of copper inliver and subsequently in other organs such as brain.Treatment for Wilson disease consists of a diet lowin copper along with lifelong administration of penicillamine,which chelates copper, is excreted in the urine,and thus depletes the body of the excess of this mineral.Another condition involving ceruloplasmin is aceruloplasminemia.In this genetic disorder, levels of ceruloplasminare very low and consequently its ferroxidase activityis markedly deficient. This leads to failure of releaseof iron from cells, and iron accumulates in certain braincells, hepatocytes, and pancreatic islet cells. Affected individualsshow severe neurologic signs and have diabetesmellitus. Use of a chelating agent or administration ofplasma or ceruloplasmin concentrate may be beneficial.Deficiency of 1 -Antiproteinase( 1 -Antitrypsin) Is AssociatedWith Emphysema & One Typeof Liver Diseaseα 1 -Antiproteinase (about 52 kDa) was formerly calledα 1 -antitrypsin, and this name is retained here. It is asingle-chain protein of 394 amino acids, contains threeoligosaccharide chains, and is the major component(> 90%) of the α 1 fraction of human plasma. It is synthesizedby hepatocytes and macrophages and is theprincipal serine protease inhibitor (serpin, or Pi) ofhuman plasma. It inhibits trypsin, elastase, and certainother proteases by forming complexes with them. Atleast 75 polymorphic forms occur, many of which canbe separated by electrophoresis. The major genotype isMM, and its phenotypic product is PiM. There are twoareas of clinical interest concerning α 1 -antitrypsin. A deficiencyof this protein has a role in certain cases (approximately5%) of emphysema. This occurs mainly insubjects with the ZZ genotype, who synthesize PiZ, andalso in PiSZ heterozygotes, both of whom secrete considerablyless protein than PiMM individuals. Considerablyless of this protein is secreted as compared withPiM. When the amount of α 1 -antitrypsin is deficientand polymorphonuclear white blood cells increase in thelung (eg, during pneumonia), the affected individuallacks a countercheck to proteolytic damage of the lungby proteases such as elastase (Figure 50–6). It is of considerableinterest that a particular methionine (residue358) of α 1 -antitrypsin is involved in its binding to proteases.Smoking oxidizes this methionine to methioninesulfoxide and thus inactivates it. As a result, affectedmolecules of α 1 -antitrypsin no longer neutralizeproteases. This is particularly devastating in patients(eg, PiZZ phenotype) who already have low levels ofα 1 -antitrypsin. The further diminution in α 1 -antitrypsinbrought about by smoking results in increased proteolyticdestruction of lung tissue, accelerating the developmentof emphysema. Intravenous administration of α 1 -antitrypsin(augmentation therapy) has been used as an adjunctin the treatment of patients with emphysema dueto α 1 -antitrypsin deficiency. Attempts are being made,using the techniques of protein engineering, to replacemethionine 358 by another residue that would not besubject to oxidation. The resulting “mutant” α 1 -antitrypsinwould thus afford protection against proteasesfor a much longer period of time than would nativeα 1 -antitrypsin. Attempts are also being made to developgene therapy for this condition. One approach is to usea modified adenovirus (a pathogen of the respiratorytract) into which the gene for α 1 -antitrypsin has beeninserted. The virus would then be introduced into therespiratory tract (eg, by an aerosol). The hope is thatpulmonary epithelial cells would express the gene andsecrete α 1 -antitrypsin locally. Experiments in animalshave indicated the feasibility of this approach.Deficiency of α 1 -antitrypsin is also implicated inone type of liver disease (α 1 -antitrypsin deficiency liverA. Active elastase + α 1 –AT → Inactive elastase: α 1 –AT complex → No proteolysis of lung → No tissue damageB. Active elastase + or no α 1 –AT → Active elastase → Proteolysis of lung → Tissue damage→Figure 50–6. Scheme illustrating (A) normal inactivation of elastase by α 1 -antitrypsin and(B) situation in which the amount of α 1 -antitrypsin is substantially reduced, resulting in proteolysisby elastase and leading to tissue damage.

590 / CHAPTER 50disease). In this condition, molecules of the ZZ phenotypeaccumulate and aggregate in the cisternae of theendoplasmic reticulum of hepatocytes. Aggregation isdue to formation of polymers of mutant α 1 -antitrypsin,the polymers forming via a strong interaction between aspecific loop in one molecule and a prominent β-pleated sheet in another (loop-sheet polymerization).By mechanisms that are not understood, hepatitis resultswith consequent cirrhosis (accumulation of massiveamounts of collagen, resulting in fibrosis). It is possiblethat administration of a synthetic peptideresembling the loop sequence could inhibit loop-sheetpolymerization. Diseases such as α 1 -antitrypsin deficiency,in which cellular pathology is primarily causedby the presence of aggregates of aberrant forms of individualproteins, have been named conformational diseases.Most appear to be due to the formation by conformationallyunstable proteins of β sheets, which inturn leads to formation of aggregates. Other membersof this group of conditions include Alzheimer disease,Parkinson disease, and Huntington disease.At present, severe α 1 -antitrypsin deficiency liver diseasecan be successfully treated by liver transplantation.In the future, introduction of the gene for normal α 1 -antitrypsin into hepatocytes may become possible, butthis would not stop production of the PiZ protein. Figure50–7 is a scheme of the causation of this disease. 2 -Macroglobulin Neutralizes ManyProteases & Targets CertainCytokines to Tissuesα 2 -Macroglobulin is a large plasma glycoprotein (720kDa) made up of four identical subunits of 180 kDa. ItGAG to AAG mutation in exon 5 of gene for α 1 -ATon chromosome 14Results in Glu 342 to Lys 342 substitution in α 1 -AT,causing formation of PiZZPiZZ accumulates in cisternaeof endoplasmic reticulum and aggregatesvia loop-sheet polymerizationLeads to hepatitis (mechanism unknown)and cirrhosis in ~10% of ZZ homozygotesFigure 50–7. Scheme of causation of α 1 -antitrypsindeficiencyliver disease. The mutation shown causesformation of PiZZ (MIM 107400). (α 1 -AT, α 1 -antitrypsin.)comprises 8–10% of the total plasma protein in humans.Approximately 10% of the zinc in plasma istransported by α 2 -macroglobulin, the remainder beingtransported by albumin. The protein is synthesized by avariety of cell types, including monocytes, hepatocytes,and astrocytes. It is the major member of a group ofplasma proteins that include complement proteins C3and C4. These proteins contain a unique internal cyclicthiol ester bond (formed between a cysteine and a glutamineresidue) and for this reason have been designatedas the thiol ester plasma protein family.α 2 -Macroglobulin binds many proteinases and isthus an important panproteinase inhibitor. The α 2 -macroglobulin-proteinase complexes are rapidly clearedfrom the plasma by a receptor located on many celltypes. In addition, α 2 -macroglobulin binds many cytokines(platelet-derived growth factor, transforminggrowth factor-β, etc) and appears to be involved in targetingthem toward particular tissues or cells. Oncetaken up by cells, the cytokines can dissociate from α 2 -macroglobulin and subsequently exert a variety of effectson cell growth and function. The binding of proteinasesand cytokines by α 2 -macroglobulin involvesdifferent mechanisms that will not be considered here.Amyloidosis Occurs by the Depositionof Fragments of Various PlasmaProteins in TissuesAmyloidosis is the accumulation of various insolublefibrillar proteins between the cells of tissues to an extentthat affects function. The fibrils generally representproteolytic fragments of various plasma proteins andpossess a β-pleated sheet structure. The term “amyloidosis”is a misnomer, as it was originally thought thatthe fibrils were starch-like in nature. Among the mostcommon precursor proteins are immunoglobulin lightchains (see below), amyloid-associated protein derivedfrom serum amyloid-associated protein (a plasma glycoprotein),and transthyretin (Table 50–2). The precursorproteins in plasma are generally either increased inamount (eg, immunoglobulin light chains in multiplemyeloma or β 2 -microglobulin in patients being maintainedon chronic dialysis) or mutant forms (eg, oftransthyretin in familial amyloidotic neuropathies).The precise factors that determine the deposition ofproteolytic fragments in tissues await elucidation.Other proteins have been found in amyloid fibrils, suchas calcitonin and amyloid β protein (not derived from aplasma protein) in Alzheimer disease; a total of about15 different proteins have been found. All fibrils have aP component associated with them, which is derivedfrom serum amyloid P component, a plasma proteinclosely related to C-reactive protein. Tissue sectionscontaining amyloid fibrils interact with Congo red stain

PLASMA PROTEINS & IMMUNOGLOBULINS / 591and display striking green birefringence when viewedby polarizing microscopy. Deposition of amyloid occursin patients with a variety of disorders; treatment ofthe underlying disorder should be provided if possible.PLASMA IMMUNOGLOBULINS PLAYA MAJOR ROLE IN THE BODY’SDEFENSE MECHANISMSThe immune system of the body consists of two majorcomponents: B lymphocytes and T lymphocytes. TheB lymphocytes are mainly derived from bone marrowcells in higher animals and from the bursa of Fabriciusin birds. The T lymphocytes are of thymic origin. TheB cells are responsible for the synthesis of circulating,humoral antibodies, also known as immunoglobulins.The T cells are involved in a variety of important cellmediatedimmunologic processes such as graft rejection,hypersensitivity reactions, and defense against malignantcells and many viruses. This section considersonly the plasma immunoglobulins, which are synthesizedmainly in plasma cells. These are specialized cellsof B cell lineage that synthesize and secrete immunoglobulinsinto the plasma in response to exposure to avariety of antigens.All Immunoglobulins Contain a Minimumof Two Light & Two Heavy ChainsImmunoglobulins contain a minimum of two identicallight (L) chains (23 kDa) and two identical heavy(H) chains (53–75 kDa), held together as a tetramer(L 2 H 2 ) by disulfide bonds The structure of IgG isshown in Figure 50–8; it is Y-shaped, with binding ofantigen occurring at both tips of the Y. Each chain canbe divided conceptually into specific domains, or regions,that have structural and functional significance.The half of the light (L) chain toward the carboxyl terminalis referred to as the constant region (C L ), whilethe amino terminal half is the variable region of thelight chain (V L ). Approximately one-quarter of theheavy (H) chain at the amino terminals is referred to asits variable region (V H ), and the other three-quarters ofthe heavy chain are referred to as the constant regions(C H 1, C H 2, C H 3) of that H chain. The portion of theimmunoglobulin molecule that binds the specific antigenis formed by the amino terminal portions (variableregions) of both the H and L chains—ie, the V H andV L domains. The domains of the protein chains consistof two sheets of antiparallel distinct stretches of aminoacids that bind antigen.As depicted in Figure 50–8, digestion of an immunoglobulinby the enzyme papain produces twoantigen-binding fragments (Fab) and one crystallizablefragment (Fc), which is responsible for functions of immunoglobulinsother than direct binding of antigens.Because there are two Fab regions, IgG molecules bindtwo molecules of antigen and are termed divalent. Thesite on the antigen to which an antibody binds istermed an antigenic determinant, or epitope. Thearea in which papain cleaves the immunoglobulin molecule—ie,the region between the C H 1 and C H 2 domains—isreferred to as the “hinge region.” The hingeregion confers flexibility and allows both Fab arms tomove independently, thus helping them to bind toantigenic sites that may be variable distances apart (eg,on bacterial surfaces). Fc and hinge regions differ in thedifferent classes of antibodies, but the overall model ofantibody structure for each class is similar to thatshown in Figure 50–8 for IgG.All Light Chains Are Either Kappaor Lambda in TypeThere are two general types of light chains, kappa (κ)and lambda (λ), which can be distinguished on thebasis of structural differences in their C L regions. Agiven immunoglobulin molecule always contains two κor two λ light chains—never a mixture of κ and λ. Inhumans, the κ chains are more frequent than λ chainsin immunoglobulin molecules.The Five Types of Heavy Chain DetermineImmunoglobulin ClassFive classes of H chain have been found in humans(Table 50–7), distinguished by differences in their C Hregions. They are designated γ, α, µδ, and ε. The µand ε chains each have four C H domains rather thanthe usual three. The type of H chain determines theclass of immunoglobulin and thus its effector function.There are thus five immunoglobulin classes: IgG, IgA,IgM, IgD, and IgE. The biologic functions of thesefive classes are summarized in Table 50–8.No Two Variable Regions Are IdenticalThe variable regions of immunoglobulin moleculesconsist of the V L and V H domains and are quite heterogeneous.In fact, no two variable regions from differenthumans have been found to have identical amino acidsequences. However, amino acid analyses have shownthat the variable regions are comprised of relatively invariableregions and other hypervariable regions (Figure50–9). L chains have three hypervariable regions (inV L ) and H chains have four (in V H ). These hypervariableregions comprise the antigen-binding site (locatedat the tips of the Y shown in Figure 50–8) and dictatethe amazing specificity of antibodies. For this reason,hypervariable regions are also termed complementar-

592 / CHAPTER 50+ H3 N+ H3 NV LFabS S S SS S S SL chainH chainC LHinge regionV HC H 1FCC H 2C H 3S SS SS SS SS SH chainH chainS S S SCOO —COO —+ H3 NFabH chainL chainS S S SS S S SS SPepsinPapainCleavage sites+ H3 NFigure 50–8. Structure of IgG. The molecule consists of two light (L) chains andtwo heavy (H) chains. Each light chain consists of a variable (V L ) and a constant (C L )region. Each heavy chain consists of a variable region (V H ) and a constant regionthat is divided into three domains (C H 1, C H 2, and C H 3). The C H 2 domain containsthe complement-binding site and the C H 3 domain contains a site that attaches toreceptors on neutrophils and macrophages. The antigen-binding site is formed bythe hypervariable regions of both the light and heavy chains, which are located inthe variable regions of these chains (see Figure 50–9). The light and heavy chainsare linked by disulfide bonds, and the heavy chains are also linked to each otherby disulfide bonds. (Reproduced, with permission, from Parslow TG et al [editors]:Medical Immunology, 10th ed. McGraw-Hill, 2001.)ity-determining regions (CDRs). About five to tenamino acids in each hypervariable region (CDR) contributeto the antigen-binding site. CDRs are locatedon small loops of the variable domains, the surroundingpolypeptide regions between the hypervariable regionsbeing termed framework regions. CDRs from bothV H and V L domains, brought together by folding of thepolypeptide chains in which they are contained, form asingle hypervariable surface comprising the antigenbindingsite. Various combinations of H and L chainCDRs can give rise to many antibodies of differentspecificities, a feature that contributes to the tremendousdiversity of antibody molecules and is termedcombinatorial diversity. Large antigens interact withall of the CDRs of an antibody, whereas small ligandsmay interact with only one or a few CDRs that form apocket or groove in the antibody molecule. The essenceof antigen-antibody interactions is mutual complementaritybetween the surfaces of CDRs and epitopes. Theinteractions between antibodies and antigens involvenoncovalent forces and bonds (electrostatic and van derWaals forces and hydrogen and hydrophobic bonds).

Table 50–7. Properties of human immunoglobulins. 1PLASMA PROTEINS & IMMUNOGLOBULINS / 593Property IgG IgA IgM IgD IgEPercentage of total immunoglo- 75 15 9 0.2 0.004bulin in serum (approximate)Serum concentration 1000 200 120 3 0.05(mg/dL) (approximate)Sedimentation coefficient 7S 7S or 11S 2 19S 7S 8SMolecular weight 150 170 or 900 180 190(× 1000) 400 2Structure Monomer Monomer or dimer Monomer or dimer Monomer MonomerH-chain symbol γ α µ δ εComplement fixation + − + − −Transplacental passage + − − ? −Mediation of allergic responses − − − − +Found in secretions − + − − −Opsonization + − − 3 − −Antigen receptor on B cell − − + ? −Polymeric form contains J chain − + + − −1 Reproduced, with permission, from Levinson W, Jawetz E: Medical Microbiology and Immunology, 7th ed. McGraw-Hill,2002.2 The 11S form is found in secretions (eg, saliva, milk, tears) and fluids of the respiratory, intestinal, and genital tracts.3 IgM opsonizes indirectly by activating complement. This produces C3b, which is an opsonin.The Constant Regions DetermineClass-Specific Effector FunctionsThe constant regions of the immunoglobulin molecules,particularly the C H 2 and C H 3 (and C H 4 of IgM andIgE), which constitute the Fc fragment, are responsiblefor the class-specific effector functions of the differentimmunoglobulin molecules (Table 50–7, bottom part),eg, complement fixation or transplacental passage.Some immunoglobulins such as immune IgG existonly in the basic tetrameric structure, while others suchas IgA and IgM can exist as higher order polymers oftwo, three (IgA), or five (IgM) tetrameric units (Figure50–10).The L chains and H chains are synthesized as separatemolecules and are subsequently assembled withinthe B cell or plasma cell into mature immunoglobulinmolecules, all of which are glycoproteins.Both Light & Heavy Chains Are Productsof Multiple GenesEach immunoglobulin light chain is the product of atleast three separate structural genes: a variable region(V L ) gene, a joining region (J) gene (bearing no relationshipto the J chain of IgA or IgM), and a constantregion (C L ) gene. Each heavy chain is the product of atleast four different genes: a variable region (V H ) gene, adiversity region (D) gene, a joining region (J) gene, anda constant region (C H ) gene. Thus, the “one gene, oneprotein” concept is not valid. The molecular mechanismsresponsible for the generation of the single immunoglobulinchains from multiple structural genes arediscussed in Chapters 36 and 39.Antibody Diversity Dependson Gene RearrangementsEach person is capable of generating antibodies directedagainst perhaps 1 million different antigens. The generationof such immense antibody diversity dependsupon a number of factors including the existence ofmultiple gene segments (V, C, J, and D segments),their recombinations (see Chapters 36 and 39), thecombinations of different L and H chains, a high frequencyof somatic mutations in immunoglobulin genes,and junctional diversity. The latter reflects the addi-

594 / CHAPTER 50Table 50–8. Major functions ofimmunoglobulins. 1ImmunoglobulinIgGIgAIgMIgDIgEMajor FunctionsMain antibody in the secondary response.Opsonizes bacteria, makingthem easier to phagocytose. Fixes complement,which enhances bacterialkilling. Neutralizes bacterial toxins andviruses. Crosses the placenta.Secretory IgA prevents attachment ofbacteria and viruses to mucous membranes.Does not fix complement.Produced in the primary response to anantigen. Fixes complement. Does notcross the placenta. Antigen receptor onthe surface of B cells.Uncertain. Found on the surface ofmany B cells as well as in serum.Mediates immediate hypersensitivity bycausing release of mediators from mastcells and basophils upon exposure toantigen (allergen). Defends againstworm infections by causing release ofenzymes from eosinophils. Does not fixcomplement. Main host defense againsthelminthic infections.1 Reproduced, with permission, from Levinson W, Jawetz E: MedicalMicrobiology and Immunology, 7th ed. McGraw-Hill, 2002.IntrachaindisulfidebondsVLC LVHC HInterchaindisulfidebondsLight chainhypervariableregionsHeavy chainhypervariableregionsFigure 50–9. Schematic model of an IgG moleculeshowing approximate positions of the hypervariable regionsin heavy and light chains. The antigen-bindingsite is formed by these hypervariable regions. The hypervariableregions are also called complementaritydeterminingregions (CDRs). (Modified and reproduced,with permission, from Parslow TG et al [editors]: MedicalImmunology, 10th ed. McGraw-Hill, 2001.)tion or deletion of a random number of nucleotideswhen certain gene segments are joined together, and introducesan additional degree of diversity. Thus, theabove factors ensure that a vast number of antibodiescan be synthesized from several hundred gene segments.Class (Isotype) Switching OccursDuring Immune ResponsesIn most humoral immune responses, antibodies withidentical specificity but of different classes are generatedin a specific chronologic order in response to the immunogen(immunizing antigen). For instance, antibodiesof the IgM class normally precede molecules of theIgG class. The switch from one class to another is designated“class or isotype switching,” and its molecularbasis has been investigated extensively. A single type ofimmunoglobulin light chain can combine with an antigen-specificµ chain to generate a specific IgM molecule.Subsequently, the same antigen-specific light chaincombines with a γ chain with an identical V H region togenerate an IgG molecule with antigen specificity identicalto that of the original IgM molecule. The samelight chain can also combine with an α heavy chain,again containing the identical V H region, to form an IgAmolecule with identical antigen specificity. These threeclasses (IgM, IgG, and IgA) of immunoglobulin moleculesagainst the same antigen have identical variable domainsof both their light (V L ) chains and heavy (V H )chains and are said to share an idiotype. (Idiotypes arethe antigenic determinants formed by the specific aminoacids in the hypervariable regions.) The different classesof these three immunoglobulins (called isotypes) arethus determined by their different C H regions, which arecombined with the same antigen-specific V H regions.Both Over- & Underproductionof Immunoglobulins May Resultin Disease StatesDisorders of immunoglobulins include increased productionof specific classes of immunoglobulins or even

PLASMA PROTEINS & IMMUNOGLOBULINS / 595HLMonomerDimerLHA. Serum lgAHLJ chainHLLHB. Secretory lgA(dimer)J chainSecretorycomponentHLFigure 50–10. Schematic representationof serum IgA, secretoryIgA, and IgM. Both IgA and IgM havea J chain, but only secretory IgA hasa secretory component. Polypeptidechains are represented by thick lines;disulfide bonds linking differentpolypeptide chains are representedby thin lines. (Reproduced, with permission,from Parslow TG et al [editors]:Medical Immunology, 10th ed.McGraw-Hill, 2001.)C. lgM(pentamer)HLHLJ chainspecific immunoglobulin molecules, the latter by clonaltumors of plasma cells called myelomas. Multiplemyeloma is a neoplastic condition; electrophoresis ofserum or urine will usually reveal a large increase of oneparticular immunoglobulin or one particular light chain(the latter termed a Bence Jones protein). Decreasedproduction may be restricted to a single class of immunoglobulinmolecules (eg, IgA or IgG) or may involveunderproduction of all classes of immunoglobulins(IgA, IgD, IgE, IgG, and IgM). A severe reductionin synthesis of an immunoglobulin class due to a geneticabnormality can result in a serious immunodeficiencydisease—eg, agammaglobulinemia, in whichproduction of IgG is markedly affected—because ofimpairment of the body’s defense against microorganisms.Hybridomas Provide Long-Term Sourcesof Highly Useful Monoclonal AntibodiesWhen an antigen is injected into an animal, the resultingantibodies are polyclonal, being synthesized by amixture of B cells. Polyclonal antibodies are directedagainst a number of different sites (epitopes or determinants)on the antigen and thus are not monospecific.However, by means of a method developed by Kohlerand Milstein, large amounts of a single monoclonal antibodyspecific for one epitope can be obtained.The method involves cell fusion, and the resultingpermanent cell line is called a hybridoma. Typically, Bcells are obtained from the spleen of a mouse (or othersuitable animal) previously injected with an antigen ormixture of antigens (eg, foreign cells). The B cells are

596 / CHAPTER 50Myeloma cellHybridoma cellHybridoma cellB cellFused in presence of PEGGrown in presence of HAT mediumHybridoma multiplies; myeloma and B cells dieFigure 50–11. Scheme of production of a hybridomacell. The myeloma cells are immortalized, donot produce antibody, and are HGPRT – (rendering thesalvage pathway of purine synthesis [Chapter 34] inactive).The B cells are not immortalized, each produces aspecific antibody, and they are HGPRT + . Polyethyleneglycol (PEG) stimulates cell fusion. The resulting hybridomacells are immortalized (via the parentalmyeloma cells), produce antibody, and are HGPRT +(both latter properties gained from the parental B cells).The B cells will die in the medium because they are notimmortalized. In the presence of HAT, the myelomacells will also die, since the aminopterin in HAT suppressespurine synthesis by the de novo pathway by inhibitingreutilization of tetrahydrofolate (Chapter 34).However, the hybridoma cells will survive, grow (becausethey are HGPRT + ), and—if cloned—producemonoclonal antibody. (HAT, hypoxanthine,aminopterin, and thymidine; HGPRT, hypoxanthineguaninephosphoribosyl transferase.)mixed with mouse myeloma cells and exposed to polyethyleneglycol, which causes cell fusion. A summary ofthe principles involved in generating hybridoma cells isgiven in Figure 50–11. Under the conditions used, onlythe hybridoma cells multiply in cell culture. This involvesplating the hybrid cells into hypoxanthineaminopterin-thymidine(HAT)-containing medium ata concentration such that each dish contains approximatelyone cell. Thus, a clone of hybridoma cells multipliesin each dish. The culture medium is harvestedand screened for antibodies that react with the originalantigen or antigens. If the immunogen is a mixture ofmany antigens (eg, a cell membrane preparation), anindividual culture dish will contain a clone of hybridomacells synthesizing a monoclonal antibody toone specific antigenic determinant of the mixture. Byharvesting the media from many culture dishes, a batteryof monoclonal antibodies can be obtained, many ofwhich are specific for individual components of the immunogenicmixture. The hybridoma cells can be frozenand stored and subsequently thawed when more of theantibody is required; this ensures its long-term supply.The hybridoma cells can also be grown in the abdomenof mice, providing relatively large supplies of antibodies.Because of their specificity, monoclonal antibodieshave become useful reagents in many areas of biologyand medicine. For example, they can be used to measurethe amounts of many individual proteins (eg,plasma proteins), to determine the nature of infectiousagents (eg, types of bacteria), and to subclassify bothnormal (eg, lymphocytes) and tumor cells (eg, leukemiccells). In addition, they are being used to direct therapeuticagents to tumor cells and also to accelerate removalof drugs from the circulation when they reachtoxic levels (eg, digoxin).The Complement System Comprises About20 Plasma Proteins & Is Involved in CellLysis, Inflammation, & Other ProcessesPlasma contains approximately 20 proteins that aremembers of the complement system. This system wasdiscovered when it was observed that addition of freshserum containing antibodies directed to a bacteriumcaused its lysis. Unlike antibodies, the factor was labilewhen heated at 56 °C. Subsequent work has resolvedthe proteins of the system and how they function; mosthave been cloned and sequenced. The major proteincomponents are designated C1–9, with C9 associatedwith the C5–8 complex (together constituting themembrane attack complex) being involved in generatinga lipid-soluble pore in the cell membrane thatcauses osmotic lysis.The details of this system are relatively complex, anda textbook of immunology should be consulted. Thebasic concept is that the normally inactive proteins ofthe system, when triggered by a stimulus, become activatedby proteolysis and interact in a specific sequencewith one or more of the other proteins of the system.This results in cell lysis and generation of peptide orpolypeptide fragments that are involved in various aspectsof inflammation (chemotaxis, phagocytosis, etc).The system has other functions, such as clearance ofantigen-antibody complexes from the circulation. Activationof the complement system is triggered by one oftwo routes, called the classic and the alternative pathways.The first involves interaction of C1 with antigenantibodycomplexes, and the second (not involving antibody)involves direct interaction of bacterial cellsurfaces or polysaccharides with a component designatedC3b.

PLASMA PROTEINS & IMMUNOGLOBULINS / 597The complement system resembles blood coagulation(Chapter 51) in that it involves both conversion ofinactive precursors to active products by proteases and acascade with amplification.SUMMARY• Plasma contains many proteins with a variety offunctions. Most are synthesized in the liver and areglycosylated.• Albumin, which is not glycosylated, is the major proteinand is the principal determinant of intravascularosmotic pressure; it also binds many ligands, such asdrugs and bilirubin.• Haptoglobin binds extracorpuscular hemoglobin,prevents its loss into the kidney and urine, and hencepreserves its iron for reutilization.• Transferrin binds iron, transporting it to sites whereit is required. Ferritin provides an intracellular storeof iron. Iron deficiency anemia is a very prevalentdisorder. Hereditary hemochromatosis has beenshown to be due to mutations in HFE, a gene encodingthe protein HFE, which appears to play an importantrole in absorption of iron.• Ceruloplasmin contains substantial amounts of copper,but albumin appears to be more important withregard to its transport. Both Wilson disease andMenkes disease, which reflect abnormalities of coppermetabolism, have been found to be due to mutationsin genes encoding copper-binding P-type ATPases.• α 1 -Antitrypsin is the major serine protease inhibitorof plasma, in particular inhibiting the elastase of neutrophils.Genetic deficiency of this protein is a causeof emphysema and can also lead to liver disease.• α 2 -Macroglobulin is a major plasma protein thatneutralizes many proteases and targets certain cytokinesto specific organs.• Immunoglobulins play a key role in the defensemechanisms of the body, as do proteins of the complementsystem. Some of the principal features ofthese proteins are described.REFERENCESAndrews NC: Disorders of iron metabolism. N Engl J Med1999;341:1986.Carrell RW, Lomas DA: Alpha 1 -antitrypsin deficiency—a modelfor conformational diseases. N Engl J Med 2002;346:45.Gabay C, Kushner I: Acute-phase proteins and other systemic responsesto inflammation. New Engl J Med 1999;340:448.Harris ED: Cellular copper transport and metabolism. Annu RevNutr 2000;20:291.Langlois MR, Delanghe JR: Biological and clinical significance ofhaptoglobin polymorphism in humans. Clin Chem 1996;2:1589.Levinson W, Jawetz E: Medical Microbiology and Immunology, 6thed. Appleton & Lange, 2000.Parslow TG et al (editors): Medical Immunology, 10th ed. Appleton& Lange, 2001.Pepys MB, Berger A: The renaissance of C reactive protein. BMJ2001;322:4.Waheed A et al: Regulation of transferrin-mediated iron uptake byHFE, the protein defective in hereditary hemochromatosis.Proc Natl Acad U S A 2002;99:3117.

Hemostasis & Thrombosis 51Margaret L. Rand, PhD, & Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEBasic aspects of the proteins of the blood coagulationsystem and of fibrinolysis are described in this chapter.Some fundamental aspects of platelet biology are alsopresented. Hemorrhagic and thrombotic states cancause serious medical emergencies, and thromboses inthe coronary and cerebral arteries are major causes ofdeath in many parts of the world. Rational managementof these conditions requires a clear understandingof the bases of blood clotting and fibrinolysis.HEMOSTASIS & THROMBOSIS HAVETHREE COMMON PHASESHemostasis is the cessation of bleeding from a cut orsevered vessel, whereas thrombosis occurs when the endotheliumlining blood vessels is damaged or removed(eg, upon rupture of an atherosclerotic plaque). Theseprocesses encompass blood clotting (coagulation) andinvolve blood vessels, platelet aggregation, and plasmaproteins that cause formation or dissolution of plateletaggregates.In hemostasis, there is initial vasoconstriction of theinjured vessel, causing diminished blood flow distal tothe injury. Then hemostasis and thrombosis share threephases:(1) Formation of a loose and temporary platelet aggregateat the site of injury. Platelets bind to collagenat the site of vessel wall injury and are activatedby thrombin (the mechanism of activationof platelets is described below), formed in the coagulationcascade at the same site, or by ADP releasedfrom other activated platelets. Upon activation,platelets change shape and, in thepresence of fibrinogen, aggregate to form the hemostaticplug (in hemostasis) or thrombus (inthrombosis).(2) Formation of a fibrin mesh that binds to theplatelet aggregate, forming a more stable hemostaticplug or thrombus.(3) Partial or complete dissolution of the hemostaticplug or thrombus by plasmin.598There Are Three Types of ThrombiThree types of thrombi or clots are distinguished. Allthree contain fibrin in various proportions.(1) The white thrombus is composed of platelets andfibrin and is relatively poor in erythrocytes. Itforms at the site of an injury or abnormal vesselwall, particularly in areas where blood flow israpid (arteries).(2) The red thrombus consists primarily of red cellsand fibrin. It morphologically resembles the clotformed in a test tube and may form in vivo inareas of retarded blood flow or stasis (eg, veins)with or without vascular injury, or it may form ata site of injury or in an abnormal vessel in conjunctionwith an initiating platelet plug.(3) A third type is a disseminated fibrin deposit invery small blood vessels or capillaries.We shall first describe the coagulation pathway leadingto the formation of fibrin. Then we shall briefly describesome aspects of the involvement of platelets andblood vessel walls in the overall process. This separationof clotting factors and platelets is artificial, since bothplay intimate and often mutually interdependent rolesin hemostasis and thrombosis, but it facilitates descriptionof the overall processes involved.Both Intrinsic & Extrinsic Pathways Resultin the Formation of FibrinTwo pathways lead to fibrin clot formation: the intrinsicand the extrinsic pathways. These pathways are notindependent, as previously thought. However, this artificialdistinction is retained in the following text to facilitatetheir description.Initiation of the fibrin clot in response to tissue injuryis carried out by the extrinsic pathway. How theintrinsic pathway is activated in vivo is unclear, but itinvolves a negatively charged surface. The intrinsic andextrinsic pathways converge in a final common pathwayinvolving the activation of prothrombin to thrombinand the thrombin-catalyzed cleavage of fibrinogento form the fibrin clot. The intrinsic, extrinsic, andfinal common pathways are complex and involve manydifferent proteins (Figure 51–1 and Table 51–1). In

HEMOSTASIS & THROMBOSIS / 599Intrinsic pathwayPKHKXIIXIIaHKXICa 2+ Ca 2+XIaExtrinsic pathwayVIICa 2+IXIXaVIIa/Tissue factorVIIIVIIIaPLXXaXFigure 51–1. The pathways ofblood coagulation. The intrinsicand extrinsic pathways are indicated.The events depicted belowfactor Xa are designated the finalcommon pathway, culminating inthe formation of cross-linked fibrin.New observations (dotted arrow)include the finding that complexesof tissue factor and factor VIIa activatenot only factor X (in the classicextrinsic pathway) but also factorIX in the intrinsic pathway. In addition,thrombin and factor Xafeedback-activate at the two sitesindicated (dashed arrows). (PK,prekallikrein; HK, HMW kininogen;PL, phospholipids.) (Reproduced,with permission, from Roberts HR,Lozier JN: New perspectives on thecoagulation cascade. Hosp Pract [OffEd] 1992 Jan;27:97.)Classic coagulationcascadePositive feedback(hypothesized)Extrinsic-to-intrinsicactivationVVaProthrombinFibrinogenCa 2+PLFibrin monomerFibrin polymerCross-linkedfibrin polymerThrombinXIIIaXIII

600 / CHAPTER 51Table 51–1. Numerical system for nomenclatureof blood clotting factors. The numbers indicatethe order in which the factors have beendiscovered and bear no relationship to the orderin which they act.FactorCommon NameI Fibrinogen These factors are usually referredII Prothrombinto by their common names.III Tissue factor These factors are usually not re-IV Ca 2+ ferred to as coagulation factors.V Proaccelerin, labile factor, accelerator (Ac-)globulinVII 1 Proconvertin, serum prothrombin conversionaccelerator (SPCA), cothromboplastinVIII Antihemophilic factor A, antihemophilic globulin(AHG)IX Antihemophilic factor B, Christmas factor, plasmathromboplastin component (PTC)X Stuart-Prower factorXI Plasma thromboplastin antecedent (PTA)XII Hageman factorXIII Fibrin stabilizing factor (FSF), fibrinoligase1 There is no factor VI.general, as shown in Table 51–2, these proteins can beclassified into five types: (1) zymogens of serine-dependentproteases, which become activated during theprocess of coagulation; (2) cofactors; (3) fibrinogen;(4) a transglutaminase, which stabilizes the fibrin clot;and (5) regulatory and other proteins.The Intrinsic Pathway Leadsto Activation of Factor XThe intrinsic pathway (Figure 51–1) involves factorsXII, XI, IX, VIII, and X as well as prekallikrein, highmolecular-weight(HMW) kininogen, Ca 2+ , and plateletphospholipids. It results in the production of factorXa (by convention, activated clotting factors arereferred to by use of the suffix a).This pathway commences with the “contact phase”in which prekallikrein, HMW kininogen, factor XII,and factor XI are exposed to a negatively charged activatingsurface. In vivo, the proteins probably assembleon endothelial cell membranes, whereas glass or kaolincan be used for in vitro tests of the intrinsic pathway.When the components of the contact phase assembleon the activating surface, factor XII is activated to factorXIIa upon proteolysis by kallikrein. This factorXIIa, generated by kallikrein, attacks prekallikrein togenerate more kallikrein, setting up a reciprocal activation.Factor XIIa, once formed, activates factor XI toTable 51–2. The functions of the proteinsinvolved in blood coagulation.Zymogens of serine proteasesFactor XII Binds to negatively charged surface at site ofvessel wall injury; activated by high-MWkininogen and kallikrein.Factor XI Activated by factor XIIa.Factor IX Activated by factor Xla in presence of Ca 2+ .Factor VII Activated thrombin in presence of Ca 2+ .Factor X Activated on surface of activated platelets bytenase complex (Ca 2+ , factors VIIIa and IXa)and by factor VIIa in presence of tissue factorand Ca 2+ .Factor II Activated on surface of activated platelets byprothrombinase complex (Ca 2+ , factors Vaand Xa).[Factors II, VII, IX, and X are Gla-containingzymogens.] (Gla = γ-carboxyglutamate.)CofactorsFactor VIIIFactor VActivated by thrombin; factor VIIIa is a cofactorin the activation of factor X byfactor IXa.Activated by thrombin; factor Va is a cofactorin the activation of prothrombin byfactor Xa.Tissue factor A glycoprotein expressed on the surface of(factor III) injured or stimulated endothelial cells toact as a cofactor for factor VIIa.FibrinogenFactor I Cleaved by thrombin to form fibrin clot.Thiol-dependent transglutaminaseFactor XIII Activated by thrombin in presence of Ca 2+ ;stabilizes fibrin clot by covalent crosslinking.Regulatory and other proteinsProtein C Activated to protein Ca by thrombin boundto thrombomodulin; then degrades factorsVIIIa and Va.Protein S Acts as a cofactor of protein C; both proteinscontain Gla (γ-carboxyglutamate)residues.Thrombo- Protein on the surface of endothelialmodulin cells; binds thrombin, which then activatesprotein C.XIa and also releases bradykinin (a nonapeptide withpotent vasodilator action) from HMW kininogen.Factor XIa in the presence of Ca 2+ activates factor IX(55 kDa, a zymogen containing vitamin K-dependentγ-carboxyglutamate [Gla] residues; see Chapter 45), tothe serine protease, factor IXa. This in turn cleaves anArg-Ile bond in factor X (56 kDa) to produce the twochainserine protease, factor Xa. This latter reaction requiresthe assembly of components, called the tenase

HEMOSTASIS & THROMBOSIS / 601complex, on the surface of activated platelets: Ca 2+ andfactor VIIIa, as well as factors IXa and X. It should benoted that in all reactions involving the Gla-containingzymogens (factors II, VII, IX, and X), the Gla residuesin the amino terminal regions of the molecules serve ashigh-affinity binding sites for Ca 2+ . For assembly of thetenase complex, the platelets must first be activated toexpose the acidic (anionic) phospholipids, phosphatidylserineand phosphatidylinositol, that arenormally on the internal side of the plasma membraneof resting, nonactivated platelets. Factor VIII (330kDa), a glycoprotein, is not a protease precursor but acofactor that serves as a receptor for factors IXa and Xon the platelet surface. Factor VIII is activated byminute quantities of thrombin to form factor VIIIa,which is in turn inactivated upon further cleavage bythrombin.The Extrinsic Pathway Also Leadsto Activation of Factor X Butby a Different MechanismFactor Xa occurs at the site where the intrinsic and extrinsicpathways converge (Figure 51–1) and lead intothe final common pathway of blood coagulation. Theextrinsic pathway involves tissue factor, factors VII andX, and Ca 2+ and results in the production of factor Xa.It is initiated at the site of tissue injury with the exposureof tissue factor (Figure 51–1) on subendothelialcells. Tissue factor interacts with and activates factorVII (53 kDa), a circulating Gla-containing glycoproteinsynthesized in the liver. Tissue factor acts as a cofactorfor factor VIIa, enhancing its enzymatic activity to activatefactor X. The association of tissue factor and factorVIIa is called tissue factor complex. Factor VIIacleaves the same Arg-Ile bond in factor X that is cleavedby the tenase complex of the intrinsic pathway. Activationof factor X provides an important link between theintrinsic and extrinsic pathways.Another important interaction between the extrinsicand intrinsic pathways is that complexes of tissue factorand factor VIIa also activate factor IX in the intrinsicpathway. Indeed, the formation of complexes betweentissue factor and factor VIIa is now consideredto be the key process involved in initiation ofblood coagulation in vivo. The physiologic significanceof the initial steps of the intrinsic pathway, inwhich factor XII, prekallikrein, and HMW kininogenare involved, has been called into question because patientswith a hereditary deficiency of these componentsdo not exhibit bleeding problems. Similarly, patientswith a deficiency of factor XI may not have bleedingproblems. The intrinsic pathway may actually be moreimportant in fibrinolysis (see below) than in coagulation,since kallikrein, factor XIIa, and factor XIa cancleave plasminogen and kallikrein can activate singlechainurokinase.Tissue factor pathway inhibitor (TFPI) is a majorphysiologic inhibitor of coagulation. It is a protein thatcirculates in the blood associated with lipoproteins.TFPI directly inhibits factor Xa by binding to the enzymenear its active site. This factor Xa-TFPI complexthen inhibits the factor VIIa-tissue factor complex.The Final Common Pathway of BloodClotting Involves Activation ofProthrombin to ThrombinIn the final common pathway, factor Xa, produced byeither the intrinsic or the extrinsic pathway, activatesprothrombin (factor II) to thrombin (factor IIa),which then converts fibrinogen to fibrin (Figure 51–1).The activation of prothrombin, like that of factor X,occurs on the surface of activated platelets and requiresthe assembly of a prothrombinase complex, consistingof platelet anionic phospholipids, Ca 2+ , factor Va, factorXa, and prothrombin.Factor V (330 kDa), a glycoprotein with homologyto factor VIII and ceruloplasmin, is synthesized in theliver, spleen, and kidney and is found in platelets aswell as in plasma. It functions as a cofactor in a mannersimilar to that of factor VIII in the tenase complex.When activated to factor Va by traces of thrombin, itbinds to specific receptors on the platelet membrane(Figure 51–2) and forms a complex with factor Xa andprothrombin. It is subsequently inactivated by furtheraction of thrombin, thereby providing a means of limitingthe activation of prothrombin to thrombin. Prothrombin(72 kDa; Figure 51–3) is a single-chain glycoproteinsynthesized in the liver. The amino terminalregion of prothrombin (1 in Figure 51–3) contains tenGla residues, and the serine-dependent active proteasesite (indicated by the arrowhead) is in the carboxyl terminalregion of the molecule. Upon binding to thecomplex of factors Va and Xa on the platelet membrane,prothrombin is cleaved by factor Xa at two sites(Figure 51–2) to generate the active, two-chain thrombinmolecule, which is then released from the plateletsurface. The A and B chains of thrombin are held togetherby a disulfide bond.Conversion of Fibrinogen to FibrinIs Catalyzed by ThrombinFibrinogen (factor I, 340 kDa; see Figures 51–1 and51–4 and Tables 51–1 and 51–2) is a soluble plasmaglycoprotein that consists of three nonidentical pairs ofpolypeptide chains (Aα,Bβγ) 2 covalently linked bydisulfide bonds. The Bβ and γ chains contain asparagine-linkedcomplex oligosaccharides. All three

602 / CHAPTER 51PrethrombinCa 2+ Ca 2+ V a X aPlateletFigure 51-2. Diagrammatic representation(not to scale) of the binding of factors Va, Xa,F-1.2Ca 2+ , and prothrombin to the plasma membrane+NH 3S-S COO – of the activated platelet. The sites of cleavage ofplasmamembraneIndicates negative chargesto which Ca 2+ binds.prothrombin by factor Xa are indicated by twoarrows. The part of prothrombin destined toform thrombin is labeled prethrombin. The Ca 2+is bound to anionic phospholipids of the plasmamembrane of the activated platelet.X aS – SGla n12AX aF-1 2Prethrombin(before Xa cleavage)Thrombin (after Xa cleavage)Figure 51–3. Diagrammatic representation (not toscale) of prothrombin. The amino terminal is to the left;region 1 contains all ten Gla residues. The sites of cleavageby factor Xa are shown and the products named.The site of the catalytically active serine residue is indicatedby the solid triangle. The A and B chains of activethrombin (shaded) are held together by the disulfidebridge.Bchains are synthesized in the liver; the three structuralgenes involved are on the same chromosome, and theirexpression is coordinately regulated in humans. Theamino terminal regions of the six chains are held inclose proximity by a number of disulfide bonds, whilethe carboxyl terminal regions are spread apart, givingrise to a highly asymmetric, elongated molecule (Figure51–4). The A and B portions of the Aα and Bβ chains,designated fibrinopeptides A (FPA) and B (FPB), respectively,at the amino terminal ends of the chains,bear excess negative charges as a result of the presenceof aspartate and glutamate residues, as well as an unusualtyrosine O-sulfate in FPB. These negative chargescontribute to the solubility of fibrinogen in plasma andalso serve to prevent aggregation by causing electrostaticrepulsion between fibrinogen molecules.Thrombin (34 kDa), a serine protease formed bythe prothrombinase complex, hydrolyzes the four Arg-Gly bonds between the fibrinopeptides and the α and βportions of the Aα and Bβ chains of fibrinogen (Figure51–5A). The release of the fibrinopeptides by thrombingenerates fibrin monomer, which has the subunit structure(α, β, γ) 2 . Since FPA and FPB contain only 16 and14 residues, respectively, the fibrin molecule retains98% of the residues present in fibrinogen. The removalof the fibrinopeptides exposes binding sites that allowthe molecules of fibrin monomers to aggregate spontaneouslyin a regularly staggered array, forming an insolublefibrin clot. It is the formation of this insoluble fibrinpolymer that traps platelets, red cells, and othercomponents to form the white or red thrombi. Thisinitial fibrin clot is rather weak, held together only bythe noncovalent association of fibrin monomers.In addition to converting fibrinogen to fibrin,thrombin also converts factor XIII to factor XIIIa. Thisfactor is a highly specific transglutaminase that covalentlycross-links fibrin molecules by forming peptidebonds between the amide groups of glutamine and theε-amino groups of lysine residues (Figure 51–5B),yielding a more stable fibrin clot with increased resistanceto proteolysis.Levels of Circulating Thrombin Must BeCarefully Controlled or Clots May FormOnce active thrombin is formed in the course of hemostasisor thrombosis, its concentration must be carefullycontrolled to prevent further fibrin formation orplatelet activation. This is achieved in two ways.Thrombin circulates as its inactive precursor, prothrombin,which is activated as the result of a cascadeof enzymatic reactions, each converting an inactive zymogento an active enzyme and leading finally to theconversion of prothrombin to thrombin (Figure 51–1).At each point in the cascade, feedback mechanismsproduce a delicate balance of activation and inhibition.The concentration of factor XII in plasma is approximately30 µg/mL, while that of fibrinogen is 3 mg/mL,with intermediate clotting factors increasing in concentrationas one proceeds down the cascade, showing thatthe clotting cascade provides amplification. The secondmeans of controlling thrombin activity is the inactivationof any thrombin formed by circulating inhibi-

HEMOSTASIS & THROMBOSIS / 603FPAFPBAα chainFigure 51–4. Diagrammatic representation(not to scale) of fibrinogen showing pairs of Aα,Bβ, and γ chains linked by disulfide bonds. (FPA,fibrinopeptide A; FPB, fibrinopeptide B.)COO –NH 3+Bβ chainγ chainCOO –tors, the most important of which is antithrombin III(see below).The Activity of Antithrombin III,an Inhibitor of Thrombin,Is Increased by HeparinFour naturally occurring thrombin inhibitors exist innormal plasma. The most important is antithrombinIII (often called simply antithrombin), which contributesapproximately 75% of the antithrombin activity.Antithrombin III can also inhibit the activities offactors IXa, Xa, XIa, XIIa, and VIIa complexed withtissue factor. 2 -Macroglobulin contributes most ofthe remainder of the antithrombin activity, with heparincofactor II and 1 -antitrypsin acting as minor inhibitorsunder physiologic conditions.The endogenous activity of antithrombin III isgreatly potentiated by the presence of acidic proteoglycanssuch as heparin (Chapter 48). These bind to aspecific cationic site of antithrombin III, inducing aconformational change and promoting its binding tothrombin as well as to its other substrates. This is thebasis for the use of heparin in clinical medicine to inhibitcoagulation. The anticoagulant effects of heparincan be antagonized by strongly cationic polypeptidessuch as protamine, which bind strongly to heparin,thus inhibiting its binding to antithrombin III. Individualswith inherited deficiencies of antithrombin III areprone to develop venous thrombosis, providing evidencethat antithrombin III has a physiologic functionand that the coagulation system in humans is normallyin a dynamic state.Thrombin is involved in an additional regulatorymechanism that operates in coagulation. It combineswith thrombomodulin, a glycoprotein present on thesurfaces of endothelial cells. The complex activates proteinC. In combination with protein S, activated proteinC (APC) degrades factors Va and VIIIa, limitingtheir actions in coagulation. A genetic deficiency of eitherprotein C or protein S can cause venous thrombosis.Furthermore, patients with factor V Leiden (whichhas a glutamine residue in place of an arginine at position506) have an increased risk of venous thromboticAThrombinNH 3+– –––Fibrinopeptide(A or B)Arg Gly COO –Fibrin chain( α or β)Figure 51-5. Formation of a fibrinclot. A: Thrombin-induced cleavageof Arg-Gly bonds of the Aα and Bβchains of fibrinogen to produce fibrinopeptides(left-hand side) andthe α and β chains of fibrin monomer(right-hand side). B: Crosslinkingof fibrin molecules by activatedfactor XIII (factor XIIIa).BFibrin CH 2 CH 2 CH 2 CH 2+NH 3 H 2 NOC CH 2 CH 2 Fibrin(Lysyl)(Glutaminyl)+NH 4 Factor XIIIa (Transglutaminase)OFibrin CH 2 CH 2 CH 2 CH 2 NH C CH 2 CH 2 Fibrin

604 / CHAPTER 51disease because factor V Leiden is resistant to inactivationby APC. This condition is termed APC resistance.Coumarin Anticoagulants Inhibit theVitamin K-Dependent Carboxylation ofFactors II, VII, IX, & XThe coumarin drugs (eg, warfarin), which are used asanticoagulants, inhibit the vitamin K-dependent carboxylationof Glu to Gla residues (see Chapter 45) inthe amino terminal regions of factors II, VII, IX, and Xand also proteins C and S. These proteins, all of whichare synthesized in the liver, are dependent on the Ca 2+ -binding properties of the Gla residues for their normalfunction in the coagulation pathways. The coumarinsact by inhibiting the reduction of the quinone derivativesof vitamin K to the active hydroquinone forms(Chapter 45). Thus, the administration of vitamin Kwill bypass the coumarin-induced inhibition and allowmaturation of the Gla-containing factors. Reversal ofcoumarin inhibition by vitamin K requires 12–24hours, whereas reversal of the anticoagulant effects ofheparin by protamine is almost instantaneous.Heparin and warfarin are widely used in the treatmentof thrombotic and thromboembolic conditions,such as deep vein thrombosis and pulmonary embolus.Heparin is administered first, because of its promptonset of action, whereas warfarin takes several days toreach full effect. Their effects are closely monitored byuse of appropriate tests of coagulation (see below) becauseof the risk of producing hemorrhage.Hemophilia A Is Due to a GeneticallyDetermined Deficiency of Factor VIIIInherited deficiencies of the clotting system that resultin bleeding are found in humans. The most common isdeficiency of factor VIII, causing hemophilia A, an Xchromosome-linked disease that has played a major rolein the history of the royal families of Europe. HemophiliaB is due to a deficiency of factor IX; its clinicalfeatures are almost identical to those of hemophilia A,but the conditions can be separated on the basis of specificassays that distinguish between the two factors.The gene for human factor VIII has been clonedand is one of the largest so far studied, measuring 186kb in length and containing 26 exons. A variety of mutationshave been detected leading to diminished activityof factor VIII; these include partial gene deletionsand point mutations resulting in premature chain termination.Prenatal diagnosis by DNA analysis afterchorionic villus sampling is now possible.In past years, treatment for patients with hemophiliaA has consisted of administration of cryoprecipitates(enriched in factor VIII) prepared from individualdonors or lyophilized factor VIII concentrates preparedfrom plasma pools of up to 5000 donors. It is now possibleto prepare factor VIII by recombinant DNAtechnology. Such preparations are free of contaminatingviruses (eg, hepatitis A, B, C, or HIV-1) found inhuman plasma but are at present expensive; their usemay increase if cost of production decreases.Fibrin Clots Are Dissolved by PlasminAs stated above, the coagulation system is normally in astate of dynamic equilibrium in which fibrin clots areconstantly being laid down and dissolved. This latterprocess is termed fibrinolysis. Plasmin, the serine proteasemainly responsible for degrading fibrin and fibrinogen,circulates in the form of its inactive zymogen,plasminogen (90 kDa), and any small amounts of plasminthat are formed in the fluid phase under physiologicconditions are rapidly inactivated by the fastactingplasmin inhibitor, α 2 -antiplasmin. Plasminogenbinds to fibrin and thus becomes incorporated in clotsas they are produced; since plasmin that is formedwhen bound to fibrin is protected from α 2 -antiplasmin,it remains active. Activators of plasminogen of varioustypes are found in most body tissues, and all cleave thesame Arg-Val bond in plasminogen to produce the twochainserine protease, plasmin (Figure 51–6).Tissue plasminogen activator (alteplase; t-PA) is aserine protease that is released into the circulation fromvascular endothelium under conditions of injury orNH 3+PlasminogenactivatorsArg–ValSSPLASMINOGEN–COO+NH 3 Arg Val–COOS SPLASMINFigure 51–6. Activation of plasminogen. The sameArg-Val bond is cleaved by all plasminogen activatorsto give the two-chain plasmin molecule. The solid triangleindicates the serine residue of the active site. Thetwo chains of plasmin are held together by a disulfidebridge.

HEMOSTASIS & THROMBOSIS / 605stress and is catalytically inactive unless bound to fibrin.Upon binding to fibrin, t-PA cleaves plasminogenwithin the clot to generate plasmin, which in turn digeststhe fibrin to form soluble degradation productsand thus dissolves the clot. Neither plasmin nor theplasminogen activator can remain bound to thesedegradation products, and so they are released into thefluid phase, where they are inactivated by their naturalinhibitors. Prourokinase is the precursor of a second activatorof plasminogen, urokinase. Originally isolatedfrom urine, it is now known to be synthesized by celltypes such as monocytes and macrophages, fibroblasts,and epithelial cells. Its main action is probably in thedegradation of extracellular matrix. Figure 51–7 indicatesthe sites of action of five proteins that influencethe formation and action of plasmin.Recombinant t-PA & Streptokinase AreUsed as Clot BustersAlteplase (t-PA), produced by recombinant DNA technology,is used therapeutically as a fibrinolytic agent, asis streptokinase. However, the latter is less selectivethan t-PA, activating plasminogen in the fluid phase(where it can degrade circulating fibrinogen) as well asplasminogen that is bound to a fibrin clot. The amountof plasmin produced by therapeutic doses of streptokinasemay exceed the capacity of the circulating α 2 -antiplasmin, causing fibrinogen as well as fibrin to bedegraded and resulting in the bleeding often encounteredduring fibrinolytic therapy. Because of its selectivityfor degrading fibrin, there is considerable therapeuticinterest in the use of recombinant t-PA to restorethe patency of coronary arteries following thrombosis.If administered early enough, before irreversible damageof heart muscle occurs (about 6 hours after onset ofthrombosis), t-PA can significantly reduce the mortalityrate from myocardial damage following coronarythrombosis. t-PA is more effective than streptokinase atrestoring full patency and also appears to result in aslightly better survival rate. Table 51–3 compares somethrombolytic features of streptokinase and t-PA.There are a number of disorders, including cancerand shock, in which the concentrations of plasminogenactivators increase. In addition, the antiplasminactivities contributed by α 1 -antitrypsin and α 2 -antiplasminmay be impaired in diseases such as cirrhosis. Sincecertain bacterial products, such as streptokinase, are capableof activating plasminogen, they may be responsiblefor the diffuse hemorrhage sometimes observed inpatients with disseminated bacterial infections.Activation of Platelets InvolvesStimulation of thePolyphosphoinositide PathwayPlatelets normally circulate in an unstimulated diskshapedform. During hemostasis or thrombosis, theybecome activated and help form hemostatic plugs orthrombi. Three major steps are involved: (1) adhesionto exposed collagen in blood vessels, (2) release of thecontents of their granules, and (3) aggregation.Platelets adhere to collagen via specific receptors onthe platelet surface, including the glycoprotein complexGPIa–IIa (α 2 β 1 integrin; Chapter 52), in a reactionthat involves von Willebrand factor. This is a glycoprotein,secreted by endothelial cells into the plasma,which stabilizes factor VIII and binds to collagen andthe subendothelium. Platelets bind to von Willebrandfactor via a glycoprotein complex (GPIb–V–IX) on theplatelet surface; this interaction is especially importantin platelet adherence to the subendothelium under conditionsof high shear stress that occur in small vesselsand stenosed arteries.Platelets adherent to collagen change shape andspread out on the subendothelium. They release thecontents of their storage granules (the dense granulesand the alpha granules); secretion is also stimulated bythrombin.Figure 51–7. Scheme of sites of actionof streptokinase, tissue plasminogen activator(t-PA), urokinase, plasminogen activatorinhibitor, and α 2 -antiplasmin (thelast two proteins exert inhibitory actions).Streptokinase forms a complex with plasminogen,which exhibits proteolytic activity;this cleaves some plasminogen to plasmin,initiating fibrinolysis.Plasminogenactivatorinhibitor–t-PAFibrinStreptokinase+PlasminogenPlasmin–Streptokinase-plasminogencomplexUrokinaseα 2 -AntiplasminFibrin degradation products

606 / CHAPTER 51Table 51–3. Comparison of some properties ofstreptokinase (SK) and tissue plasminogenactivator (t-PA) with regard to their use asthrombolytic agents. 1 SK t-PASelective for fibrin clot − +Produces plasminemia + −Reduces mortality + +Causes allergic reaction + −Causes hypotension + −Cost per treatment Relatively low Relatively high(approximate)1 Data from Webb J, Thompson C: Thrombolysis for acute myocardialinfarction. Can Fam Physician 1992;38:1415.Thrombin, formed from the coagulation cascade, isthe most potent activator of platelets and initiatesplatelet activation by interacting with its receptor onthe plasma membrane (Figure 51–8). The furtherevents leading to platelet activation are examples oftransmembrane signaling, in which a chemical messengeroutside the cell generates effector molecules insidethe cell. In this instance, thrombin acts as the externalchemical messenger (stimulus or agonist). Theinteraction of thrombin with its receptor stimulates theactivity of an intracellular phospholipase C. This enzymehydrolyzes the membrane phospholipid phosphatidylinositol4,5-bisphosphate (PIP 2 , a polyphosphoinositide)to form the two internal effector molecules,1,2-diacylglycerol and 1,4,5-inositol trisphosphate.Hydrolysis of PIP 2 is also involved in the action ofmany hormones and drugs. Diacylglycerol stimulatesCollagenProstacyclin TxA 2 Thrombin ADPAggregationFibrinogenR 1 R 2 R 3 R 4 R 5+++ ++PLCβ PIP 2AC PLA 2 PLGPllb-lllaPlasmamembranecAMPArachidonic acid+PKCSignalingeventsTxA 2–IP 3DAGActinCa 2+ Phosphorylationof light chain ofmyosinPhosphorylationof pleckstrinRelease of contentsof platelet granules(dense and alpha),including ADP;signaling eventsActomyosinChange of shapeFigure 51–8. Diagrammatic representation of platelet activation. The external environment,the plasma membrane, and the inside of a platelet are depicted from top to bottom.Thrombin and collagen are the two most important platelet activators. ADP is considereda weak agonist; it causes aggregation but not granule release. (GP, glycoprotein; R 1 –R 5 ,various receptors; AC, adenylyl cyclase; PLA 2 , phospholipase A 2 ; PL, phospholipids; PLCβ,phospholipase Cβ; PIP 2 , phosphatidylinositol 4,5-bisphosphate; cAMP, cyclic AMP; PKC,protein kinase C; TxA 2 , thromboxane A 2 ; IP 3 , inositol 1,4,5-trisphosphate; DAG, 1,2-diacylglycerol.The G proteins that are involved are not shown.)

HEMOSTASIS & THROMBOSIS / 607protein kinase C, which phosphorylates the proteinpleckstrin (47 kDa). This results in aggregation and releaseof the contents of the storage granules. ADP releasedfrom dense granules can also activate platelets,resulting in aggregation of additional platelets. IP 3causes release of Ca 2+ into the cytosol mainly from thedense tubular system (or residual smooth endoplasmicreticulum from the megakaryocyte), which then interactswith calmodulin and myosin light chain kinase,leading to phosphorylation of the light chains ofmyosin. These chains then interact with actin, causingchanges of platelet shape.Collagen-induced activation of a platelet phospholipaseA 2 by increased levels of cytosolic Ca 2+ results inliberation of arachidonic acid from platelet phospholipids,leading to the formation of thromboxane A 2(Chapter 23), which in turn, in a receptor-mediatedfashion, can further activate phospholipase C, promotingplatelet aggregation.Activated platelets, besides forming a platelet aggregate,are required, via newly expressed anionic phospholipidson the membrane surface, for acceleration ofthe activation of factors X and II in the coagulation cascade(Figure 51–1).All of the aggregating agents, including thrombin,collagen, ADP, and others such as platelet-activatingfactor, modify the platelet surface so that fibrinogencan bind to a glycoprotein complex, GPIIb–IIIa(α IIb β 3 integrin; Chapter 52), on the activated plateletsurface. Molecules of divalent fibrinogen then link adjacentactivated platelets to each other, forming a plateletaggregate. Some agents, including epinephrine, serotonin,and vasopressin, exert synergistic effects withother aggregating agents.Endothelial Cells Synthesize Prostacyclin& Other Compounds That AffectClotting & ThrombosisThe endothelial cells in the walls of blood vessels makeimportant contributions to the overall regulation of hemostasisand thrombosis. As described in Chapter 23,these cells synthesize prostacyclin (PGI 2 ), a potent inhibitorof platelet aggregation, opposing the action ofthromboxane A 2 . Prostacyclin acts by stimulating theactivity of adenylyl cyclase in the surface membranes ofplatelets. The resulting increase of intraplatelet cAMPopposes the increase in the level of intracellular Ca 2+produced by IP 3 and thus inhibits platelet activation(Figure 51–8). Endothelial cells play other roles in theregulation of thrombosis. For instance, these cells possessan ADPase, which hydrolyzes ADP, and thus opposesits aggregating effect on platelets. In addition,these cells appear to synthesize heparan sulfate, an anticoagulant,and they also synthesize plasminogen activators,which may help dissolve thrombi. Table 51–4 listssome molecules produced by endothelial cells that affectthrombosis and fibrinolysis. Endothelium-derivedrelaxing factor (nitric oxide) is discussed in Chapter 49.Analysis of the mechanisms of uptake of atherogeniclipoproteins, such as LDL, by endothelial, smooth muscle,and monocytic cells of arteries, along with detailedstudies of how these lipoproteins damage such cells is akey area of study in elucidating the mechanisms of atherosclerosis(Chapter 26).Aspirin Is an Effective Antiplatelet DrugCertain drugs (antiplatelet drugs) modify the behaviorof platelets. The most important is aspirin (acetylsalicylicacid), which irreversibly acetylates and thus inhibitsthe platelet cyclooxygenase system involved information of thromboxane A 2 (Chapter 14), a potentaggregator of platelets and also a vasoconstrictor.Platelets are very sensitive to aspirin; as little as 30 mg/d(one aspirin tablet usually contains 325 mg) effectivelyeliminates the synthesis of thromboxane A 2 . Aspirinalso inhibits production of prostacyclin (PGI 2 , whichopposes platelet aggregation and is a vasodilator) by en-Table 51–4. Molecules synthesized byendothelial cells that play a role in the regulationof thrombosis and fibrinolysis. 1MoleculeADPase (an ectoenzyme)Endothelium-derived relaxingfactor (nitric oxide)Heparan sulfate (a glycosaminoglycan)Prostacyclin (PGI 2 , a prostaglandin)Thrombomodulin (a glycoprotein)ActionDegrades ADP (an aggregatingagent of platelets) to AMP + P iInhibits platelet adhesion andaggregation by elevating levelsof cGMPAnticoagulant; combines withantithrombin III to inhibitthrombinInhibits platelet aggregation byincreasing levels of cAMPBinds protein C, which is thencleaved by thrombin to yieldactivated protein C; this incombination with protein Sdegrades factors Va and VIIIa,limiting their actionsActivates plasminogen to plas-min, which digests fibrin; theaction of t-PA is opposed byplasminogen activator inhibitor-1(PAI-1)Tissue plasminogen activator(t-PA, a protease)1 Adapted from Wu KK: Endothelial cells in hemostasis, thrombosisand inflammation. Hosp Pract (Off Ed) 1992 Apr; 27:145.

608 / CHAPTER 51dothelial cells, but unlike platelets, these cells regeneratecyclooxygenase within a few hours. Thus, the overallbalance between thromboxane A 2 and prostacyclin canbe shifted in favor of the latter, opposing platelet aggregation.Indications for treatment with aspirin thus includemanagement of angina and evolving myocardialinfarction and also prevention of stroke and death inpatients with transient cerebral ischemic attacks.Laboratory Tests Measure Coagulation& ThrombolysisA number of laboratory tests are available to measurethe phases of hemostasis described above. The tests includeplatelet count, bleeding time, activated partialthromboplastin time (aPTT or PTT), prothrombin time(PT), thrombin time (TT), concentration of fibrinogen,fibrin clot stability, and measurement of fibrindegradation products. The platelet count quantitatesthe number of platelets, and the bleeding time is anoverall test of platelet function. aPTT is a measure ofthe intrinsic pathway and PT of the extrinsic pathway.PT is used to measure the effectiveness of oral anticoagulantssuch as warfarin, and aPTT is used to monitorheparin therapy. The reader is referred to a textbook ofhematology for a discussion of these tests.SUMMARY• Hemostasis and thrombosis are complex processesinvolving coagulation factors, platelets, and bloodvessels.• Many coagulation factors are zymogens of serine proteases,becoming activated during the overall process.• Both intrinsic and extrinsic pathways of coagulationexist, the latter initiated by tissue factor. The pathwaysconverge at factor Xa, embarking on the commonfinal pathway resulting in thrombin-catalyzedconversion of fibrinogen to fibrin, which is strengthenedby cross-linking, catalyzed by factor XIII.• Genetic disorders of coagulation factors occur, andthe two most common involve factors VIII (hemophiliaA) and IX (hemophilia B).• An important natural inhibitor of coagulation is antithrombinIII; genetic deficiency of this protein canresult in thrombosis.• For activity, factors II, VII, IX, and X and proteins Cand S require vitamin K-dependent γ-carboxylationof certain glutamate residues, a process that is inhibitedby the anticoagulant warfarin.• Fibrin is dissolved by plasmin. Plasmin exists as aninactive precursor, plasminogen, which can be activatedby tissue plasminogen activator (t-PA). Botht-PA and streptokinase are widely used to treat earlythrombosis in the coronary arteries.• Thrombin and other agents cause platelet aggregation,which involves a variety of biochemical andmorphologic events. Stimulation of phospholipase Cand the polyphosphoinositide pathway is a key eventin platelet activation, but other processes are also involved.• Aspirin is an important antiplatelet drug that acts byinhibiting production of thromboxane A 2 .REFERENCESBennett JS: Mechanisms of platelet adhesion and aggregation: anupdate. Hosp Pract (Off Ed) 1992;27:124.Broze GJ: Tissue factor pathway inhibitor and the revised theory ofcoagulation. Annu Rev Med 1995;46:103.Clemetson KJ: Platelet activation: signal transduction via membranereceptors. Thromb Haemost 1995;74:111.Collen D, Lijnen HR: Basic and clinical aspects of fibrinolysis andthrombolysis. Blood 1991;78:3114.Handin RI: Anticoagulant, fibrinolytic and antiplatelet therapy.In: Harrison’s Principles of Internal Medicine, 15th ed. BraunwaldE et al (editors). McGraw-Hill, 2001.Handin RI: Disorders of coagulation and thrombosis. In: Harrison’sPrinciples of Internal Medicine, 15th ed. Braunwald E etal (editors). McGraw-Hill, 2001.Handin RI: Disorders of the platelet and vessel wall. In: Harrison’sPrinciples of Internal Medicine, 15th ed. Braunwald E et al(editors). McGraw-Hill, 2001.Kroll MH, Schafer AI: Biochemical mechanisms of platelet activation.Blood 1989;74:1181.Roberts HR, Lozier JN: New perspectives on the coagulation cascade.Hosp Pract (Off Ed) 1992;27:97.Roth GJ, Calverley DC: Aspirin, platelets, and thrombosis: theoryand practice. Blood 1994;83:885.Schmaier AH: Contact activation: a revision. Thromb Haemost1997;78:101.Wu KK: Endothelial cells in hemostasis, thrombosis and inflammation.Hosp Pract (Off Ed) 1992;27:145.

Red & White Blood Cells 52Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEBlood cells have been studied intensively because theyare obtained easily, because of their functional importance,and because of their involvement in many diseaseprocesses. The structure and function of hemoglobin,the porphyrias, jaundice, and aspects of iron metabolismare discussed in previous chapters. Reduction ofthe number of red blood cells and of their content ofhemoglobin is the cause of the anemias, a diverse andimportant group of conditions, some of which are seenvery commonly in clinical practice. Certain of theblood group systems, present on the membranes oferythrocytes and other blood cells, are of extreme importancein relation to blood transfusion and tissuetransplantation. Table 52–1 summarizes the causes of anumber of important diseases affecting red blood cells;some are discussed in this chapter, and the remainderare discussed elsewhere in this text. Every organ in thebody can be affected by inflammation; neutrophils playa central role in acute inflammation, and other whiteblood cells, such as lymphocytes, play important rolesin chronic inflammation. Leukemias, defined as malignantneoplasms of blood-forming tissues, can affectprecursor cells of any of the major classes of whiteblood cells; common types are acute and chronic myelocyticleukemia, affecting precursors of the neutrophils;and acute and chronic lymphocytic leukemias.Combination chemotherapy, using combinations ofvarious chemotherapeutic agents, all of which act at oneor more biochemical loci, has been remarkably effectivein the treatment of certain of these types of leukemias.Understanding the role of red and white cells in healthand disease requires a knowledge of certain fundamentalaspects of their biochemistry.THE RED BLOOD CELL IS SIMPLE INTERMS OF ITS STRUCTURE & FUNCTIONThe major functions of the red blood cell are relativelysimple, consisting of delivering oxygen to the tissuesand of helping in the disposal of carbon dioxide andprotons formed by tissue metabolism. Thus, it has amuch simpler structure than most human cells, beingessentially composed of a membrane surrounding a solutionof hemoglobin (this protein forms about 95% ofthe intracellular protein of the red cell). There are no609intracellular organelles, such as mitochondria, lysosomes,or Golgi apparatus. Human red blood cells, likemost red cells of animals, are nonnucleated. However,the red cell is not metabolically inert. ATP is synthesizedfrom glycolysis and is important in processes thathelp the red blood cell maintain its biconcave shapeand also in the regulation of the transport of ions (eg,by the Na + -K + ATPase and the anion exchange protein[see below]) and of water in and out of the cell. The biconcaveshape increases the surface-to-volume ratio ofthe red blood cell, thus facilitating gas exchange. Thered cell contains cytoskeletal components (see below)that play an important role in determining its shape.About Two Million Red Blood Cells Enterthe Circulation per SecondThe life span of the normal red blood cell is 120 days;this means that slightly less than 1% of the populationof red cells (200 billion cells, or 2 million per second) isreplaced daily. The new red cells that appear in the circulationstill contain ribosomes and elements of the endoplasmicreticulum. The RNA of the ribosomes can bedetected by suitable stains (such as cresyl blue), and cellscontaining it are termed reticulocytes; they normallynumber about 1% of the total red blood cell count. Thelife span of the red blood cell can be dramatically shortenedin a variety of hemolytic anemias. The number ofreticulocytes is markedly increased in these conditions,as the bone marrow attempts to compensate for rapidbreakdown of red blood cells by increasing the amountof new, young red cells in the circulation.Erythropoietin Regulates Productionof Red Blood CellsHuman erythropoietin is a glycoprotein of 166 aminoacids (molecular mass about 34 kDa). Its amount inplasma can be measured by radioimmunoassay. It is themajor regulator of human erythropoiesis. Erythropoietinis synthesized mainly by the kidney and is released in responseto hypoxia into the bloodstream, in which ittravels to the bone marrow. There it interacts with progenitorsof red blood cells via a specific receptor. The receptoris a transmembrane protein consisting of two differentsubunits and a number of domains. It is not atyrosine kinase, but it stimulates the activities of specific

610 / CHAPTER 52Table 52–1. Summary of the causes of someimportant disorders affecting red blood cells.DisorderSole or Major CauseIron deficiency anemia Inadequate intake or excessive lossof ironMethemoglobinemia Intake of excess oxidants (variouschemicals and drugs)Genetic deficiency in the NADHdependentmethemoglobin reductasesystem (MIM 250800)Inheritance of HbM (MIM 141800)Sickle cell anemia Sequence of codon 6 of the β chain(MIM 141900)changed from GAG in the normalgene to GTG in the sickle cellgene, resulting in substitution ofvaline for glutamic acidα-Thalassemias Mutations in the α-globin genes,(MIM 141800)mainly unequal crossing-over andlarge deletions and less commonlynonsense and frameshiftmutationsβ-ThalassemiaA very wide variety of mutations in(MIM 141900)the β-globin gene, including deletions,nonsense and frameshiftmutations, and others affectingevery aspect of its structure (eg,splice sites, promoter mutants)Megaloblastic anemias Decreased absorption of B 12 , oftenDeficiency ofdue to a deficiency of intrinsic facvitaminB 12tor, normally secreted by gastricparietal cellsDeficiency of folic Decreased intake, defective absorpacidtion, or increased demand (eg, inpregnancy) for folateHereditaryDeficiencies in the amount or in thespherocytosis 1structure of α or β spectrin,ankyrin, band 3 or band 4.1Glucose-6-phosphate A variety of mutations in the genedehydrogenase (X-linked) for G6PD, mostly single(G6PD) deficiency 1 point mutations(MIM 305900)Pyruvate kinase (PK) Presumably a variety of mutationsdeficiency 1in the gene for the R (red cell) iso-(MIM 255200)zyme of PKParoxysmal nocturnal Mutations in the PIG-A gene, affecthemoglobinemia1 ing synthesis of GPI-anchored(MIM 311770)proteins1 The last four disorders cause hemolytic anemias, as do a numberof the other disorders listed. Most of the above conditions are discussedin other chapters of this text. MIM numbers apply only todisorders with a genetic basis.members of this class of enzymes involved in downstreamsignal transduction. Erythropoietin interactswith a red cell progenitor, known as the burst-formingunit-erythroid (BFU-E), causing it to proliferate anddifferentiate. In addition, it interacts with a later progenitorof the red blood cell, called the colony-formingunit-erythroid (CFU-E), also causing it to proliferateand further differentiate. For these effects, erythropoietinrequires the cooperation of other factors (eg, interleukin-3and insulin-like growth factor; Figure 52–1).The availability of a cDNA for erythropoietin hasmade it possible to produce substantial amounts of thishormone for analysis and for therapeutic purposes; previouslythe isolation of erythropoietin from humanurine yielded very small amounts of the protein. Themajor use of recombinant erythropoietin has been inthe treatment of a small number of anemic states, suchas that due to renal failure.MANY GROWTH FACTORS REGULATEPRODUCTION OF WHITE BLOOD CELLSA large number of hematopoietic growth factors havebeen identified in recent years in addition to erythropoietin.This area of study adds to knowledge about thedifferentiation of blood cells, provides factors that maybe useful in treatment, and also has implications for understandingof the abnormal growth of blood cells (eg,the leukemias). Like erythropoietin, most of the growthfactors isolated have been glycoproteins, are very activein vivo and in vitro, interact with their target cells viaspecific cell surface receptors, and ultimately (via intracellularsignals) affect gene expression, thereby promotingdifferentiation. Many have been cloned, permittingtheir production in relatively large amounts. Two ofparticular interest are granulocyte- and granulocytemacrophagecolony-stimulating factors (G-CSF andGM-CSF, respectively). G-CSF is relatively specific, inducingmainly granulocytes. GM-CSF affects a varietyof progenitor cells and induces granulocytes, macrophages,and eosinophils. When the production of neutrophilsis severely depressed, this condition is referredto as neutropenia. It is particularly likely to occur inpatients treated with certain chemotherapeutic regimensand after bone marrow transplantation. These patientsare liable to develop overwhelming infections.G-CSF has been administered to such patients to boostproduction of neutrophils.THE RED BLOOD CELL HAS A UNIQUE &RELATIVELY SIMPLE METABOLISMVarious aspects of the metabolism of the red cell, manyof which are discussed in other chapters of this text, aresummarized in Table 52–2.

RED & WHITE BLOOD CELLS / 611Pluripotentstem cellInterleukinsGM-CSFCFU-GEMMBFU-E(earlyand late)InterleukinsGM-CSFEpoErythroidprecursorMatureRBCsFigure 52–1. Greatly simplified scheme of differentiation of stem cells to redblood cells. Various interleukins (ILs), such as IL-3, IL-4, IL-9, and IL-11, are involvedat different steps of the overall process. Erythroid precursors include the pronormoblast,basophilic, polychromatophilic, and orthochromatophilic normoblasts,and the reticulocyte. Epo acts on basophilic normoblasts but not on later erythroidcells. (CFU-GEMM, colony-forming unit whose cells give rise to granulocytes,erythrocytes, macrophages, and megakaryocytes; BFU-E, burst-formingunit-erythroid; GM-CSF, granulocyte-macrophage colony-stimulating factor; Epo,erythropoietin; RBC, red blood cell.)The Red Blood Cell Has a GlucoseTransporter in Its MembraneThe entry rate of glucose into red blood cells is fargreater than would be calculated for simple diffusion.Rather, it is an example of facilitated diffusion (Chapter41). The specific protein involved in this process iscalled the glucose transporter or glucose permease.Some of its properties are summarized in Table 52–3.The process of entry of glucose into red blood cells is ofmajor importance because it is the major fuel supply forthese cells. About seven different but related glucosetransporters have been isolated from various tissues; unlikethe red cell transporter, some of these are insulindependent(eg, in muscle and adipose tissue). There isconsiderable interest in the latter types of transporterbecause defects in their recruitment from intracellularsites to the surface of skeletal muscle cells may help explainthe insulin resistance displayed by patients withtype 2 diabetes mellitus.Reticulocytes Are Activein Protein SynthesisThe mature red blood cell cannot synthesize protein.Reticulocytes are active in protein synthesis. Once reticulocytesenter the circulation, they lose their intracellularorganelles (ribosomes, mitochondria, etc) withinabout 24 hours, becoming young red blood cells andconcomitantly losing their ability to synthesize protein.Extracts of rabbit reticulocytes (obtained by injectingrabbits with a chemical—phenylhydrazine—that causesa severe hemolytic anemia, so that the red cells are almostcompletely replaced by reticulocytes) are widelyused as an in vitro system for synthesizing proteins. EndogenousmRNAs present in these reticulocytes aredestroyed by use of a nuclease, whose activity can be inhibitedby addition of Ca 2+ . The system is then programmedby adding purified mRNAs or whole-cell extractsof mRNAs, and radioactive proteins are synthesizedin the presence of 35 S-labeled L-methionine orother radiolabeled amino acids. The radioactive proteinssynthesized are separated by SDS-PAGE and detectedby radioautography.Superoxide Dismutase, Catalase,& Glutathione Protect Blood CellsFrom Oxidative Stress & DamageSeveral powerful oxidants are produced during thecourse of metabolism, in both blood cells and mostother cells of the body. These include superoxide (O 2−⋅),hydrogen peroxide (H 2 O 2 ), peroxyl radicals (ROO • and hydroxyl radicals (OH • ). The last is a particularlyreactive molecule and can react with proteins, nucleicacids, lipids, and other molecules to alter their structureand produce tissue damage. The reactions listed inTable 52–4 play an important role in forming these oxidantsand in disposing of them; each of these reactionswill now be considered in turn.Superoxide is formed (reaction 1) in the red bloodcell by the auto-oxidation of hemoglobin to methemoglobin(approximately 3% of hemoglobin in human redblood cells has been calculated to auto-oxidize per day);in other tissues, it is formed by the action of enzymessuch as cytochrome P450 reductase and xanthine oxidase.When stimulated by contact with bacteria, neutrophilsexhibit a respiratory burst (see below) and producesuperoxide in a reaction catalyzed by NADPHoxidase (reaction 2). Superoxide spontaneously dismutatesto form H 2 O 2 and O 2 ; however, the rate of thissame reaction is speeded up tremendously by the actionof the enzyme superoxide dismutase (reaction 3). Hydrogenperoxide is subject to a number of fates. The enzymecatalase, present in many types of cells, converts

612 / CHAPTER 52Table 52–2. Summary of important aspects ofthe metabolism of the red blood cell.• The RBC is highly dependent upon glucose as its energysource; its membrane contains high affinity glucose transporters.• Glycolysis, producing lactate, is the site of production ofATP.• Because there are no mitochondria in RBCs, there is no productionof ATP by oxidative phosphorylation.• The RBC has a variety of transporters that maintain ionicand water balance.• Production of 2,3-bisphosphoglycerate, by reactions closelyassociated with glycolysis, is important in regulating theability of Hb to transport oxygen.• The pentose phosphate pathway is operative in the RBC (itmetabolizes about 5–10% of the total flux of glucose) andproduces NADPH; hemolytic anemia due to a deficiency ofthe activity of glucose-6-phosphate dehydrogenase is common.• Reduced glutathione (GSH) is important in the metabolismof the RBC, in part to counteract the action of potentiallytoxic peroxides; the RBC can synthesize GSH and requiresNADPH to return oxidized glutathione (G-S-S-G) to the reducedstate.• The iron of Hb must be maintained in the ferrous state; ferriciron is reduced to the ferrous state by the action of anNADH-dependent methemoglobin reductase system involvingcytochrome b 5 reductase and cytochrome b 5 .• Synthesis of glycogen, fatty acids, protein, and nucleic acidsdoes not occur in the RBC; however, some lipids (eg, cholesterol)in the red cell membrane can exchange with correspondingplasma lipids.• The RBC contains certain enzymes of nucleotide metabolism(eg, adenosine deaminase, pyrimidine nucleotidase,and adenylyl kinase); deficiencies of these enzymes are involvedin some cases of hemolytic anemia.• When RBCs reach the end of their life span, the globin is degradedto amino acids (which are reutilized in the body),the iron is released from heme and also reutilized, and thetetrapyrrole component of heme is converted to bilirubin,which is mainly excreted into the bowel via the bile.8), which also produces OH • and OH − . Superoxide canrelease iron ions from ferritin. Thus, production ofOH • may be one of the mechanisms involved in tissueinjury due to iron overload (eg, hemochromatosis;Chapter 50).Chemical compounds and reactions capable of generatingpotential toxic oxygen species can be referred toas pro-oxidants. On the other hand, compounds andreactions disposing of these species, scavenging them,suppressing their formation, or opposing their actionsare antioxidants and include compounds such asNADPH, GSH, ascorbic acid, and vitamin E. In a normalcell, there is an appropriate pro-oxidant:antioxidantbalance. However, this balance can be shifted towardthe pro-oxidants when production of oxygenspecies is increased greatly (eg, following ingestion ofcertain chemicals or drugs) or when levels of antioxidantsare diminished (eg, by inactivation of enzymes involvedin disposal of oxygen species and by conditionsthat cause low levels of the antioxidants mentionedabove). This state is called “oxidative stress” and canresult in serious cell damage if the stress is massive orprolonged.Oxygen species are now thought to play an importantrole in many types of cellular injury (eg, resultingfrom administration of various toxic chemicals or fromischemia), some of which can result in cell death. Indirectevidence supporting a role for these species in genitto H 2 O and O 2 (reaction 4). Neutrophils possess aunique enzyme, myeloperoxidase, that uses H 2 O 2 andhalides to produce hypohalous acids (reaction 5); thissubject is discussed further below. The seleniumcontainingenzyme glutathione peroxidase (Chapter 20)will also act on reduced glutathione (GSH) and H 2 O 2to produce oxidized glutathione (GSSG) and H 2 O (reaction6); this enzyme can also use other peroxides assubstrates. OH • and OH − can be formed from H 2 O 2 ina nonenzymatic reaction catalyzed by Fe 2+ (the Fentonreaction, reaction 7). O 2−⋅ and H 2 O 2 are the substratesin the iron-catalyzed Haber-Weiss reaction (reactionTable 52–3. Some properties of the glucosetransporter of the membrane of the redblood cell.• It accounts for about 2% of the protein of the membrane ofthe RBC.• It exhibits specificity for glucose and related D-hexoses(L-hexoses are not transported).• The transporter functions at approximately 75% of its V maxat the physiologic concentration of blood glucose, is saturableand can be inhibited by certain analogs of glucose.• At least seven similar but distinct glucose transporters havebeen detected to date in mammalian tissues, of which thered cell transporter is one.• It is not dependent upon insulin, unlike the correspondingcarrier in muscle and adipose tissue.• Its complete amino acid sequence (492 amino acids) hasbeen determined.• It transports glucose when inserted into artificial liposomes.• It is estimated to contain 12 transmembrane helical segments.• It functions by generating a gated pore in the membrane topermit passage of glucose; the pore is conformationally dependenton the presence of glucose and can oscillaterapidly (about 900 times/s).

RED & WHITE BLOOD CELLS / 613Table 52–4. Reactions of importance in relation to oxidative stress in blood cells andvarious tissues.(1) Production of superoxide (by-product of various reactions) O 2 + e − → O 2− ⋅(2) NADPH-oxidase 2 O 2 + NADPH → 2 O 2− ⋅ + NADP + H +(3) Superoxide dismutase O 2− ⋅ + O 2− ⋅ + 2 H + → H 2 O 2 + O 2(4) Catalase H 2 O 2 → 2 H 2 O + O 2(5) Myeloperoxidase H 2 O 2 + X − + H + → HOX + H 2 O (X − = Cl − , Br − , SCN − )(6) Glutathione peroxidase (Se-dependent) 2 GSH + R-O-OH → GSSG + H 2 O + ROH(7) Fenton reaction Fe 2+ + H 2 O 2 → Fe 3+ + OH ⋅ + OH −(8) Iron-catalyzed Haber-Weiss reaction O 2− ⋅ + H 2 O 2 → O 2 + OH ⋅ + OH −(9) Glucose-6-phosphate dehydrogenase (G6PD) G6P + NADP → 6 Phosphogluconate + NADPH + H +(10) Glutathione reductase G-S-S-G + NADPH + H + → 2 GSH + NADPerating cell injury is provided if administration of anenzyme such as superoxide dismutase or catalase isfound to protect against cell injury in the situationunder study.Deficiency of Glucose-6-PhosphateDehydrogenase Is Frequent in CertainAreas & Is an Important Causeof Hemolytic AnemiaNADPH, produced in the reaction catalyzed by theX-linked glucose-6-phosphate dehydrogenase (Table52–4, reaction 9) in the pentose phosphate pathway(Chapter 20), plays a key role in supplying reducingequivalents in the red cell and in other cells such as thehepatocyte. Because the pentose phosphate pathway isvirtually its sole means of producing NADPH, the redblood cell is very sensitive to oxidative damage if thefunction of this pathway is impaired (eg, by enzyme deficiency).One function of NADPH is to reduce GSSGto GSH, a reaction catalyzed by glutathione reductase(reaction 10).Deficiency of the activity of glucose-6-phosphatedehydrogenase, owing to mutation, is extremely frequentin some regions of the world (eg, tropical Africa,the Mediterranean, certain parts of Asia, and in NorthAmerica among blacks). It is the most common of allenzymopathies (diseases caused by abnormalities of enzymes),and over 300 genetic variants of the enzymehave been distinguished; at least 100 million people aredeficient in this enzyme owing to these variants. Thedisorder resulting from deficiency of glucose-6-phosphatedehydrogenase is hemolytic anemia. Consumptionof broad beans (Vicia faba) by individuals deficientin activity of the enzyme can precipitate an attack ofhemolytic anemia (most likely because the beans containpotential oxidants). In addition, a number of drugs(eg, the antimalarial drug primaquine [the conditioncaused by intake of primaquine is called primaquinesensitivehemolytic anemia] and sulfonamides) andchemicals (eg, naphthalene) precipitate an attack, becausetheir intake leads to generation of H 2 O 2 or O 2−⋅.Normally, H 2 O 2 is disposed of by catalase and glutathioneperoxidase (Table 52–4, reactions 4 and 6),the latter causing increased production of GSSG. GSHis regenerated from GSSG by the action of the enzymeglutathione reductase, which depends on the availabilityof NADPH (reaction 10). The red blood cells of individualswho are deficient in the activity of glucose-6-phosphate dehydrogenase cannot generate sufficientNADPH to regenerate GSH from GSSG, which inturn impairs their ability to dispose of H 2 O 2 and ofoxygen radicals. These compounds can cause oxidationof critical SH groups in proteins and possibly peroxidationof lipids in the membrane of the red cell, causinglysis of the red cell membrane. Some of the SH groupsof hemoglobin become oxidized, and the protein precipitatesinside the red blood cell, forming Heinz bodies,which stain purple with cresyl violet. The presenceof Heinz bodies indicates that red blood cells have beensubjected to oxidative stress. Figure 52–2 summarizesthe possible chain of events in hemolytic anemia due todeficiency of glucose-6-phosphate dehydrogenase.Methemoglobin Is Uselessin Transporting OxygenThe ferrous iron of hemoglobin is susceptible to oxidationby superoxide and other oxidizing agents, formingmethemoglobin, which cannot transport oxygen. Onlya very small amount of methemoglobin is present innormal blood, as the red blood cell possesses an effectivesystem (the NADH-cytochrome b 5 methemoglobinreductase system) for reducing heme Fe 3+ back to theFe 2+ state. This system consists of NADH (generatedby glycolysis), a flavoprotein named cytochrome b 5 reductase(also known as methemoglobin reductase), andcytochrome b 5 . The Fe 3+ of methemoglobin is reduced

614 / CHAPTER 52Mutations in the gene for G6PDDecreased activity of G6PDDecreased levels of NADPHDecreased regeneration of GSH from GSSG byglutathione reductase (which uses NADPH)Oxidation, due to decreased levels of GSH andincreased levels of intracellular oxidants (eg, O 2– • ),of SH groups of Hb (forming Heinz bodies), and ofmembrane proteins, altering membrane structureand increasing susceptibility to ingestionby macrophages (peroxidative damage to lipidsin the membrane also possible)HemolysisFigure 52–2. Summary of probable events causinghemolytic anemia due to deficiency of the activity ofglucose-6-phosphate dehydrogenase (G6PD) (MIM305900).back to the Fe 2+ state by the action of reduced cytochromeb 5 :3+ 2+Hb - Fe + Cyt b5red → Hb - Fe + Cyt b5 oxReduced cytochrome b 5 is then regenerated by the actionof cytochrome b 5 reductase:Cyt b5 ox + NADH → Cyt b5 red + NADMethemoglobinemia Is Inheritedor AcquiredMethemoglobinemia can be classified as either inheritedor acquired by ingestion of certain drugs and chemicals.Neither type occurs frequently, but physicians must beaware of them. The inherited form is usually due to deficientactivity of methemoglobin reductase, transmittedin an autosomal recessive manner. Certain abnormal hemoglobins(Hb M) are also rare causes of methemoglobinemia.In Hb M, mutation changes the amino acidresidue to which heme is attached, thus altering its affinityfor oxygen and favoring its oxidation. Ingestion ofcertain drugs (eg, sulfonamides) or chemicals (eg, aniline)can cause acquired methemoglobinemia. Cyanosis(bluish discoloration of the skin and mucous mem-branes due to increased amounts of deoxygenated hemoglobinin arterial blood, or in this case due to increasedamounts of methemoglobin) is usually the presentingsign in both types and is evident when over 10%of hemoglobin is in the “met” form. Diagnosis is madeby spectroscopic analysis of blood, which reveals thecharacteristic absorption spectrum of methemoglobin.Additionally, a sample of blood containing methemoglobincannot be fully reoxygenated by flushing oxygenthrough it, whereas normal deoxygenated blood can.Electrophoresis can be used to confirm the presence ofan abnormal hemoglobin. Ingestion of methylene blueor ascorbic acid (reducing agents) is used to treat mildmethemoglobinemia due to enzyme deficiency. Acutemassive methemoglobinemia (due to ingestion of chemicals)should be treated by intravenous injection ofmethylene blue.MORE IS KNOWN ABOUT THE MEMBRANEOF THE HUMAN RED BLOOD CELL THANABOUT THE SURFACE MEMBRANE OFANY OTHER HUMAN CELLA variety of biochemical approaches have been used tostudy the membrane of the red blood cell. These includeanalysis of membrane proteins by SDS-PAGE,the use of specific enzymes (proteinases, glycosidases,and others) to determine the location of proteins andglycoproteins in the membrane, and various techniquesto study both the lipid composition and disposition ofindividual lipids. Morphologic (eg, electron microscopy,freeze-fracture electron microscopy) and othertechniques (eg, use of antibodies to specific components)have also been widely used. When red bloodcells are lysed under specific conditions, their membraneswill reseal in their original orientation to formghosts (right-side-out ghosts). By altering the conditions,ghosts can also be made to reseal with their cytosolicaspect exposed on the exterior (inside-outghosts). Both types of ghosts have been useful in analyzingthe disposition of specific proteins and lipids inthe membrane. In recent years, cDNAs for many proteinsof this membrane have become available, permittingthe deduction of their amino sequences and domains.All in all, more is known about the membraneof the red blood cell than about any other membrane ofhuman cells (Table 52–5).Analysis by SDS-PAGE Resolvesthe Proteins of the Membraneof the Red Blood CellWhen the membranes of red blood cells are analyzedby SDS-PAGE, about ten major proteins are resolved

RED & WHITE BLOOD CELLS / 615Table 52–5. Summary of biochemicalinformation about the membrane of the humanred blood cell.• The membrane is a bilayer composed of about 50% lipidand 50% protein.• The major lipid classes are phospholipids and cholesterol;the major phospholipids are phosphatidylcholine (PC),phosphatidylethanolamine (PE), and phosphatidylserine(PS) along with sphingomyelin (Sph).• The choline-containing phospholipids, PC and Sph, predominatein the outer leaflet and the amino-containingphospholipids (PE and PS) in the inner leaflet.• Glycosphingolipids (GSLs) (neutral GSLs, gangliosides, andcomplex species, including the ABO blood group substances)constitute about 5–10% of the total lipid.• Analysis by SDS-PAGE shows that the membrane containsabout 10 major proteins and more than 100 minor species.• The major proteins (which include spectrin, ankyrin, theanion exchange protein, actin, and band 4.1) have beenstudied intensively, and the principal features of their disposition(eg, integral or peripheral), structure, and functionhave been established.• Many of the proteins are glycoproteins (eg, the glycophorins)containing O- or N-linked (or both) oligosaccharidechains located on the external surface of the membrane.(Figure 52–3), several of which have been shown to beglycoproteins. Their migration on SDS-PAGE wasused to name these proteins, with the slowest migrating(and hence highest molecular mass) being designatedband 1 or spectrin. All these major proteins have beenisolated, most of them have been identified, and considerableinsight has been obtained about their functions(Table 52–6). Many of their amino acid sequencesalso have been established. In addition, it hasbeen determined which are integral or peripheral membraneproteins, which are situated on the external surface,which are on the cytosolic surface, and which spanthe membrane (Figure 52–4). Many minor componentscan also be detected in the red cell membrane byuse of sensitive staining methods or two-dimensionalgel electrophoresis. One of these is the glucose transporterdescribed above.The Major Integral Proteins of the RedBlood Cell Membrane Are the AnionExchange Protein & the GlycophorinsThe anion exchange protein (band 3) is a transmembraneglycoprotein, with its carboxyl terminal end onthe external surface of the membrane and its amino terminalend on the cytoplasmic surface. It is an exampleof a multipass membrane protein, extending across theSpectrinAnkyrinandisoforms122.12.22.32.6Anion exchange protein 34.14.2ActinG3PD567GlobinMembraneCoomassieblue stainSkeletonMembranePAS stainGlycophorinsFigure 52–3. Diagrammatic representation of themajor proteins of the membrane of the human redblood cell separated by SDS-PAGE. The bands detectedby staining with Coomassie blue are shown in the twoleft-hand channels, and the glycoproteins detected bystaining with periodic acid-Schiff (PAS) reagent areshown in the right-hand channel. (Reproduced, withpermission, from Beck WS, Tepper RI: Hemolytic anemiasIII: membrane disorders. In: Hematology, 5th ed. Beck WS[editor]. The MIT Press, 1991.)bilayer at least ten times. It probably exists as a dimer inthe membrane, in which it forms a tunnel, permittingthe exchange of chloride for bicarbonate. Carbon dioxide,formed in the tissues, enters the red cell as bicarbonate,which is exchanged for chloride in the lungs,where carbon dioxide is exhaled. The amino terminalend binds many proteins, including hemoglobin, proteins4.1 and 4.2, ankyrin, and several glycolytic enzymes.Purified band 3 has been added to lipid vesiclesin vitro and has been shown to perform its transportfunctions in this reconstituted system.Glycophorins A, B, and C are also transmembraneglycoproteins but of the single-pass type, extendingacross the membrane only once. A is the major glycophorin,is made up of 131 amino acids, and is heavilyglycosylated (about 60% of its mass). Its amino terminalend, which contains 16 oligosaccharide chains (15 ofwhich are O-glycans), extrudes out from the surface of

616 / CHAPTER 52Table 52–6. Principal proteins of the red cell membrane. 1Integral (I) or ApproximateBand Number 2 Protein Peripheral (P) Molecular Mass (kDa)1 Spectrin (α) P 2402 Spectrin (β) P 2202.1 Ankyrin P 2102.2 “ P 1952.3 “ P 1752.6 “ P 1453 Anion exchange protein I 1004.1 Unnamed P 805 Actin P 436 Glyceraldehyde-3-phosphate dehydrogenase P 357 Tropomyosin P 298 Unnamed P 23Glycophorins A, B, and C I 31, 23, and 281 Adapted from Lux DE, Becker PS: Disorders of the red cell membrane skeleton: hereditary spherocytosis andhereditary elliptocytosis. Chapter 95 in: The Metabolic Basis of Inherited Disease, 6th ed. Scriver CR et al (editors).McGraw-Hill, 1989.2 The band number refers to the position of migration on SDS-PAGE (see Figure 52–3). The glycophorins are detectedby staining with the periodic acid-Schiff reagent. A number of other components (eg, 4.2 and 4.9) are notlisted. Native spectrin is α 2 β 2 .OutsideSPECTRIN-ANKYRIN-3INTERACTIONInside 3AnkyrinGlycophorinLipid bilayerSPECTRINSELF-ASSOCIATIONAlphaBetaSpectrinSPECTRIN-ACTIN-4.1INTERACTIONActinFigure 52–4. Diagrammatic representation of theinteraction of cytoskeletal proteins with each other andwith certain integral proteins of the membrane of thered blood cell. (Reproduced, with permission, from BeckWS, Tepper RI: Hemolytic anemias III: membrane disorders.In: Hematology, 5th ed. Beck WS [editor]. The MITPress, 1991.)4.1the red blood cell. Approximately 90% of the sialic acidof the red cell membrane is located in this protein. Itstransmembrane segment (23 amino acids) is α-helical.The carboxyl terminal end extends into the cytosol andbinds to protein 4.1, which in turn binds to spectrin.Polymorphism of this protein is the basis of the MNblood group system (see below). Glycophorin A containsbinding sites for influenza virus and for Plasmodiumfalciparum, the cause of one form of malaria. Intriguingly,the function of red blood cells of individualswho lack glycophorin A does not appear to be affected.Spectrin, Ankyrin, & Other PeripheralMembrane Proteins Help Determine theShape & Flexibility of the Red Blood CellThe red blood cell must be able to squeeze throughsome tight spots in the microcirculation during its numerouspassages around the body; the sinusoids of thespleen are of special importance in this regard. For thered cell to be easily and reversibly deformable, its membranemust be both fluid and flexible; it should alsopreserve its biconcave shape, since this facilitates gas exchange.Membrane lipids help determine membranefluidity. Attached to the inner aspect of the membraneof the red blood cell are a number of peripheral cytoskeletalproteins (Table 52–6) that play importantroles in respect to preserving shape and flexibility; thesewill now be described.

RED & WHITE BLOOD CELLS / 617Spectrin is the major protein of the cytoskeleton. Itis composed of two polypeptides: spectrin 1 (α chain)and spectrin 2 (β chain). These chains, measuring approximately100 nm in length, are aligned in an antiparallelmanner and are loosely intertwined, forming adimer. Both chains are made up of segments of 106amino acids that appear to fold into triple-strandedα-helical coils joined by nonhelical segments. Onedimer interacts with another, forming a head-to-headtetramer. The overall shape confers flexibility on theprotein and in turn on the membrane of the red bloodcell. At least four binding sites can be defined in spectrin:(1) for self-association, (2) for ankyrin (bands 2.1,etc), (3) for actin (band 5), and (4) for protein 4.1.Ankyrin is a pyramid-shaped protein that bindsspectrin. In turn, ankyrin binds tightly to band 3, securingattachment of spectrin to the membrane. Ankyrinis sensitive to proteolysis, accounting for the appearanceof bands 2.2, 2.3, and 2.6, all of which are derivedfrom band 2.1.Actin (band 5) exists in red blood cells as short, double-helicalfilaments of F-actin. The tail end of spectrindimers binds to actin. Actin also binds to protein 4.1.Protein 4.1, a globular protein, binds tightly to thetail end of spectrin, near the actin-binding site of thelatter, and thus is part of a protein 4.1-spectrin-actinternary complex. Protein 4.1 also binds to the integralproteins, glycophorins A and C, thereby attaching theternary complex to the membrane. In addition, protein4.1 may interact with certain membrane phospholipids,thus connecting the lipid bilayer to the cytoskeleton.Certain other proteins (4.9, adducin, and tropomyosin)also participate in cytoskeletal assembly.Abnormalities in the Amount or Structureof Spectrin Cause HereditarySpherocytosis & ElliptocytosisHereditary spherocytosis is a genetic disease, transmittedas an autosomal dominant, that affects about 1:5000North Americans. It is characterized by the presence ofspherocytes (spherical red blood cells, with a low surface-to-volumeratio) in the peripheral blood, by a hemolyticanemia, and by splenomegaly. The spherocytesare not as deformable as are normal red blood cells, andthey are subject to destruction in the spleen, thus greatlyshortening their life in the circulation. Hereditary spherocytosisis curable by splenectomy because the spherocytescan persist in the circulation if the spleen is absent.The spherocytes are much more susceptible to osmoticlysis than are normal red blood cells. This is assessedin the osmotic fragility test, in which red bloodcells are exposed in vitro to decreasing concentrationsof NaCl. The physiologic concentration of NaCl is0.85 g/dL. When exposed to a concentration of NaClof 0.5 g/dL, very few normal red blood cells are hemolyzed,whereas approximately 50% of spherocyteswould lyse under these conditions. The explanation isthat the spherocyte, being almost circular, has little potentialextra volume to accommodate additional waterand thus lyses readily when exposed to a slightly lowerosmotic pressure than is normal.One cause of hereditary spherocytosis (Figure 52–5)is a deficiency in the amount of spectrin or abnormalitiesof its structure, so that it no longer tightly binds theother proteins with which it normally interacts. Thisweakens the membrane and leads to the spherocyticshape. Abnormalities of ankyrin and of bands 3 and 4.1are involved in other cases.Hereditary elliptocytosis is a genetic disorder thatis similar to hereditary spherocytosis except that affectedred blood cells assume an elliptic, disk-like shape,recognizable by microscopy. It is also due to abnormalitiesin spectrin; some cases reflect abnormalities of band4.1 or of glycophorin C.THE BIOCHEMICAL BASES OF THEABO BLOOD GROUP SYSTEMHAVE BEEN ESTABLISHEDAt least 21 human blood group systems are recognized,the best known of which are the ABO, Rh (Rhesus),and MN systems. The term “blood group” applies to adefined system of red blood cell antigens (blood groupsubstances) controlled by a genetic locus having a variablenumber of alleles (eg, A, B, and O in the ABO system).The term “blood type” refers to the antigenicphenotype, usually recognized by the use of appropriateMutations in DNA affecting the amount or structureof α or β spectrin or of certain other cytoskeletalproteins (eg, ankyrin, band 3, band 4.1)Weakens interactions among the peripheral andintegral proteins of the red cell membraneWeakens the structure of the red cell membraneAdopts spherocytic shape and is subject todestruction in the spleenHemolytic anemiaFigure 52–5. Summary of the causation of hereditaryspherocytosis (MIM 182900).

618 / CHAPTER 52antibodies. For purposes of blood transfusion, it is particularlyimportant to know the basics of the ABO andRh systems. However, knowledge of blood group systemsis also of biochemical, genetic, immunologic, anthropologic,obstetric, pathologic, and forensic interest.Here, we shall discuss only some key features of theABO system. From a biochemical viewpoint, the majorinterests in the ABO substances have been in isolatingand determining their structures, elucidating theirpathways of biosynthesis, and determining the naturesof the products of the A, B, and O genes.The ABO System Is of Crucial Importancein Blood TransfusionThis system was first discovered by Landsteiner in 1900when investigating the basis of compatible and incompatibletransfusions in humans. The membranes of thered blood cells of most individuals contain one bloodgroup substance of type A, type B, type AB, or type O.Individuals of type A have anti-B antibodies in theirplasma and will thus agglutinate type B or type ABblood. Individuals of type B have anti-A antibodies andwill agglutinate type A or type AB blood. Type ABblood has neither anti-A nor anti-B antibodies and hasbeen designated the universal recipient. Type O bloodhas neither A nor B substances and has been designatedthe universal donor. The explanation of these findingsis related to the fact that the body does not usually produceantibodies to its own constituents. Thus, individualsof type A do not produce antibodies to their ownblood group substance, A, but do possess antibodies tothe foreign blood group substance, B, possibly becausesimilar structures are present in microorganisms towhich the body is exposed early in life. Since individualsof type O have neither A nor B substances, they possessantibodies to both these foreign substances. Theabove description has been simplified considerably; eg,there are two subgroups of type A: A 1 and A 2 .The genes responsible for production of the ABOsubstances are present on the long arm of chromosome9. There are three alleles, two of which arecodominant (A and B) and the third (O) recessive;these ultimately determine the four phenotypic products:the A, B, AB, and O substances.The ABO Substances AreGlycosphingolipids & GlycoproteinsSharing Common Oligosaccharide ChainsThe ABO substances are complex oligosaccharides presentin most cells of the body and in certain secretions.On membranes of red blood cells, the oligosaccharidesthat determine the specific natures of the ABO substancesappear to be mostly present in glycosphingolipids,whereas in secretions the same oligosaccharidesare present in glycoproteins. Their presence in secretionsis determined by a gene designated Se (for secretor),which codes for a specific fucosyl (Fuc)transferase in secretory organs, such as the exocrineglands, but which is not active in red blood cells. Individualsof SeSe or Sese genotypes secrete A or B antigens(or both), whereas individuals of the sese genotype donot secrete A or B substances, but their red blood cellscan express the A and B antigens.H Substance Is the Biosynthetic Precursorof Both the A & B SubstancesThe ABO substances have been isolated and their structuresdetermined; simplified versions, showing onlytheir nonreducing ends, are presented in Figure 52–6.It is important to first appreciate the structure of the Hsubstance, since it is the precursor of both the A and Bsubstances and is the blood group substance found inpersons of type O. H substance itself is formed by theaction of a fucosyltransferase, which catalyzes the additionof the terminal fucose in α1 → 2 linkage ontothe terminal Gal residue of its precursor:GDP -Fuc + Gal-β-R → Fuc-α12, -Gal-β-R + GDPPrecursorH substanceThe H locus codes for this fucosyltransferase. The h alleleof the H locus codes for an inactive fucosyltransferase;therefore, individuals of the hh genotype cannotgenerate H substance, the precursor of the A and Bantigens. Thus, individuals of the hh genotype will havered blood cells of type O, even though they may possessthe enzymes necessary to make the A or B substances(see below).The A Gene Encodes a GalNAc Transferase,the B Gene a Gal Transferase, & the O Genean Inactive ProductIn comparison with H substance (Figure 52–6), A substancecontains an additional GalNAc and B substancean additional Gal, linked as indicated. Anti-A antibodiesare directed to the additional GalNAc residue foundin the A substance, and anti-B antibodies are directedtoward the additional Gal residue found in the B substance.Thus, GalNAc is the immunodominant sugar(ie, the one determining the specificity of the antibodyformed) of blood group A substance, whereas Gal is theimmunodominant sugar of the B substance. In view ofthe structural findings, it is not surprising that A substancecan be synthesized in vitro from O substance in areaction catalyzed by a GalNAc transferase, employingUDP-GalNAc as the sugar donor. Similarly, blood

RED & WHITE BLOOD CELLS / 619Fucα1 2Galβ1 4GlcNAc – RH (or O) substanceGalNAc transferaseGal transferaseFucα1 2Galβ1 4GlcNAc – Rα1 3GalNAcA substanceFucα1 2Galβ1 4GlcNAc – RGalα1 3B substanceFigure 52–6. Diagrammatic representation of the structures of the H, A, and Bblood group substances. R represents a long complex oligosaccharide chain,joined either to ceramide where the substances are glycosphingolipids, or to thepolypeptide backbone of a protein via a serine or threonine residue where thesubstances are glycoproteins. Note that the blood group substances are biantennary;ie, they have two arms, formed at a branch point (not indicated) between theGlcNAc—R, and only one arm of the branch is shown. Thus, the H, A, and B substanceseach contain two of their respective short oligosaccharide chains shownabove. The AB substance contains one type A chain and one type B chain.group B can be synthesized from O substance by theaction of a Gal transferase, employing UDP-Gal. It iscrucial to appreciate that the product of the A gene isthe GalNAc transferase that adds the terminal GalNActo the O substance. Similarly, the product of the B geneis the Gal transferase adding the Gal residue to the Osubstance. Individuals of type AB possess both enzymesand thus have two oligosaccharide chains (Figure52–6), one terminated by a GalNAc and the other by aGal. Individuals of type O apparently synthesize an inactiveprotein, detectable by immunologic means; thus,H substance is their ABO blood group substance.In 1990, a study using cloning and sequencing technologydescribed the nature of the differences betweenthe glycosyltransferase products of the A, B, and Ogenes. A difference of four nucleotides is apparently responsiblefor the distinct specificities of the A and Bglycosyltransferases. On the other hand, the O allele hasa single base-pair mutation, causing a frameshift mutationresulting in a protein lacking transferase activity.HEMOLYTIC ANEMIAS ARE CAUSEDBY ABNORMALITIES OUTSIDE,WITHIN, OR INSIDE THE REDBLOOD CELL MEMBRANECauses outside the membrane include hypersplenism, acondition in which the spleen is enlarged from a varietyof causes and red blood cells become sequestered in it.Immunologic abnormalities (eg, transfusion reactions,the presence in plasma of warm and cold antibodiesthat lyse red blood cells, and unusual sensitivity to complement)also fall in this class, as do toxins released byvarious infectious agents, such as certain bacteria (eg,clostridium). Some snakes release venoms that act tolyse the red cell membrane (eg, via the action of phospholipasesor proteinases).Causes within the membrane include abnormalitiesof proteins. The most important conditions are hereditaryspherocytosis and hereditary elliptocytosis, principallycaused by abnormalities in the amount or structureof spectrin (see above).Causes inside the red blood cell include hemoglobinopathiesand enzymopathies. Sickle cell anemia isthe most important hemoglobinopathy. Abnormalitiesof enzymes in the pentose phosphate pathway and inglycolysis are the most frequent enzymopathies involved,particularly the former. Deficiency of glucose-6-phosphate dehydrogenase is prevalent in certain partsof the world and is a frequent cause of hemolytic anemia(see above). Deficiency of pyruvate kinase is notfrequent, but it is the second commonest enzyme deficiencyresulting in hemolytic anemia; the mechanismappears to be due to impairment of glycolysis, resultingin decreased formation of ATP, affecting various aspectsof membrane integrity.Laboratory investigations that aid in the diagnosis ofhemolytic anemia are listed in Table 52–7.

620 / CHAPTER 52Table 52–7. Laboratory investigations that assistin the diagnosis of hemolytic anemia.General tests and findingsIncreased nonconjugated (indirect) bilirubinShortened red cell survival time as measured by injectionof autologous 51 Cr-labeled red cellsReticulocytosisHemoglobinemiaLow level of plasma haptoglobinSpecific tests and findingsHb electrophoresis (eg, HbS)Red cell enzymes (eg, G6PD or PK deficiency)Osmotic fragility (eg, hereditary spherocytosis)Coombs test 1Cold agglutinins1 The direct Coombs test detects the presence of antibodies onred cells, whereas the indirect test detects the presence of circulatingantibodies to antigens present on red cells.NEUTROPHILS HAVE AN ACTIVEMETABOLISM & CONTAIN SEVERALUNIQUE ENZYMES & PROTEINSThe major biochemical features of neutrophils are summarizedin Table 52–8. Prominent features are activeaerobic glycolysis, active pentose phosphate pathway,moderately active oxidative phosphorylation (becausemitochondria are relatively sparse), and a high contentof lysosomal enzymes. Many of the enzymes listed inTable 52–4 are also of importance in the oxidative metabolismof neutrophils (see below). Table 52–9 summarizesthe functions of some proteins that are relativelyunique to neutrophils.Neutrophils Are Key Players in the Body’sDefense Against Bacterial InfectionNeutrophils are motile phagocytic cells that play a keyrole in acute inflammation. When bacteria enter tissues,a number of phenomena result that are collectivelyTable 52–8. Summary of major biochemicalfeatures of neutrophils.• Active glycolysis• Active pentose phosphate pathway• Moderate oxidative phosphorylation• Rich in lysosomes and their degradative enzymes• Contain certain unique enzymes (eg, myeloperoxidase andNADPH-oxidase) and proteins• Contain CD 11/CD18 integrins in plasma membraneknown as the “acute inflammatory response.” They include(1) increase of vascular permeability, (2) entry ofactivated neutrophils into the tissues, (3) activation ofplatelets, and (4) spontaneous subsidence (resolution) ifthe invading microorganisms have been dealt with successfully.A variety of molecules are released from cells andplasma proteins during acute inflammation whose netoverall effect is to increase vascular permeability, resultingin tissue edema (Table 52–10).In acute inflammation, neutrophils are recruited fromthe bloodstream into the tissues to help eliminate the foreigninvaders. The neutrophils are attracted into the tissuesby chemotactic factors, including complementfragment C5a, small peptides derived from bacteria (eg,N-formyl-methionyl-leucyl-phenylalanine), and a numberof leukotrienes. To reach the tissues, circulating neutrophilsmust pass through the capillaries. To achievethis, they marginate along the vessel walls and then adhereto endothelial (lining) cells of the capillaries.Integrins Mediate Adhesion of Neutrophilsto Endothelial CellsAdhesion of neutrophils to endothelial cells employsspecific adhesive proteins (integrins) located on theirsurface and also specific receptor proteins in the endothelialcells. (See also the discussion of selectins inChapter 47.)The integrins are a superfamily of surface proteinspresent on a wide variety of cells. They are involved inthe adhesion of cells to other cells or to specific componentsof the extracellular matrix. They are heterodimers,containing an α and a β subunit linked noncovalently.The subunits contain extracellular, transmembrane,and intracellular segments. The extracellular segmentsbind to a variety of ligands such as specific proteins ofthe extracellular matrix and of the surfaces of othercells. These ligands often contain Arg-Gly-Asp (R-G-D) sequences. The intracellular domains bind to variousproteins of the cytoskeleton, such as actin and vinculin.The integrins are proteins that link the outsidesof cells to their insides, thereby helping to integrate responsesof cells (eg, movement, phagocytosis) tochanges in the environment.Three subfamilies of integrins were recognized initially.Members of each subfamily were distinguishedby containing a common β subunit, but they differedin their α subunits. However, more than three β subunitshave now been identified, and the classification ofintegrins has become rather complex. Some integrins ofspecific interest with regard to neutrophils are listed inTable 52–11.A deficiency of the β 2 subunit (also designatedCD18) of LFA-1 and of two related integrins found in

Table 52–9. Some important enzymes and proteins of neutrophils. 1RED & WHITE BLOOD CELLS / 621Enzyme or Protein Reaction Catalyzed or Function CommentMyeloperoxidase H 2 O 2 +X − (halide) + H + → HOX + H 2 O Responsible for the green color of pus(MPO) (where X − = Cl − , HOX = hypochlorous Genetic deficiency can cause recurrent infectionsacid)NADPH-oxidase 2O 2 + NADPH → 2O 2− ⋅ + NADP + H + Key component of the respiratory burstDeficient in chronic granulomatous diseaseLysozyme Hydrolyzes link between N-acetylmura- Abundant in macrophagesmic acid and N-acetyl-D-glucosaminefound in certain bacterial cell wallsDefensins Basic antibiotic peptides of 20–33 amino Apparently kill bacteria by causing membraneacidsdamageLactoferrin Iron-binding protein May inhibit growth of certain bacteria by bindingiron and may be involved in regulation of proliferationof myeloid cellsCD11a/CD18, CD11b/CD18, Adhesion molecules (members of the Deficient in leukocyte adhesion deficiency type ICD11c/CD18 2 integrin family) (MIM 116920)Receptors for Fc fragments of IgGs Bind Fc fragments of IgG molecules Target antigen-antibody complexes to myeloidand lymphoid cells, eliciting phagocytosis andother responses1 The expression of many of these molecules has been studied during various stages of differentiation of normal neutrophils and also ofcorresponding leukemic cells employing molecular biology techniques (eg, measurements of their specific mRNAs). For the majority,cDNAs have been isolated and sequenced, amino acid sequences deduced, genes have been localized to specific chromosomal locations,and exons and intron sequences have been defined. Some important proteinases of neutrophils are listed in Table 52–12.2 CD = cluster of differentiation. This refers to a uniform system of nomenclature that has been adopted to name surface markers of leukocytes.A specific surface protein (marker) that identifies a particular lineage or differentiation stage of leukocytes and that is recognized bya group of monoclonal antibodies is called a member of a cluster of differentiation. The system is particularly helpful in categorizing subclassesof lymphocytes. Many CD antigens are involved in cell-cell interactions, adhesion, and transmembrane signaling.neutrophils and macrophages, Mac-1 (CD11b/CD18)and p150,95 (CD11c/CD18), causes type 1 leukocyteadhesion deficiency, a disease characterized by recurrentbacterial and fungal infections. Among various resultsof this deficiency, the adhesion of affected whiteblood cells to endothelial cells is diminished, and lowernumbers of neutrophils thus enter the tissues to combatinfection.Once having passed through the walls of smallblood vessels, the neutrophils migrate toward the highestconcentrations of the chemotactic factors, encounterthe invading bacteria, and attempt to attack and destroythem. The neutrophils must be activated in orderto turn on many of the metabolic processes involved inphagocytosis and killing of bacteria.Activation of Neutrophils Is Similarto Activation of Platelets& Involves Hydrolysis ofPhosphatidylinositol BisphosphateThe mechanisms involved in platelet activation are discussedin Chapter 51 (see Figure 51–8). The process involvesinteraction of the stimulus (eg, thrombin) with areceptor, activation of G proteins, stimulation of phospholipaseC, and liberation from phosphatidylinositolTable 52–10. Sources of biomolecules with vasoactive properties involved in acute inflammation.Mast Cells and Basophils Platelets Neutrophils Plasma ProteinsHistamine Serotonin Platelet-activating factor (PAF) C3a, C4a, and C5a from the complement systemEicosanoids (various prostaglan- Bradykinin and fibrin split products from the coagudinsand leukotrienes)lation system

622 / CHAPTER 52Table 52–11. Examples of integrins that are important in the function of neutrophils, of other whiteblood cells, and of platelets. 1Integrin Cell Subunit Ligand FunctionVLA-1 (CD49a) WBCs, others α1β1 Collagen, laminin Cell-ECM adhesionVLA-5 (CD49e) WBCs, others α5β1 Fibronectin Cell-ECM adhesionVLA-6 (CD49f) WBCs, others α6β1 Laminin Cell-ECM adhesionLFA-1 (CD11a) WBCs αLβ2 ICAM-1 Adhesion of WBCsGlycoprotein llb/llla Platelets αllbβ3 ICAM-2 Platelet adhesion and aggregationFibrinogen, fibronectin, von Willebrandfactor1 LFA-1, lymphocyte function-associated antigen 1; VLA, very late antigen; CD, cluster of differentiation; ICAM, intercellular adhesion molecule;ECM, extracellular matrix. A deficiency of LFA-1 and related integrins is found in type I leukocyte adhesion deficiency (MIM 116290).A deficiency of platelet glycoprotein llb/llla complex is found in Glanzmann thrombasthenia (MIM 273800), a condition characterized by ahistory of bleeding, a normal platelet count, and abnormal clot retraction. These findings illustrate how fundamental knowledge of cellsurface adhesion proteins is shedding light on the causation of a number of diseases.bisphosphate of inositol triphosphate and diacylglycerol.These two second messengers result in an elevationof intracellular Ca 2+ and activation of protein kinaseC. In addition, activation of phospholipase A 2produces arachidonic acid that can be converted to avariety of biologically active eicosanoids.The process of activation of neutrophils is essentiallysimilar. They are activated, via specific receptors, by interactionwith bacteria, binding of chemotactic factors,or antibody-antigen complexes. The resultant rise in intracellularCa 2+ affects many processes in neutrophils,such as assembly of microtubules and the actin-myosinsystem. These processes are respectively involved in secretionof contents of granules and in motility, whichenables neutrophils to seek out the invaders. The activatedneutrophils are now ready to destroy the invadersby mechanisms that include production of active derivativesof oxygen.The Respiratory Burst of PhagocyticCells Involves NADPH Oxidase& Helps Kill BacteriaWhen neutrophils and other phagocytic cells engulfbacteria, they exhibit a rapid increase in oxygen consumptionknown as the respiratory burst. This phenomenonreflects the rapid utilization of oxygen (followinga lag of 15–60 seconds) and production from itof large amounts of reactive derivatives, such as O 2−⋅,H 2 O 2 , OH • , and OCl − (hypochlorite ion). Some ofthese products are potent microbicidal agents.The electron transport chain system responsiblefor the respiratory burst (named NADPH oxidase) iscomposed of several components. One is cytochromeb 558 , located in the plasma membrane; it is a heterodimer,containing two polypeptides of 91 kDa and22 kDa. When the system is activated (see below), twocytoplasmic polypeptides of 47 kDa and 67 kDa are recruitedto the plasma membrane and, together with cytochromeb 558 , form the NADPH oxidase responsiblefor the respiratory burst. The reaction catalyzed byNADPH oxidase, involving formation of superoxideanion, is shown in Table 52–4 (reaction 2). This systemcatalyzes the one-electron reduction of oxygen to superoxideanion. The NADPH is generated mainly by thepentose phosphate cycle, whose activity increases markedlyduring phagocytosis.The above reaction is followed by the spontaneousproduction (by spontaneous dismutation) of hydrogenperoxide from two molecules of superoxide:O −⋅ 2 + O −⋅ +2 + 2 H → H2O2 + O2The superoxide ion is discharged to the outside ofthe cell or into phagolysosomes, where it encounters ingestedbacteria. Killing of bacteria within phagolysosomesappears to depend on the combined action ofelevated pH, superoxide ion, or further oxygen derivatives(H 2 O 2 , OH • , and HOCl [hypochlorous acid; seebelow]) and on the action of certain bactericidal peptides(defensins) and other proteins (eg, cathepsin G andcertain cationic proteins) present in phagocytic cells.Any superoxide that enters the cytosol of the phagocyticcell is converted to H 2 O 2 by the action of superoxidedismutase, which catalyzes the same reaction as thespontaneous dismutation shown above. In turn, H 2 O 2 isused by myeloperoxidase (see below) or disposed of bythe action of glutathione peroxidase or catalase.NADPH oxidase is inactive in resting phagocyticcells and is activated upon contact with various ligands(complement fragment C5a, chemotactic peptides, etc)

RED & WHITE BLOOD CELLS / 623with receptors in the plasma membrane. The events resultingin activation of the oxidase system have beenmuch studied and are similar to those described abovefor the process of activation of neutrophils. They involveG proteins, activation of phospholipase C, andgeneration of inositol 1,4,5-triphosphate (IP 3 ). Thelast mediates a transient increase in the level of cytosolicCa 2+ , which is essential for induction of the respiratoryburst. Diacylglycerol is also generated and induces thetranslocation of protein kinase C into the plasma membranefrom the cytosol, where it catalyzes the phosphorylationof various proteins, some of which are componentsof the oxidase system. A second pathway ofactivation not involving Ca 2+ also operates.Mutations in the Genes for Componentsof the NADPH Oxidase System CauseChronic Granulomatous DiseaseThe importance of the NADPH oxidase system wasclearly shown when it was observed that the respiratoryburst was defective in chronic granulomatous disease, arelatively uncommon condition characterized by recurrentinfections and widespread granulomas (chronic inflammatorylesions) in the skin, lungs, and lymphnodes. The granulomas form as attempts to wall offbacteria that have not been killed, owing to genetic deficienciesin the NADPH oxidase system. The disorderis due to mutations in the genes encoding the fourpolypeptides that constitute the NADPH oxidase system.Some patients have responded to treatment withgamma interferon, which may increase transcription ofthe 91-kDa component if it is affected. The probablesequence of events involved in the causation of chronicgranulomatous disease is shown in Figure 52–7.Neutrophils Contain Myeloperoxidase,Which Catalyzes the Productionof Chlorinated OxidantsThe enzyme myeloperoxidase, present in large amountsin neutrophil granules and responsible for the green colorof pus, can act on H 2 O 2 to produce hypohalous acids:MYELOPEROXIDASEH 2 O 2 + X — + H +HOX + H 2 O(X — = Cl — , Br — , I — or SCN — ; HOCl = hypochlorous acid)The H 2 O 2 used as substrate is generated by theNADPH oxidase system. Cl – is the halide usually employed,since it is present in relatively high concentrationin plasma and body fluids. HOCl, the active ingredientof household liquid bleach, is a powerful oxidantand is highly microbicidal. When applied to normal tissues,its potential for causing damage is diminished be-Mutations in genes for the polypeptide componentsof the NADPH oxidase systemDiminished production of superoxide ionand other active derivatives of oxygenDiminished killing of certain bacteriaRecurrent infections and formation of tissuegranulomas in order to wall off surviving bacteriaFigure 52–7. Simplified scheme of the sequence ofevents involved in the causation of chronic granulomatousdisease (MIM 306400). Mutations in any of thegenes for the four polypeptides involved (two are componentsof cytochrome b 558 and two are derived fromthe cytoplasm) can cause the disease. The polypeptideof 91 kDa is encoded by a gene in the X chromosome;approximately 60% of cases of chronic granulomatousdisease are X-linked, with the remainder being inheritedin an autosomal recessive fashion.cause it reacts with primary or secondary amines presentin neutrophils and tissues to produce various nitrogen-chlorinederivatives; these chloramines are also oxidants,though less powerful than HOCl, and act asmicrobicidal agents (eg, in sterilizing wounds) withoutcausing tissue damage.The Proteinases of NeutrophilsCan Cause Serious Tissue DamageIf Their Actions Are Not CheckedNeutrophils contain a number of proteinases (Table52–12) that can hydrolyze elastin, various types of collagens,and other proteins present in the extracellularmatrix. Such enzymatic action, if allowed to proceedunopposed, can result in serious damage to tissues.Most of these proteinases are lysosomal enzymes andexist mainly as inactive precursors in normal neutrophils.Small amounts of these enzymes are releasedinto normal tissues, with the amounts increasingmarkedly during inflammation. The activities of elastaseand other proteinases are normally kept in checkby a number of antiproteinases (also listed in Table52–12) present in plasma and the extracellular fluid.Each of them can combine—usually forming a noncovalentcomplex—with one or more specific proteinasesand thus cause inhibition. In Chapter 50 it was shownthat a genetic deficiency of 1 -antiproteinase inhibitor(α 1 -antitrypsin) permits elastase to act unopposedand digest pulmonary tissue, thereby participating in

624 / CHAPTER 52Table 52–12. Proteinases of neutrophils andantiproteinases of plasma and tissues. 1ProteinasesElastaseCollagenaseGelatinaseCathepsin GPlasminogen activatorAntiproteinasesα 1 -Antiproteinase (α 1 -antitrypsin)α 2 -MacroglobulinSecretory leukoproteinase inhibitorα 1 -AntichymotrypsinPlasminogen activator inhibitor–1Tissue inhibitor of metalloproteinase1 The table lists some of the important proteinases of neutrophilsand some of the proteins that can inhibit their actions. Most ofthe proteinases listed exist inside neutrophils as precursors. Plasminogenactivator is not a proteinase, but it is included because itinfluences the activity of plasmin, which is a proteinase. The proteinaseslisted can digest many proteins of the extracellular matrix,causing tissue damage. The overall balance of proteinase:antiproteinaseaction can be altered by activating the precursors ofthe proteinases, or by inactivating the antiproteinases. The lattercan be caused by proteolytic degradation or chemical modification,eg, Met-358 of α 1 -antiproteinase inhibitor is oxidized by cigarettesmoke.the causation of emphysema. 2 -Macroglobulin is aplasma protein that plays an important role in thebody’s defense against excessive action of proteases; itcombines with and thus neutralizes the activities of anumber of important proteases (Chapter 50).When increased amounts of chlorinated oxidants areformed during inflammation, they affect the proteinase:antiproteinaseequilibrium, tilting it in favor ofthe former. For instance, certain of the proteinaseslisted in Table 52–12 are activated by HOCl, whereascertain of the antiproteinases are inactivated by thiscompound. In addition, the tissue inhibitor of metalloproteinasesand α 1 -antichymotrypsin can be hydrolyzedby activated elastase, and α 1 -antiproteinase inhibitorcan be hydrolyzed by activated collagenase and gelatinase.In most circumstances, an appropriate balanceof proteinases and antiproteinases is achieved. However,in certain instances, such as in the lung whenα 1 -antiproteinase inhibitor is deficient or when largeamounts of neutrophils accumulate in tissues because ofinadequate drainage, considerable tissue damage can resultfrom the unopposed action of proteinases.RECOMBINANT DNA TECHNOLOGYHAS HAD A PROFOUND IMPACTON HEMATOLOGYRecombinant DNA technology has had a major impacton many aspects of hematology. The bases of the thalassemiasand of many disorders of coagulation (Chapter51) have been greatly clarified by investigationsusing cloning and sequencing. The study of oncogenesand chromosomal translocations has advanced understandingof the leukemias. As discussed above, cloningtechniques have made available therapeutic amounts oferythropoietin and other growth factors. Deficiency ofadenosine deaminase, which affects lymphocytes particularly,is the first disease to be treated by gene therapy.Like many other areas of biology and medicine, hematologyhas been and will continue to be revolutionizedby this technology.SUMMARY• The red blood cell is simple in terms of its structureand function, consisting principally of a concentratedsolution of hemoglobin surrounded by a membrane.• The production of red cells is regulated by erythropoietin,whereas other growth factors (eg, granulocyte-and granulocyte-macrophage colony-stimulatingfactors) regulate the production of white bloodcells.• The red cell contains a battery of cytosolic enzymes,such as superoxide dismutase, catalase, and glutathioneperoxidase, to dispose of powerful oxidantsgenerated during its metabolism.• Genetically determined deficiency of the activity ofglucose-6-phosphate dehydrogenase, which producesNADPH, is an important cause of hemolytic anemia.• Methemoglobin is unable to transport oxygen; bothgenetic and acquired causes of methemoglobinemiaare recognized. Considerable information has accumulatedconcerning the proteins and lipids of the redcell membrane. A number of cytoskeletal proteins,such as spectrin, ankyrin, and actin, interact with specificintegral membrane proteins to help regulate theshape and flexibility of the membrane.• Deficiency of spectrin results in hereditary spherocytosis,another important cause of hemolytic anemia.• The ABO blood group substances in the red cellmembrane are complex glycosphingolipids; the immunodominantsugar of A substance is N-acetylgalactosamine,whereas that of the B substance isgalactose.• Neutrophils play a major role in the body’s defensemechanisms. Integrins on their surface membranesdetermine specific interactions with various cell andtissue components.• Leukocytes are activated on exposure to bacteria andother stimuli; NADPH oxidase plays a key role in theprocess of activation (the respiratory burst). Mutationsin this enzyme and associated proteins causechronic granulomatous disease.

RED & WHITE BLOOD CELLS / 625• The proteinases of neutrophils can digest many tissueproteins; normally, this is kept in check by a batteryof antiproteinases. However, this defense mechanismcan be overcome in certain circumstances, resultingin extensive tissue damage.• The application of recombinant DNA technology isrevolutionizing the field of hematology.REFERENCESBorregaard N, Cowland JB: Granules of the human neutrophilicpolymorphonuclear leukocyte. Blood 1997;89:3503.Daniels G: A century of human blood groups. Wien Klin Wochenschr2001;113:781.Goodnough LT et al: Erythropoietin, iron, and erythropoiesis.Blood 2000;96:823.Hirono A et al: Pyruvate kinase deficiency and other enzymopathiesof the erythrocyte. In: The Metabolic and MolecularBases of Inherited Disease, 8th ed. Scriver CR et al (editors).McGraw-Hill, 2001.Israels LG, Israels ED: Mechanisms in Hematology, 2nd ed. UnivManitoba Press, 1997. (Includes an excellent interactive CD.)Jaffe ER, Hultquist DE: Cytochrome b 5 reductase deficiency andenzymopenic hereditary methemoglobinemia. In: The Metabolicand Molecular Bases of Inherited Disease, 8th ed. ScriverCR et al (editors). McGraw-Hill, 2001.Lekstrom-Hunes JA, Gallin JI: Immunodeficiency diseases causedby defects in granulocytes. N Engl J Med 2000;343:1703.Luzzato L et al: Glucose-6-phosphate dehydrogenase. In: TheMetabolic and Molecular Bases of Inherited Disease, 8th ed.Scriver CR et al (editors). McGraw-Hill, 2001.Rosse WF et al: New Views of Sickle Cell Disease Pathophysiologyand Treatment. The American Society of Hematology.www.asheducationbook.orgTse WT, Lux SE: Hereditary spherocytosis and hereditary elliptocytosis.In: The Molecular Bases of Inherited Disease, 8th ed.Scriver CR et al (editors). McGraw-Hill, 2001.Weatherall DJ et al: The hemoglobinopathies. In: The Metabolicand Molecular Bases of Inherited Disease, 8th ed. Scriver CR etal (editors). McGraw-Hill, 2001.

Metabolism of Xenobiotics 53Robert K. Murray, MD, PhDBIOMEDICAL IMPORTANCEIncreasingly, humans are subjected to exposure to variousforeign chemicals (xenobiotics)—drugs, food additives,pollutants, etc. The situation is well summarizedin the following quotation from Rachel Carson: “Ascrude a weapon as the cave man’s club, the chemicalbarrage has been hurled against the fabric of life.” Understandinghow xenobiotics are handled at the cellularlevel is important in learning how to cope with thechemical onslaught.Knowledge of the metabolism of xenobiotics is basicto a rational understanding of pharmacology and therapeutics,pharmacy, toxicology, management of cancer,and drug addiction. All these areas involve administrationof, or exposure to, xenobiotics.HUMANS ENCOUNTER THOUSANDSOF XENOBIOTICS THAT MUST BEMETABOLIZED BEFORE BEING EXCRETEDA xenobiotic (Gk xenos “stranger”) is a compound thatis foreign to the body. The principal classes of xenobioticsof medical relevance are drugs, chemical carcinogens,and various compounds that have found their wayinto our environment by one route or another, such aspolychlorinated biphenyls (PCBs) and certain insecticides.More than 200,000 manufactured environmentalchemicals exist. Most of these compounds are subject tometabolism (chemical alteration) in the human body,with the liver being the main organ involved; occasionally,a xenobiotic may be excreted unchanged. At least30 different enzymes catalyze reactions involved inxenobiotic metabolism; however, this chapter will onlycover a selected group of them.It is convenient to consider the metabolism of xenobioticsin two phases. In phase 1, the major reaction involvedis hydroxylation, catalyzed by members of aclass of enzymes referred to as monooxygenases or cytochromeP450s. Hydroxylation may terminate the actionof a drug, though this is not always the case. In additionto hydroxylation, these enzymes catalyze a widerange of reactions, including those involving deamination,dehalogenation, desulfuration, epoxidation, peroxygenation,and reduction. Reactions involving hydrolysis(eg, catalyzed by esterases) and certain othernon-P450-catalyzed reactions also occur in phase 1.626In phase 2, the hydroxylated or other compoundsproduced in phase 1 are converted by specific enzymesto various polar metabolites by conjugation with glucuronicacid, sulfate, acetate, glutathione, or certainamino acids, or by methylation.The overall purpose of the two phases of metabolismof xenobiotics is to increase their water solubility(polarity) and thus excretion from the body. Very hydrophobicxenobiotics would persist in adipose tissuealmost indefinitely if they were not converted to morepolar forms. In certain cases, phase 1 metabolic reactionsconvert xenobiotics from inactive to biologicallyactive compounds. In these instances, the originalxenobiotics are referred to as “prodrugs” or “procarcinogens.”In other cases, additional phase 1 reactions(eg, further hydroxylation reactions) convert the activecompounds to less active or inactive forms prior to conjugation.In yet other cases, it is the conjugation reactionsthemselves that convert the active products ofphase 1 reactions to less active or inactive species, whichare subsequently excreted in the urine or bile. In a veryfew cases, conjugation may actually increase the biologicactivity of a xenobiotic.The term “detoxification” is sometimes used formany of the reactions involved in the metabolism ofxenobiotics. However, the term is not always appropriatebecause, as mentioned above, in some cases the reactionsto which xenobiotics are subject actually increasetheir biologic activity and toxicity.ISOFORMS OF CYTOCHROMEP450 HYDROXYLATE A MYRIADOF XENOBIOTICS IN PHASE 1OF THEIR METABOLISMHydroxylation is the chief reaction involved in phase1. The responsible enzymes are called monooxygenasesor cytochrome P450s; the human genome encodes atleast 14 families of these enzymes. Estimates of thenumber of distinct cytochrome P450s in human tissuesrange from approximately 35 to 60. The reaction catalyzedby a monooxygenase (cytochrome P450) is asfollows:+RH + O2 + NADPH + H → R — OH + H2O + NADP

METABOLISM OF XENOBIOTICS / 627RH above can represent a very wide variety of xenobiotics,including drugs, carcinogens, pesticides, petroleumproducts, and pollutants (such as a mixture ofPCBs). In addition, endogenous compounds, such ascertain steroids, eicosanoids, fatty acids, and retinoids,are also substrates. The substrates are generally lipophilicand are rendered more hydrophilic by hydroxylation.Cytochrome P450 is considered the most versatilebiocatalyst known. The actual reaction mechanism iscomplex and has been briefly described previously (Figure11–6). It has been shown by the use of 18 O 2 thatone atom of oxygen enters R⎯OH and one atom enterswater. This dual fate of the oxygen accounts for theformer naming of monooxygenases as “mixedfunctionoxidases.” The reaction catalyzed by cytochromeP450 can also be represented as follows:Reduced cytochrome P450 Oxidized cytochrome P450RH + O 2 → R—OH + H 2 OThe major monooxygenases in the endoplasmic reticulumare cytochrome P450s—so named because the enzymewas discovered when it was noted that preparationsof microsomes that had been chemically reducedand then exposed to carbon monoxide exhibited a distinctpeak at 450 nm. Among reasons that this enzymeis important is the fact that approximately 50% of thedrugs humans ingest are metabolized by isoforms of cytochromeP450; these enzymes also act on various carcinogensand pollutants.Isoforms of Cytochrome P450 Make Up aSuperfamily of Heme-Containing EnzymesThe following are important points concerning cytochromeP450s.(1) Because of the large number of isoforms (about150) that have been discovered, it became important tohave a systematic nomenclature for isoforms of P450and for their genes. This is now available and in wideuse and is based on structural homology. The abbreviatedroot symbol CYP denotes a cytochrome P450.This is followed by an Arabic number designating thefamily; cytochrome P450s are included in the samefamily if they exhibit 40% or more sequence identity.The Arabic number is followed by a capital letter indicatingthe subfamily, if two or more members exist;P450s are in the same subfamily if they exhibit greaterthan 55% sequence identity. The individual P450sare then arbitrarily assigned Arabic numerals. Thus,CYP1A1 denotes a cytochrome P450 that is a memberof family 1 and subfamily A and is the first individualmember of that subfamily. The nomenclature for thegenes encoding cytochrome P450s is identical to thatdescribed above except that italics are used; thus, thegene encoding CYP1A1 is CYP1A1.(2) Like hemoglobin, they are hemoproteins.(3) They are widely distributed across species. Bacteriapossess cytochrome P450s, and P450 cam (involvedin the metabolism of camphor) of Pseudomonas putidais the only P450 isoform whose crystal structure hasbeen established.(4) They are present in highest amount in liver andsmall intestine but are probably present in all tissues.In liver and most other tissues, they are present mainlyin the membranes of the smooth endoplasmic reticulum,which constitute part of the microsomal fractionwhen tissue is subjected to subcellular fractionation. Inhepatic microsomes, cytochrome P450s can compriseas much as 20% of the total protein. P450s are foundin most tissues, though often in low amounts comparedwith liver. In the adrenal, they are found in mitochondriaas well as in the endoplasmic reticulum; the varioushydroxylases present in that organ play an importantrole in cholesterol and steroid biosynthesis. Themitochondrial cytochrome P450 system differs fromthe microsomal system in that it uses an NADPHlinkedflavoprotein, adrenodoxin reductase, and anonheme iron-sulfur protein, adrenodoxin. In addition,the specific P450 isoforms involved in steroidbiosynthesis are generally much more restricted in theirsubstrate specificity.(5) At least six isoforms of cytochrome P450 arepresent in the endoplasmic reticulum of human liver,each with wide and somewhat overlapping substratespecificities and acting on both xenobiotics and endogenouscompounds. The genes for many isoforms ofP450 (from both humans and animals such as the rat)have been isolated and studied in detail in recent years.(6) NADPH, not NADH, is involved in the reactionmechanism of cytochrome P450. The enzyme thatuses NADPH to yield the reduced cytochrome P450,shown at the left-hand side of the above equation, iscalled NADPH-cytochrome P450 reductase. Electronsare transferred from NADPH to NADPHcytochromeP450 reductase and then to cytochromeP450. This leads to the reductive activation of molecularoxygen, and one atom of oxygen is subsequentlyinserted into the substrate. Cytochrome b 5 , anotherhemoprotein found in the membranes of the smoothendoplasmic reticulum (Chapter 11), may be involvedas an electron donor in some cases.(7) Lipids are also components of the cytochromeP450 system. The preferred lipid is phosphatidylcholine,which is the major lipid found in membranesof the endoplasmic reticulum.(8) Most isoforms of cytochrome P450 are inducible.For instance, the administration of phenobarbitalor of many other drugs causes hypertrophy of the

628 / CHAPTER 53smooth endoplasmic reticulum and a three- to fourfoldincrease in the amount of cytochrome P450 within 4–5days. The mechanism of induction has been studied extensivelyand in most cases involves increased transcriptionof mRNA for cytochrome P450. However, certaincases of induction involve stabilization of mRNA, enzymestabilization, or other mechanisms (eg, an effecton translation).Induction of cytochrome P450 has important clinicalimplications, since it is a biochemical mechanism ofdrug interaction. A drug interaction has occurredwhen the effects of one drug are altered by prior, concurrent,or later administration of another. As an illustration,consider the situation when a patient is takingthe anticoagulant warfarin to prevent blood clotting.This drug is metabolized by CYP2C9. Concomitantly,the patient is started on phenobarbital (an inducer ofthis P450) to treat a certain type of epilepsy, but thedose of warfarin is not changed. After 5 days or so, thelevel of CYP2C9 in the patient’s liver will be elevatedthree- to fourfold. This in turn means that warfarin willbe metabolized much more quickly than before, and itsdosage will have become inadequate. Therefore, thedose must be increased if warfarin is to be therapeuticallyeffective. To pursue this example further, a problemcould arise later on if the phenobarbital is discontinuedbut the increased dosage of warfarin stays thesame. The patient will be at risk of bleeding, since thehigh dose of warfarin will be even more active than before,because the level of CYP2C9 will decline oncephenobarbital has been stopped.Another example of enzyme induction involvesCYP2E1, which is induced by consumption ofethanol. This is a matter for concern, because thisP450 metabolizes certain widely used solvents and alsocomponents found in tobacco smoke, many of whichare established carcinogens. Thus, if the activity ofCYP2E1 is elevated by induction, this may increase therisk of carcinogenicity developing from exposure tosuch compounds.(9) Certain isoforms of cytochrome P450 (eg,CYP1A1) are particularly involved in the metabolism ofpolycyclic aromatic hydrocarbons (PAHs) and relatedmolecules; for this reason they were formerly called aromatichydrocarbon hydroxylases (AHHs). This enzymeis important in the metabolism of PAHs and in carcinogenesisproduced by these agents. For example, inthe lung it may be involved in the conversion of inactivePAHs (procarcinogens), inhaled by smoking, to activecarcinogens by hydroxylation reactions. Smokershave higher levels of this enzyme in some of their cellsand tissues than do nonsmokers. Some reports have indicatedthat the activity of this enzyme may be elevated(induced) in the placenta of a woman who smokes, thuspotentially altering the quantities of metabolites ofPAHs (some of which could be harmful) to which thefetus is exposed.(10) Certain cytochrome P450s exist in polymorphicforms (genetic isoforms), some of which exhibitlow catalytic activity. These observations are one importantexplanation for the variations in drug responsesnoted among many patients. One P450 exhibitingpolymorphism is CYP2D6, which is involved in themetabolism of debrisoquin (an antihypertensive drug;see Table 53–2) and sparteine (an antiarrhythmic andoxytocic drug). Certain polymorphisms of CYP2D6cause poor metabolism of these and a variety of otherdrugs so that they can accumulate in the body, resultingin untoward consequences. Another interesting polymorphismis that of CYP2A6, which is involved in themetabolism of nicotine to conitine. Three CYP2A6 alleleshave been identified: a wild type and two null orinactive alleles. It has been reported that individualswith the null alleles, who have impaired metabolism ofnicotine, are apparently protected against becoming tobacco-dependentsmokers (Table 53–2). These individualssmoke less, presumably because their blood andbrain concentrations of nicotine remain elevated longerthan those of individuals with the wild-type allele. Ithas been speculated that inhibiting CYP2A6 may be anovel way to help prevent and to treat smoking.Table 53–1 summarizes some principal features ofcytochrome P450s.CONJUGATION REACTIONS PREPAREXENOBIOTICS FOR EXCRETION INPHASE 2 OF THEIR METABOLISMIn phase 1 reactions, xenobiotics are generally convertedto more polar, hydroxylated derivatives. In phase2 reactions, these derivatives are conjugated with moleculessuch as glucuronic acid, sulfate, or glutathione.This renders them even more water-soluble, and theyare eventually excreted in the urine or bile.Five Types of Phase 2 ReactionsAre Described HereA. GLUCURONIDATIONThe glucuronidation of bilirubin is discussed in Chapter32; the reactions whereby xenobiotics are glucuronidatedare essentially similar. UDP-glucuronicacid is the glucuronyl donor, and a variety of glucuronosyltransferases,present in both the endoplasmicreticulum and cytosol, are the catalysts. Molecules suchas 2-acetylaminofluorene (a carcinogen), aniline, benzoicacid, meprobamate (a tranquilizer), phenol, and

METABOLISM OF XENOBIOTICS / 629Table 53–1. Some properties of humancytochrome P450s.• Involved in phase I of the metabolism of innumerablexenobiotics, including perhaps 50% of the drugs administeredto humans• Involved in the metabolism of many endogenous compounds(eg, steroids)• All are hemoproteins• Often exhibit broad substrate specificity, thus acting onmany compounds; consequently, different P450s may catalyzeformation of the same product• Extremely versatile catalysts, perhaps catalyzing about 60types of reactions• However, basically they catalyze reactions involving introductionof one atom of oxygen into the substrate and oneinto water• Their hydroxylated products are more water-soluble thantheir generally lipophilic substrates, facilitating excretion• Liver contains highest amounts, but found in most if notall tissues, including small intestine, brain, and lung• Located in the smooth endoplasmic reticulum or in mitochondria(steroidogenic hormones)• In some cases, their products are mutagenic or carcinogenic• Many have a molecular mass of about 55 kDa• Many are inducible, resulting in one cause of drug interactions• Many are inhibited by various drugs or their metabolicproducts, providing another cause of drug interactions• Some exhibit genetic polymorphisms, which can result inatypical drug metabolism• Their activities may be altered in diseased tissues (eg, cirrhosis),affecting drug metabolism• Genotyping the P450 profile of patients (eg, to detectpolymorphisms) may in the future permit individualizationof drug therapymany steroids are excreted as glucuronides. The glucuronidemay be attached to oxygen, nitrogen, or sulfurgroups of the substrates. Glucuronidation is probablythe most frequent conjugation reaction.B. SULFATIONSome alcohols, arylamines, and phenols are sulfated.The sulfate donor in these and other biologic sulfationreactions (eg, sulfation of steroids, glycosaminoglycans,glycolipids, and glycoproteins) is adenosine 3-phosphate-5-phosphosulfate(PAPS) (Chapter 24); thiscompound is called “active sulfate.”C. CONJUGATION WITH GLUTATHIONEGlutathione (γ-glutamyl-cysteinylglycine) is a tripeptideconsisting of glutamic acid, cysteine, and glycine(Figure 3–3). Glutathione is commonly abbreviatedGSH (because of the sulfhydryl group of its cysteine,which is the business part of the molecule). A numberof potentially toxic electrophilic xenobiotics (such ascertain carcinogens) are conjugated to the nucleophilicGSH in reactions that can be represented as follows:R + GSH → R — S — Gwhere R = an electrophilic xenobiotic. The enzymescatalyzing these reactions are called glutathione S-transferases and are present in high amounts in livercytosol and in lower amounts in other tissues. A varietyof glutathione S-transferases are present in human tissue.They exhibit different substrate specificities andcan be separated by electrophoretic and other techniques.If the potentially toxic xenobiotics were notconjugated to GSH, they would be free to combine covalentlywith DNA, RNA, or cell protein and couldthus lead to serious cell damage. GSH is therefore animportant defense mechanism against certain toxiccompounds, such as some drugs and carcinogens. If thelevels of GSH in a tissue such as liver are lowered (ascan be achieved by the administration to rats of certaincompounds that react with GSH), then that tissue canbe shown to be more susceptible to injury by variouschemicals that would normally be conjugated to GSH.Glutathione conjugates are subjected to further metabolismbefore excretion. The glutamyl and glycinylgroups belonging to glutathione are removed by specificenzymes, and an acetyl group (donated by acetyl-CoA) is added to the amino group of the remainingcysteinyl moiety. The resulting compound is a mercapturicacid, a conjugate of L-acetylcysteine, which isthen excreted in the urine.Glutathione has other important functions inhuman cells apart from its role in xenobiotic metabolism.1. It participates in the decomposition of potentiallytoxic hydrogen peroxide in the reaction catalyzedby glutathione peroxidase (Chapter 20).2. It is an important intracellular reductant, helpingto maintain essential SH groups of enzymes intheir reduced state. This role is discussed in Chapter20, and its involvement in the hemolytic anemiacaused by deficiency of glucose-6-phosphatedehydrogenase is discussed in Chapters 20 and52.3. A metabolic cycle involving GSH as a carrier hasbeen implicated in the transport of certainamino acids across membranes in the kidney.The first reaction of the cycle is shown below.Amino acid+ GSH →γ- Glutamyl amino acid+Cysteinylglycine

630 / CHAPTER 53This reaction helps transfer certain amino acids acrossthe plasma membrane, the amino acid being subsequentlyhydrolyzed from its complex with GSH andthe GSH being resynthesized from cysteinylglycine.The enzyme catalyzing the above reaction is -glutamyltransferase(GGT). It is present in the plasmamembrane of renal tubular cells and bile ductule cells,and in the endoplasmic reticulum of hepatocytes. Theenzyme has diagnostic value because it is released intothe blood from hepatic cells in various hepatobiliarydiseases.D. OTHER REACTIONSThe two most important other reactions are acetylationand methylation.1. Acetylation—Acetylation is represented byX + Acetyl-CoA → Acetyl-X + CoAwhere X represents a xenobiotic. As for other acetylationreactions, acetyl-CoA (active acetate) is the acetyldonor. These reactions are catalyzed by acetyltransferasespresent in the cytosol of various tissues, particularlyliver. The drug isoniazid, used in the treatment oftuberculosis, is subject to acetylation. Polymorphictypes of acetyltransferases exist, resulting in individualswho are classified as slow or fast acetylators, and influencethe rate of clearance of drugs such as isoniazidfrom blood. Slow acetylators are more subject to certaintoxic effects of isoniazid because the drug persistslonger in these individuals.2. Methylation—A few xenobiotics are subject tomethylation by methyltransferases, employing S-adenosylmethionine(Figure 30–17) as the methyl donor.THE ACTIVITIES OF XENOBIOTIC-METABOLIZING ENZYMES AREAFFECTED BY AGE, SEX,& OTHER FACTORSVarious factors affect the activities of the enzymes metabolizingxenobiotics. The activities of these enzymesmay differ substantially among species. Thus, for example,the possible toxicity or carcinogenicity of xenobioticscannot be extrapolated freely from one species toanother. There are significant differences in enzyme activitiesamong individuals, many of which appear to bedue to genetic factors. The activities of some of theseenzymes vary according to age and sex.Intake of various xenobiotics such as phenobarbital,PCBs, or certain hydrocarbons can cause enzyme induction.It is thus important to know whether or notan individual has been exposed to these inducing agentsin evaluating biochemical responses to xenobiotics.Metabolites of certain xenobiotics can inhibit or stimulatethe activities of xenobiotic-metabolizing enzymes.Again, this can affect the doses of certain drugs that areadministered to patients. Various diseases (eg, cirrhosisof the liver) can affect the activities of drug-metabolizingenzymes, sometimes necessitating adjustment ofdosages of various drugs for patients with these disorders.RESPONSES TO XENOBIOTICSINCLUDE PHARMACOLOGIC,TOXIC, IMMUNOLOGIC,& CARCINOGENIC EFFECTSXenobiotics are metabolized in the body by the reactionsdescribed above. When the xenobiotic is a drug,phase 1 reactions may produce its active form or maydiminish or terminate its action if it is pharmacologicallyactive in the body without prior metabolism. Thediverse effects produced by drugs comprise the area ofstudy of pharmacology; here it is important to appreciatethat drugs act primarily through biochemical mechanisms.Table 53–2 summarizes four important reactionsto drugs that reflect genetically determineddifferences in enzyme and protein structure among individuals—partof the field of study known as pharmacogenetics(see below).Table 53–2. Some important drug reactions dueto mutant or polymorphic forms of enzymes orproteins. 1Enzyme or ProteinAffectedReaction or ConsequenceGlucose-6-phosphate Hemolytic anemia following indehydrogenase(G6PD) gestion of drugs such as prim-[mutations] (MIM 305900) aquineCa 2+ release channel (ryan- Malignant hyperthermia (MIModine receptor) in the 145600) following administrasarcoplasmicreticulum tion of certain anesthetics (eg,[mutations] (MIM 180901) halothane)CYP2D6 [polymorphisms](MIM 124030)CYP2A6 [polymorphisms](MIM 122720)Slow metabolism of certaindrugs (eg, debrisoquin), resultingin their accumulationImpaired metabolism of nicotine,resulting in protectionagainst becoming a tobaccodependentsmoker1 G6PD deficiency is discussed in Chapters 20 and 52 and malignanthyperthermia in Chapter 49. At least one gene other thanthat encoding the ryanodine receptor is involved in certain casesof malignant hypertension. Many other examples of drug reactionsbased on polymorphism or mutation are available.

METABOLISM OF XENOBIOTICS / 631Certain xenobiotics are very toxic even at low levels(eg, cyanide). On the other hand, there are few xenobiotics,including drugs, that do not exert some toxic effectsif sufficient amounts are administered. The toxiceffects of xenobiotics cover a wide spectrum, but themajor effects can be considered under three generalheadings (Figure 53–1).The first is cell injury (cytotoxicity), which can besevere enough to result in cell death. There are manymechanisms by which xenobiotics injure cells. The oneconsidered here is covalent binding to cell macromoleculesof reactive species of xenobiotics produced bymetabolism. These macromolecular targets includeDNA, RNA, and protein. If the macromolecule towhich the reactive xenobiotic binds is essential forshort-term cell survival, eg, a protein or enzyme involvedin some critical cellular function such as oxidativephosphorylation or regulation of the permeabilityof the plasma membrane, then severe effects on cellularfunction could become evident quite rapidly.Second, the reactive species of a xenobiotic maybind to a protein, altering its antigenicity. The xenobioticis said to act as a hapten, ie, a small molecule thatby itself does not stimulate antibody synthesis but willcombine with antibody once formed. The resulting antibodiescan then damage the cell by several immunologicmechanisms that grossly perturb normal cellularbiochemical processes.Third, reactions of activated species of chemical carcinogenswith DNA are thought to be of great importancein chemical carcinogenesis. Some chemicals (eg,benzo[α]pyrene) require activation by monooxygenasesin the endoplasmic reticulum to become carcinogenic(they are thus called indirect carcinogens). The activitiesof the monooxygenases and of other xenobioticmetabolizingenzymes present in the endoplasmic reticulumthus help to determine whether such compoundsbecome carcinogenic or are “detoxified.” Other chemicals(eg, various alkylating agents) can react directly (directcarcinogens) with DNA without undergoing intracellularchemical activation.The enzyme epoxide hydrolase is of interest becauseit can exert a protective effect against certain carcinogens.The products of the action of certainmonooxygenases on some procarcinogen substrates areepoxides. Epoxides are highly reactive and mutagenicor carcinogenic or both. Epoxide hydrolase—like cytochromeP450, also present in the membranes of theendoplasmic reticulum—acts on these compounds,converting them into much less reactive dihydrodiols.The reaction catalyzed by epoxide hydrolase can be representedas follows:C C + H 2 OC COEpoxideHO OHDihydrodiolPHARMACOGENOMICS WILL DRIVE THEDEVELOPMENT OF NEW & SAFER DRUGSAs indicated above, pharmacogenetics is the study ofthe roles of genetic variations in the responses to drugs.As a result of the progress made in sequencing theXenobioticCytochrome P450Reactive metaboliteGSH S-transferase orepoxide hydrolaseNontoxic metaboliteCovalent binding tomacromoleculesCell injuryHaptenMutationAntibody productionCancerCell injuryFigure 53–1. Simplified scheme showing how metabolism of a xenobiotic can result in cell injury, immunologicdamage, or cancer. In this instance, the conversion of the xenobiotic to a reactive metabolite is catalyzedby a cytochrome P450, and the conversion of the reactive metabolite (eg, an epoxide) to a nontoxic metaboliteis catalyzed either by a GSH S-transferase or by epoxide hydrolase.

632 / CHAPTER 53human genome, a new field of study—pharmacogenomics—hasdeveloped recently. It includes pharmacogeneticsbut covers a much wider sphere of activity. Informationfrom genomics, proteomics, bioinformatics,and other disciplines such as biochemistry and toxicologywill be integrated to make possible the synthesis ofnewer and safer drugs. As the sequences of all our genesand their encoded proteins are determined, this will revealmany new targets for drug actions. It will also revealpolymorphisms (this term is briefly discussed inChapter 50) of enzymes and proteins related to drugmetabolism, action, and toxicity. DNA probes capableof detecting them will be synthesized, permittingscreening of individuals for potentially harmful polymorphismsprior to the start of drug therapy. As thestructures of relevant proteins and their polymorphismsare revealed, model building and other techniques willpermit the design of drugs that take into account bothnormal protein targets and their polymorphisms. Atleast to some extent, drugs will be tailor-made for individualsbased on their genetic profiles. A new era of rationaldrug design built on information derived fromgenomics and proteomics has already commenced.SUMMARY• Xenobiotics are chemical compounds foreign to thebody, such as drugs, food additives, and environmentalpollutants; more than 200,000 have been identified.• Xenobiotics are metabolized in two phases. Themajor reaction of phase 1 is hydroxylation catalyzedby a variety of monooxygenases, also known as thecytochrome P450s. In phase 2, the hydroxylatedspecies are conjugated with a variety of hydrophiliccompounds such as glucuronic acid, sulfate, or glutathione.The combined operation of these twophases renders lipophilic compounds into watersolublecompounds that can be eliminated from thebody.• Cytochrome P450s catalyze reactions that introduceone atom of oxygen derived from molecular oxygeninto the substrate, yielding a hydroxylated product.NADPH and NADPH-cytochrome P450 reductaseare involved in the complex reaction mechanism.• All cytochrome P450s are hemoproteins and generallyhave a wide substrate specificity, acting on manyexogenous and endogenous substrates. They representthe most versatile biocatalyst known.• Members of at least 11 families of cytochrome P450are found in human tissue.• Cytochrome P450s are generally located in the endoplasmicreticulum of cells and are particularly enrichedin liver.• Many cytochrome P450s are inducible. This has importantimplications in phenomena such as drug interaction.• Mitochondrial cytochrome P450s also exist and areinvolved in cholesterol and steroid biosynthesis.They use a nonheme iron-containing sulfur protein,adrenodoxin, not required by microsomal isoforms.• Cytochrome P450s, because of their catalytic activities,play major roles in the reactions of cells tochemical compounds and in chemical carcinogenesis.• Phase 2 reactions are catalyzed by enzymes such asglucuronosyltransferases, sulfotransferases, and glutathioneS-transferases, using UDP-glucuronic acid,PAPS (active sulfate), and glutathione, respectively,as donors.• Glutathione not only plays an important role inphase 2 reactions but is also an intracellular reducingagent and is involved in the transport of certainamino acids into cells.• Xenobiotics can produce a variety of biologic effects,including pharmacologic responses, toxicity, immunologicreactions, and cancer.• Catalyzed by the progress made in sequencing thehuman genome, the new field of pharmacogenomicsoffers the promise of being able to make available ahost of new rationally designed, safer drugs.REFERENCESEvans WE, Johnson JA: Pharmacogenomics: the inherited basis forinterindividual differences in drug response. Annu Rev GenomicsHum Genet 2001;2:9.Guengerich FP: Common and uncommon cytochrome P450 reactionsrelated to metabolism and chemical toxicity. Chem ResToxicol 2001;14:611.Honkakakoski P, Negishi M: Regulation of cytochrome P450(CYP) genes by nuclear receptors. Biochem J 2000;347:321.Kalow W, Grant DM: Pharmacogenetics. In: The Metabolic andMolecular Bases of Inherited Disease, 8th ed. Scriver CR et al(editors). McGraw-Hill, 2001.Katzung BG (editor): Basic & Clinical Pharmacology, 8th ed. Mc-Graw-Hill, 2001.McLeod HL, Evans WE: Pharmacogenomics: unlocking thehuman genome for better drug therapy. Annu Rev PharmacolToxicol 2001;41:101.Nelson DR et al: P450 superfamily: update on new sequences, genemapping, accession numbers and nomenclature. Pharmacogenetics1996;6:1.

The Human Genome Project 54Robert K. Murray, MD, PhDBIOMEDICAL SIGNIFICANCEThe information deriving from determination of the sequencesof the human genome and those of other organismswill change biology and medicine for all time.For example, with reference to the human genome, newinformation on our origins, on disease genes, on diagnosis,and possible approaches to therapy are alreadyflooding in. Progress in fields such as genomics, proteomics,bioinformatics, biotechnology, and pharmacogenomicsis accelerating rapidly.The aims of this chapter are to briefly summarizethe major findings of the Human Genome Project(HGP) and indicate their implications for biology andmedicine.633THE HUMAN GENOME PROJECTHAS A VARIETY OF GOALSThe HGP, which started in 1990, is an internationaleffort whose principal goals were to sequence the entirehuman genome and the genomes of several other modelorganisms that have been basic to the study of genetics(eg, Escherichia coli, Saccharomyces cerevisiae [a yeast],Drosophila melanogaster [the fruit fly], Caenorhabditiselegans [the roundworm], and Mus musculus [the commonhouse mouse]). Most of these goals have been accomplished.In the United States, the National Centerfor Human Genome Research (NCHGR) was establishedin 1989, initially directed by James D. Watsonand subsequently by Francis Collins. The NCHGRplayed a leading role in directing the United States effortin the HGP. In 1997, it became the NationalHuman Genome Research Institute (NHGRI). The internationalcollaboration—involving groups from theUSA, UK, Japan, France, Germany, and China—cameto be known as the International Human Genome SequencingConsortium (IHGSC). Initially, a number ofshort-term goals were established for the United Stateseffort—eg, producing a human genetic map with markers2–5 centimorgans (cM) apart and constructing aphysical map of all 24 human chromosomes (22 autosomalplus X and Y) with markers spaced at approximately100,000 base pairs (bp). Figure 54–1 summarizesthe differences between a genetic map, a cytogeneticmap, and a physical map of a chromosome. Theseand other initial goals were achieved and surpassed bythe mid-nineties. In 1998, new goals for the UnitedStates wing of the HGP were announced. These includedthe aim of completing the entire sequence bythe end of 2003 or sooner. Other specific objectivesconcerned sequencing technology, comparative genomics,bioinformatics, ethical considerations, andother issues. By the fall of 1998, about 6% of thehuman genome sequence had been completed and thefoundations for future work laid. Further progress wascatalyzed by the announcement that a second group,the private company Celera Genomics, led by CraigVenter, had undertaken the objective of sequencing thehuman genome. Venter and colleagues had publishedin 1995 the entire genome sequences of Haemophilusinfluenzae and Mycoplasma genitalium, the first of manyspecies to have their genomic sequences determined. Animportant factor in the success of these workers was theuse of a shotgun approach, ie, sonicating the DNA, sequencingthe fragments, and reassembling the sequence,based on overlaps. For comparison, a variety ofapproaches that have been used at different times tostudy normal and disease genes are listed in Table 54–1.A Draft Sequence of the Human GenomeWas Announced in June 2000In June 2000, leaders of the IHGSC and the personnelat Celera Genomics announced completion of workingdrafts of the sequence of the human genome, coveringmore than 90% of it. The principal findings of the twogroups were published separately in February 2001 inspecial issues of Nature (the IHGSC) and Science (Celera).The draft published by the Consortium was theproduct of at least 10 years of work involving 20 sequencingcenters located in six countries. That publishedby Celera and associates was the product of some3 years or less of work; it relied in part on data obtainedby the IHGSC. The combined achievement has beenhailed, among other descriptions, as providing a Libraryof Life, supplying a Periodic Table of Life, and findingthe Holy Grail of Human Genetics.

634 / CHAPTER 5420 30 30 20 25cMGenetic mapCytogeneticmapPhysical map25 50 75 100 125 150 MbEcoRIHind III NotIRestriction mapSTS mapContig mapFigure 54–1. Principal methods used to identify and isolate normaland disease genes. For the genetic map, the positions of several hypotheticalgenetic markers are shown, along with the genetic distances incentimorgans between them. The circle shows the position of the centromere.For the cytogenetic map, the classic banding pattern of a hypotheticalchromosome is shown. For the physical map, the approximatephysical positions of the above genetic markers are shown, along withthe relative physical distances in megabase pairs. Examples of a restrictionmap, a contig mark, and an STS map are also shown. (Reproduced,with permission, from Green ED, Waterston RH: The Human GenomeProject: Prospects and implications for clinical medicine. JAMA 1991;266:1966. Copyright © 1991 by the American Medical Association.)Different Approaches Were Usedby the Two GroupsWe shall summarize the major findings reported in thetwo drafts and comment on their implications. Whilethere are differences between the drafts, they will not bedwelt on here, as the areas of general agreement aremuch more extensive. It is worthwhile, however, tosummarize the different approaches used by the twogroups. Basically, the IHGSC employed a map first,sequence later approach. In part, this was because sequencingwas a slow process when the public projectstarted, and the strategy of the Consortium evolvedover time as advances were made in sequencing andother techniques. The overall approach, referred to ashierarchical shotgun sequencing, consisted of fragmentingthe entire genome into pieces of approximately100–200 kb and inserting them into bacterialartificial chromosomes (BACs). The BACs were thenpositioned on individual chromosomes by looking formarker sequences known as sequence-tagged sites(STSs), whose locations had been already determined.STSs are short (usually < 500 bp), unique genomic locifor which a PCR assay is available. Clones of the BACswere then broken into small fragments (shotgunning).Each fragment was then sequenced, and computer algorithmswere used that recognized matching sequenceinformation from overlapping fragments to piece togetherthe complete sequence.Celera used the whole genome shotgun approach,in effect bypassing the mapping step. Shotgun fragmentswere assembled by algorithms onto large scaffolds,and the correct position of these scaffolds in thegenome was determined using STSs. A scaffold is a seriesof “contigs” that are in the right order but not necessarilyconnected in one continuous sequence. Contigsare contiguous sequences of DNA made by assemblingoverlapping sequenced fragments of a natural chromosomeor a BAC. The availability of high-throughputsequenators, powerful computer programs, the elementof competition, and other factors accounted for therapid progress made by both groups from 1998 onward.

THE HUMAN GENOME PROJECT / 635Table 54–1. Principal methods used to identify and isolate normal and disease genes.ProcedureCommentsDetection of specific cytogenetic For instance, a small deletion of band Xp21.2 was important in cloning the gene involved inabnormalitiesDuchenne muscular dystrophy.Extensive linkage studiesLarge families with defined pedigrees are desirable. Dominant genes are easier to recognizethan recessives.Use of probes to define marker Probes identify STSs, RFLPs, SNPs, 1 etc; thousands, covering all the chromosomes, are nowlociavailable. It is desirable to flank the gene on both sides, clearly delineating it.Radiation hybrid mapping 2 Now the most rapid method of localizing a gene or DNA fragment to a subregion of a humanchromosome and constructing a physical map.Use of rodent or human somatic Permits assignment of a gene to one specific chromosome but not to a subregion.cell hybridsFluorescence in situ hybridization Permits localization of a gene to one chromosomal band.Use of pulsed-field gelPermits isolation of large DNA fragments obtained by use of restriction endonucleases (rareelectrophoresis (PFGE) to cutters) that result in very limited cutting of DNA.separate large DNA fragmentsChromosome walkingInvolves repeated cloning of overlapping DNA segments; the procedure is laborious and canusually cover only 100–200 kb.Chromosome jumpingBy cutting DNA into relatively large fragments and circularizing it, one can move more quicklyand cover greater lengths of DNA than with chromosomal walking.Cloning via YACs, BACs, cosmids, Permits isolation of fragments of varying lengths.phages, and plasmidsDetection of expression of The mRNA should be expressed in affected tissues.mRNAs in tissues by Northernblotting using one or morefragments of the gene as aprobePCRCan be used to amplify fragments of the gene; also many other applications.DNA sequencingEstablishes the highest resolution physical map. Identifies open reading frame. Facilities withmany high throughput instruments could sequence millions of base pairs per day.DatabasesComparison of DNA and protein sequences obtained from unknown gene with known sequencesin databases can facilitate gene identification.Abbreviations: STS, sequence tagged site; RFLP, restriction fragment linked polymorphism; SNP, single nucleotide polymorphism; YAC,yeast artificial chromosome; BAC, bacterial artificial chromosome; PCR, polymerase chain reaction.1 Many single nucleotide polymorphisms (SNPs) are being detected and catalogued. These are stable and frequent, and their detectioncan be automated. It is anticipated that they will be particularly useful for mapping complex traits such as diabetes mellitus.2 Radiation hybrid mapping (consult http://compgen.rutgers.edu/rhmap/ for a detailed bibliography of this technique) makes use of apanel of somatic cell hybrids, with each cell line containing a random set of irradiated human genomic DNA in a hamster background.Briefly, the radiation fragments the DNA into small pieces of variable length; if a gene is located close to another known gene, it is likelythat the two will remain linked (compare genetic linkage) on the same fragment. An STS marker is typed against a radiation hybrid panelby using its two oligonucleotide primers to perform a PCR assay against the DNA from each hybrid cell line of the panel. If enough markersare typed on one panel, continuous linkage can be established along each arm of a chromosome, and the markers can be assembledinto the map as a single linkage group.

636 / CHAPTER 54DETERMINATION OF THE SEQUENCE OFTHE HUMAN GENOME HAS PRODUCED AWEALTH OF NEW FINDINGSOnly a small fraction of the findings can be coveredhere. The interested reader is referred to the original articles.Table 54–2 summarizes a number of the highlights,which can now be described.Most of the Human GenomeHas Been SequencedOver 90% of the human genome had been sequencedby July 2000. This is by far the largest genome sequenced,with an estimated size of approximately 3.2gigabases (Gb). Prior to the human genome, that of thefruit fly had been the largest (~180 Mb) sequenced.Gaps still exist, small and large, and the quality of someof the sequencing data will be refined since some of thefindings are probably not exactly right.The Human Genome Is Estimated toEncode About 30,000–40,000 ProteinsThe greatest surprise provided by the results to date hasbeen the apparently low number of genes encoding proteins,estimated to lie between 30,000 and 40,000. Thehigher number could increase as new data are obtained.This number is approximately twice that found in theroundworm (19,099) and three times that of the fruitTable 54–2. Major findings reported in the roughdrafts of the human genome.• More than 90% of the genome has been sequenced; gaps,large and small, remain to be filled in.• Estimated number of protein-coding genes ranges from30,000 to 40,000.• Only 1.1–1.5% of the genome codes for proteins.• There are wide variations in features of individual chromosomes(eg, in gene number per Mb, SNP density, GC content,numbers of transposable elements and CpG islands,recombination rate).• Human genes do more work than those of the roundwormor fruit fly (eg, alternative splicing is used more frequently).• The human proteome is more complex than that found ininvertebrates.• Repeat sequences probably constitute more than 50% ofthe genome.• Approximately 100 coding regions have been copied andmoved by RNA-based transposons.• Approximately 200 genes may be derived from bacteria bylateral transfer.• More than 3 million SNPs have been identified.fly (13,061). The figures suggest that the complexity ofhumans compared with that of the two simpler organismsmust have explanations other than strictly genenumber.Only 1.1–1.5% of the HumanGenome Encodes ProteinsAnalyses of the available data reveal that 1.1–1.5% ofthe genome consists of exons. About 24% consists ofintrons, and 75% of sequences lying between genes (intergenic).Comparisons with the data on the roundwormand fruit fly have shown that exon size across thethree species is relatively constant (mean size of 145 bpin humans). However, intron size in humans is muchmore variable (mean size of over 3300 bp), resulting ingreat variation in gene size.The Landscape of Human ChromosomesVaries WidelyThere are marked differences among individual chromosomesin many features, such as gene number permegabase, density of single nucleotide polymorphisms(SNPs), GC content, number of transposable elementsand CpG islands, and recombination rate. To take oneexample, chromosome 19 has the richest gene content(23 genes per megabase), whereas chromosome 13 andthe Y chromosome have the sparsest content (5 genesper megabase). Explanations for these variations are notapparent at this time.Human Genes Do More Work ThanThose of Simpler OrganismsAlternative splicing appears to be more prevalent in humans,involving at least 35% of their genes. Data indicatethat the average number of distinct transcripts pergene for chromosomes 22 and 19 were 2.6 and 3.2, respectively.These figures are higher than for the roundworm,where only 12.2% of genes appear to be alternativelyspliced and only 1.34 splice variants per genewere noted.The Human Proteome Is More ComplexThan That of InvertebratesRelatively few new protein domains appear to haveemerged among vertebrates. However, the number ofdistinct domain architectures (~1800) in human proteinsis 1.8 times that of the roundworm or fruit fly.About 90 vertebrate-specific families of proteins havebeen identified, and these have been found to be enrichedin proteins of the immune and nervous systems.

THE HUMAN GENOME PROJECT / 637The results of the two drafts are rich in informationabout protein families and classes. One example isshown in Table 54–3, in which the major classes ofproteins encoded by human genes are listed. As can beseen, the largest class is “unknown.” Identification ofthese unknown proteins will be a major focus of activityfor many laboratories.Repeat Sequences Probably ConstituteMore Than 50% of the Human GenomeRepeat sequences probably account for at least half ofthe genome. They fall into five classes: (1) transposonderivedrepeats (interspersed repeats); (2) processedpseudogenes; (3) simple sequence repeats; (4) segmentalduplications, made up of 10–300 kb that have beencopied from one region of the genome into another;and (5) blocks of tandemly repeated sequences, foundat centromeres, telomeres, and other areas. Considerableinformation on most of the above classes of repeatsequences—of great value in understanding the architectureand development of the human genome—is reportedin the drafts. Only two points of interest will bementioned here. It is speculated that Alu elements, themost prominent members (about 10% of the totalgenome) of the short interspersed elements (SINEs),may be present in GC-rich areas because of positive selection,implying that they are of benefit to the host.Table 54–3. Major classes of proteins encoded byhuman genes. 1Class of Protein Number (%) 2Unknown 12,809 (41%)Nucleic acid enzymes 2,308 (7.5%)Transcription factors 1,850 (6%)Receptors 1,543 (5%)Hydrolases 1,227 (4.0%)Select regulatory molecules (eg, 988 (3.2%)G proteins, cell cycle regulators)Protooncogenes 902 (2.9%)Cytoskeletal structural proteins 876 (2.8%)Kinases 868 (2.8%)1 Data from Venter JC et al: The sequence of the human genome.Science 2001;291:1304.2 The percentages are derived from a total of 26,383 genes reportedin the rough draft by Celera Genomics. Classes containingmore than 2.5% of the total proteins encoded by the genes identifiedwhen this rough draft was written are arbitrarily listed asmajor.Segmental duplications have been found to be muchmore common than in the roundworm or fruit fly. It ispossible that these structures may be involved in exonshuffling and the increased diversity of proteins foundin humans.Other Findings of InterestThe last three major points of interest listed in Table54–2 will be briefly described together.Approximately 100 coding regions are estimated tohave been copied and moved by RNA-based transposons(retrotransposons). It is possible that some ofthese genes may adopt new roles in the course of time.A surprising finding is that over 200 genes may be derivedfrom bacteria by lateral transfer. None of thesegenes are present in nonvertebrate eukaryotes. Morethan 3 million SNPs have been identified. It is likelythat they will prove invaluable for certain aspects ofgene mapping.It is stressed that the findings listed here are only afew of those reported in the drafts, and the reader isurged to consult the original reports (see References,below).FURTHER WORK IS PLANNED ON THEHUMAN & OTHER GENOMESThe IHGSC has indicated that it will determine thecomplete sequence, it is hoped, by 2003. The task involvesfilling in the gaps and identifying new genes,their locations, and functions. Regulatory regions willbe identified, and the sequences of other large genomes(eg, of the house mouse; of Rattus norvegicus, the Norwayrat; of Danio rerio, the zebra fish; of Fugu rubripes,the tiger puffer fish; and of one or more primates) willbe obtained; indeed, a draft version of the genome ofthe tiger puffer fish was published in 2002. AdditionalSNPs will be identified; a complete catalog of thesevariants is expected to be of great value in mappinggenes associated with complex traits and for other usesas well. Along with the above, existing databases will beadded to as new information flows in, and new databaseswill probably be established to serve specific purposes.A variety of studies in functional genomics (ie,the study of genomes to determine the functions of alltheir genes and their products) will also be undertaken.IMPLICATIONS FOR PROTEOMICS,BIOTECHNOLOGY, & BIOINFORMATICSMany fields will be influenced by knowledge of thehuman genome. Only a few are briefly discussed here.Proteomics (see Chapter 4) in its broadest sense isthe study of all the proteins encoded in an organism (ie,

638 / CHAPTER 54the proteome), including their structures, modifications,functions, and interactions. In a narrower sense,it involves the identification and study of multiple proteinslinked through cellular actions—but not necessarilythe entire proteome. With regard to humans, manyindividual proteins will be identified and characterized;their interactions and levels will be determined in physiologicand pathologic states, and the resulting informationwill be entered into appropriate databases. Techniquessuch as two-dimensional electrophoresis, avariety of modes of mass spectrometry, and antibodyarrays will be central to expansion of this rapidly growingfield. Overall, proteomics will greatly advance ourknowledge of proteins at the basic level and will alsonourish biotechnology as new proteins that are likelyto have diagnostic, therapeutic, and other uses are discoveredand methods for their economic production aredeveloped. Specialists in bioinformatics will be in demand,as this field rapidly gears up to manage, analyze,and utilize the flood of data from genomic and proteomicstudies.IMPLICATIONS FOR MEDICINEPractically every area of medicine will be affected by thenew information accruing from knowledge of thehuman genome. The tracking of disease genes will beenormously facilitated. As mentioned above, SNP mapsshould greatly assist determination of genes involved incomplex diseases. Probes for any gene will be availableif needed, leading to improved diagnostic testing fordisease susceptibility genes and for genes directly involvedin the causation of specific diseases. The field ofpharmacogenomics (see Chapter 53) is already expandinggreatly, and it is possible that in the futuredrugs will be tailored to accommodate the variations inenzymes and other proteins involved in drug action andmetabolism found among individuals. Studies of genesinvolved in behavior may lead to new insights into thecausation and possible treatment of psychiatric disorders.Many ethical issues—eg, privacy concerns andthe use of genomic information for commercial purposes—willhave to be addressed. It will also be importantthat medical and economic benefits accrue to individualsin Third World countries from the anticipatedeffects on health services and the diagnosis and treatmentof disease.SUMMARY• Determination of the complete sequence of thehuman genome, now almost completed, is one of themost significant scientific achievements of all time.• Many important findings have already emerged. Theone to date that has generated the most discussion isthat the number of human genes may be only two tothree times that estimated for the roundworm andthe fruit fly.• Information flowing from the Human Genome Projectis having major influences in fields such as proteomics,bioinformatics, biotechnology, and pharmacogenomicsas well as all areas of biology andmedicine.• It is hoped that the knowledge derived will be usedwisely and fairly and that the benefits that will ensueregarding health, disease, and other matters will bemade available to all people everywhere.REFERENCESCollins FS, McKusick VA: Implications of the Human GenomeProject for medical science. JAMA 2001;285:540. (The February7, 2001, issue describes opportunities for medical researchin the 21st century. Many articles of interest.)Hedges SB, Kumar S: Vertebrate genomes compared. Science2002;297:1283. (The same issue—No. 5585, August 23—contains a draft version of the genome of the tiger pufferfish.)McKusick VA: The anatomy of the human genome: a neo-Vesalianbasis for medicine in the 21st century. JAMA 2001;286:2289. (The November 14, 2001, issue contains a number ofother excellent articles—eg, on clinical proteomics, pharmacogenomics—relatingto the Human Genome Project and itsimpact on medicine.)Nature 2001;409(6822) (February 15), and Science 2001;291(5507) (February 16). (These two issues present the roughdrafts prepared by the IHGSC and Celera, respectively, alongwith many other articles analyzing the meaning and significanceof the findings.)Science 2001;294(5540) (October 5). (This issue contains a numberof articles under the title Genome: Unlocking Biology’sStorehouse. They describe new ideas, approaches, and researchrelated to genome information.)

APPENDIXSELECTED WORLD WIDE WEB SITESThe following is a list of Web sites that readers mayfind useful. The sites have been visited at various timesby one of the authors (RKM). Most are located in theUSA, but many provide extensive links to internationalsites and to databases (eg, for protein and nucleic acidsequences) and online journals. RKM would be gratefulif readers who find other useful sites would notify himof their URLs by e-mail (rmurray6745@rogers. com) sothat they may be considered for inclusion in future editionsof this text.Readers should note that URLs may change or ceaseto exist.ACCESS TO THE BIOMEDICALLITERATUREHighWire Press: http://highwire.stanford.edu(Extensive lists of various classes of journals—biology, medicine,etc—and offers also the most extensive list of journals withfree online access.)National Library of Medicine: http://www.nlm.nih.gov/(Free access to Medline via PubMed.)GENERAL RESOURCE SITESThe Biology Project (from the University of Arizona): http://www.biology.arizona.edu/default.htmlHarvard Department of Molecular & Cellular Biology Links:http://mcb.harvard.edu/BioLinks.htmlSITES ON SPECIFIC TOPICSAmerican Heart Association: http://www.americanheart.org(Useful information on nutrition, on the role of various biomolecules—eg,cholesterol, lipoproteins—in heart disease, and onthe major cardiovascular diseases.)Cancer Genome Anatomy Project (CGAP): http://www.ncbi.nlm.nih.gov/ncicgap(An interdisciplinary program to generate the information andtechnical tools needed to decipher the molecular anatomy ofthe cancer cell.)European Bioinformatics Institute: http://www.ebi.ac.uk/ebi_home.html(Maintains the EMBL Nucleotide and SWISS-PROT databases aswell as other databases.)GeneCards: http://bioinformatics.weizmann.ac.il/cards/(A database of human genes, their products, and their involvementsin diseases. From the Weizmann Institute of Science.)GeneTestsGeneClinics: http://www.geneclinics.org/(A medical genetics information resource with comprehensive articleson many genetic diseases.)Genes and Disease: http://www.ncbi.nlm.nih.gov/disease/(Coverage of the genetic basis of many different types of diseases.)The Glycoscience Network (TGN): http://www.vei.co.uk/TGN/tgn_side.htm(TGN is an informal worldwide grouping of scientists who share aninterest in carbohydrates. The site contains considerable informationon carbohydrates and an extensive list of links toother sites dealing with sugar-containing molecules).Howard Hughes Medical Institute: http://www.hhmi.org/(An excellent site for following current biomedical research. Containsa comprehensive Research News Archive.)The Human Gene Mutation Database: http://archive.uwcm.ac.uk/uwcm/mg/hgmd0.html(An extensive tabulation of mutations in human genes from the Instituteof Medical Genetics in Cardiff, Wales.)Human Genome Project Information: http://www.ornl.gov/hgmis/(From the Human Genome Program of the United States Departmentof Energy.)The Institute for Genetic Research: http://www.tigr.org/(Sequences of various bacterial genomes and other information.)Karolinska Institute Nutritional and Metabolic Diseases: http://www.mic.ki.se/Diseases/c18.html(Access to information on many nutritional and metabolic disorders.)MITOMAP: http://www.mitomap.org/(A human mitochondrial genome database.)National Center for Biotechnology Information: http://ncbi.nlm.nih.gov/(Information on molecular biology and how molecular processes affecthuman health and disease.)National Human Genome Research Institute: http://www.genome.gov/(Extensive information about the Human Genome Project.)National Institutes of Health (NIH): http://www.nih.gov/(Includes links to the separate Institutes and Centers that constituteNIH, covering a wide range of biomedical research.)Neuroscience (Biosciences): http://neuro.med.cornell.edu/VL/(A comprehensive list of neuroscience resources; part of the World-Wide Web Virtual Library.)639

640 / APPENDIXOffice of Rare Diseases: http://rarediseases.info.nih.gov/index_main.html(Access to information on more than 7000 rare diseases, includingcurrent research.)OMIM Home Page—Online Mendelian Inheritance in Man:http://www.ncbi.nlm.nih.gov/omim/(An extensive catalog of human genetic disorders, updated daily.Lists access to various allied resources.)Protein Data Bank (PDB): http://www.rcsb.org/pdb/(A worldwide repository for the processing and distribution ofthree-dimensional biologic macromolecular structure data.)The Protein Kinase Resource: http://pkr.sdsc.edu/html/index.shtml(Information on the protein kinase family of enzymes.)The Protein Society: http://www.faseb.org/protein/index.html(An extensive list of Web resources for protein scientists.)Signaling Update: http://www.signaling-update.org/(A one-stop overview for the specialist or nonspecialist of what ishappening in cell signaling.)Society for Endocrinology: http://www.endocrinology.org(Aims to promote advancement of public education in endocrinology.Contains a number of articles on endocrinology and alist of links to other relevant sites.)tbase—the Transgenic/Targeted Mutation Database at the JacksonLaboratory, Bar Harbor, Maine: http://tbase.jax.org/(An attempt to organize information on transgenic animals and targetedmutations generated and analyzed worldwide.)The Wellcome Trust Sanger Institute: http://www.sanger.ac.uk(A genome research center whose purpose it is to increase knowledgeof genomes, particularly through large-scale sequencingand analysis,)Whitehead Institute/MIT Center for Genome Research: http://www.genome.wi.mit.edu/(Access to various databases and articles entitled “What’s New inGenome Research.”)Chapter 2http://www.geocities.com/bioelectrochemistry/sorensen.htmChapter 3http: //www.bio.cmu.edu/Courses/03231http: //www.bcbp.gu.se/~orjan/bmstruct/Chapter 4http://www.lundberg.bcbp.gu.se/~orjan/bmstruct/Chapter 5http://www.umass.edu/microbio/chime/explorer/http://molvis.sdsc.edu/protexpl/index.htmhttp://www.umass.edu/microbio/rasmol/http://www.cryst.bbk.ac.uk/PPS2/course/section10/membrane.htmlhttp://molbio.info.nih.gov/cgi-bin/pdb/http://www.mc.vanderbilt.edu/peds/pidl/genetic/ehlers.htmChapter 6http://sickle.bwh.harvard.edu/http://globin.cse.psu.edu/Chapter 7http://s02.middlebury.edu/CH441A/EnzymeTutorials.htmlhttp://www.i-a-s.de/IAS/botanik/e18/18.htmChapter 8http://www.indstate.edu/thcme/mwking/enzyme-kinetics.htmlhttp://ntri.tamuk.edu/cell/kinetics.htmlChapter 9http://users.wmin.ac.uk/~mellerj/physiology/bernard.htmhttp://www.cm.utexas.edu/academic/courses/Fall2001/CH369/LEC06/Lec6.htmhttp://www.cellularsignaling.orghttp://arethusa.unh.edu/bchm752/ppthtml/Jan27/sld015.htmChapter 22http://www.auhs.edu/netbiochem/NetWelco.htmChapter 28http://www.people.virginia.edu/~rjh9u/scurvy.htmlhttp://www.mc.vanderbilt.edu/biolib/hc/journeys/scurvy.htmlhttp://opbs.okstate.edu/~melcher/MG/MGW2/MG2411.htmlChapter 29http://www.nucdf.org/whatis.htmChapter 30http://www.pkunetwork.org/http://www.rarediseases.org/search/rdblist.htmlhttp://www.msud-support.org/overv.htmChapter 34http://www.rarediseases.org/search/rdblist.htmlhttp://www.rheumatology.org/patients/factsheet/gout.htmlhttp://www.merck.com/pubs/mmanual/section5/chapter55/55a.htmhttp://www.nlm.nih.gov/medlineplus/goutandpseudogout.htmlhttp://www.amg.gda.pl/~essppmm/ppd/ppd_py_umps.html

APPENDIX / 641BIOCHEMICAL JOURNALS AND REVIEWSThe following is a partial list of biochemistry journalsand review series and of some biomedical journals thatcontain biochemical articles. Biochemistry and biologyjournals now usually have Web sites, often with usefullinks, and some journals are fully accessible withoutcharge. The reader can obtain the URLs for the followingby using a search engine.Annual Reviews of Biochemistry, Cell and DevelopmentalBiology, Genetics, Genomics and HumanGeneticsArchives of Biochemistry and Biophysics (ArchBiochem Biophys)Biochemical and Biophysical Research Communications(Biochem Biophys Res Commun)Biochemical Journal (Biochem J)Biochemistry (Biochemistry)Biochemistry (Moscow) (Biochemistry [Mosc])Biochimica et Biophysica Acta (Biochim BiophysActa)Biochimie (Biochimie)European Journal of Biochemistry (Eur J Biochem)Indian Journal of Biochemistry and Biophysics (IndianJ Biochem Biophys)Journal of Biochemistry (Tokyo) (J Biochem[Tokyo])Journal of Biological Chemistry (J Biol Chem)Journal of Clinical Investigation (J Clin Invest)Journal of Lipid Research (J Lipid Res)Nature (Nature)Nature Genetics (Nat Genet)Proceedings of the National Academy of SciencesUSA (Proc Natl Acad Sci USA)Science (Science)Trends in Biochemical Sciences (Trends BiochemSci)

IndexNote: Page numbers in bold face type indicate a major discussion. A t following a page number indicates tabular materialand an f following a page number indicates a figure.A bands, 556, 557f, 558fA blood group substance, 618, 619fA cyclins, 333, 334f, 335tA gene, GalNAc transferase encoded by,618–619A (aminoacyl/acceptor) site, aminoacyltRNAbinding to, in proteinsynthesis, 368, 368fABC-1. See ATP-binding cassettetransporter-1Abetalipoproteinemia, 207, 228tABO blood group system, biochemical basisof, 617–619, 619fAbsorption, 474–480Absorption chromatography, forprotein/peptide purification, 22Absorption spectra, of porphyrins,273–274, 277fAbsorptive pinocytosis, 430ACAT (acyl-CoA:cholesterol acyltransferase),223Accelerator (Ac-) globulin (factor V), 600t,601, 602fAcceptor (A/aminoacyl) site, aminoacyltRNAbinding to, in proteinsynthesis, 368, 368fAcceptor arm, of tRNA, 310, 312f, 360,361fAceruloplasminemia, 589ACEs. See Angiotensin-converting enzymeinhibitorsAcetal links, 105–106Acetic acid, 112tpK/pK a value of, 12tAcetoacetate, 183–184, 184fin tyrosine catabolism, 254f, 255Acetoacetyl-CoA synthetase, in mevalonatesynthesis, 219, 220fAcetone, 183Acetone bodies. See Ketone bodiesAcetylationin covalent modification, mass increasesand, 27tof xenobiotics, 630Acetyl-CoA, 122, 122fcarbohydrate metabolism and, 122, 122f,123fcatabolism of, 130–135, 131f, 132f. Seealso Citric acid cyclecholesterol synthesis and, 219–220, 220f,221f, 222ffatty acid oxidation to, 123–124, 123f,181–183, 181f, 182fformation of, 254f, 255–259, 255f, 256f,257f, 258f, 259flipogenesis and, 173–177, 174f, 175fas fatty acid building block, 176–177pyruvate dehydrogenase regulated by,141–142, 142f, 178pyruvate oxidation to, 134, 135f,140–142, 141f, 142f, 143txenobiotic metabolism and, 630Acetyl-CoA carboxylase, 156t, 179in lipogenesis regulation, 156t, 173,174f, 178, 178f, 179N-Acetylgalactosamine (GalNAc), inglycoproteins, 515, 516tN-Acetylglucosamine (GlcNAc), inglycoproteins, 516tN-Acetylglucosamine phosphotransferase(GlcNAc phosphotransferase)in I-cell disease, 532in pseudo-Hurler polydystrophy, 532N-Acetylglutamate, in urea biosynthesis,245, 246fAcetyl hexosamines, in glycoproteins, 109tN-Acetyl lactosamines, on N-linked glycanchains, 521Acetyl (acyl)-malonyl enzyme, 173, 175fN-Acetyl neuraminic acid, 169, 171fin gangliosides, 201, 203fin glycoproteins, 169, 171f, 515, 516tin mucins, 519f, 520Acetyl transacylase, 173, 174f, 175fAcetyltransferases, xenobiotic metabolismand, 630Acholuric jaundice, 282Achondroplasia, 432t, 551t, 553–554, 554fAcid anhydride bonds, 287Acid anhydrides, group transfer potentialfor, 289–290, 289f, 290f, 290tAcid-base balance, ammonia metabolism in,245Acid-base catalysis, 51–52HIV protease in, 52, 53fα 1 -Acid glycoprotein (orosomucoid), 583tAcid hydrolysis, for polypeptide cleavage,26tAcid phosphatase, diagnostic significanceof, 57tAcidemia, isovaleric, 259, 259–262Acidosislactic. See Lactic acidosismetabolic, ammonia in, 245Acidsconjugate, 10643molecular structure affecting strength of,12, 12tpolyfunctional, nucleotides as, 290as proton donors, 9strong, 9weak. See Weak acidsAciduriadicarboxylic, 188methylmalonic, 155orotic, 300, 301urocanic, 250Aconitase (aconitate hydratase), 130ACP. See Acyl carrier proteinAcrosomal reaction, glycoproteins in, 528ACTH. See Adrenocorticotropic hormoneActin, 557, 559, 560decoration of, 561, 561ffibronectin receptor interacting with,540, 541fin muscle contraction, 557–559, 558f,561–562, 561f, 562fregulation of striated muscle and,562–563in nonmuscle cells, 576–577in red cell membranes, 615f, 616f, 616t,617in striated vs. smooth muscle, 572tβ-Actin, 577F-Actin, 559, 559f, 561in nonmuscle cells, 576, 577G-Actin, 559, 559fin nonmuscle cells, 576γ-Actin, 577Actin-filament capping protein, 540, 541fActin (thin) filaments, 557, 558f, 559fα-Actinin, 540, 566tActivated protein C, in blood coagulation,603Activation energy, 61, 63Activation energy barrier, enzymes affecting,63Activation function 1, 460Activation function 2, 460Activation reaction, 456, 458fActivator-recruited cofactor (ARC), 472t,473Activatorsin regulation of gene expression, 374,376. See also Enhancerstranscription, 351, 351tActive chromatin, 316–318, 318f, 383Active site, 51, 51f. See also Catalytic site∆G F and, 63

644 / INDEXActive sulfate (adenosine 3′-phosphate-5′-phosphosulfate), 289, 289f, 629Active transport, 423, 423t, 424f, 426–427,427–428, 428fin bilirubin secretion, 280, 281fActivity (physical), energy expenditure and,478Actomyosin, 560ACTR coactivator, 472, 472tAcute fatty liver of pregnancy, 188Acute inflammatory response, neutrophilsin, 620Acute phase proteins, 583, 583tnegative, vitamin A as, 483–484Acylcarnitine, 180–181, 181fAcyl carrier protein (ACP), 173, 174fsynthesis of, from pantothenic acid, 173,495Acyl-CoA:cholesterol acyltransferase(ACAT), 223Acyl-CoA dehydrogenase, 87, 181, 182fmedium-chain, deficiency of, 188Acyl-CoA synthetase (thiokinase)in fatty acid activation, 180, 181fin triacylglycerol synthesis, 199, 214f, 215Acylglycerols, 197metabolism of, 197–201catabolism, 197clinical aspects of, 202synthesis, 197–201, 197f, 198fin endoplasmic reticulum, 126, 127fAdapter proteins, in absorptive pinocytosis,430Adenine, 288f, 288tAdenosine, 287f, 288tbase pairing of in DNA, 303, 304, 305fin uric acid formation, 299, 299fAdenosine deaminasedeficiency of, 300localization of gene for, 407tAdenosine diphosphate. See ADPAdenosine monophosphate. See AMPAdenosine 3′-phosphate-5′-phosphosulfate,289, 289f, 629Adenosine triphosphate. See ATPS-Adenosylmethionine, 258f, 259, 264,266f, 289, 290f, 290tAdenylic acid, as second messenger, 457Adenylyl cyclasein cAMP-dependent signal transduction,458–459, 460tcAMP derived from, 147in lipolysis, 215, 216fAdenylyl kinase (myokinase), 84deficiencies of, 151–152in gluconeogenesis regulation, 157as source of ATP in muscle, 573, 575fAdhesion molecules, 529, 529t. See also CelladhesionAdipose tissue, 111, 214–215, 214fbrown, 217, 217fmetabolism in, 214–215, 214f, 235tcontrol of, 216–217ADP, 287ffree energy of hydrolysis of, 82tmitochondrial respiratory rate and,94–95, 97t, 98fmyosin, muscle contraction and, 561,561fin platelet activation, 606f, 607in respiratory control, 94–95, 97, 97t,98f, 134–135structure of, 83fADP/ATP transporter, 95, 98fADPase, 607, 607tADP-chaperone complex, 508. See alsoChaperonesADP-ribose, NAD as source of, 490ADP-ribosylation, 490Adrenal cortical hormones. See also specifichormone and Glucocorticoids;Mineralocorticoidssynthesis of, 438–442, 440f, 441fAdrenal gland, cytochrome P450 isoformsin, 627Adrenal medulla, catecholamines producedin, 445Adrenergic receptors, in glycogenolysis, 148Adrenocorticotropic hormone (ACTH),437, 438, 439f, 453, 453fAdrenodoxin, 627Adrenodoxin reductase, 627Adrenogenital syndrome, 442Adrenoleukodystrophy, neonatal, 503, 503tAerobic glycolysis, 139as muscle ATP source, 575, 575f, 575tAerobic respirationcitric acid cycle and, 130AF-1. See Activation function 1AF-2. See Activation function 2AF-2 domain, 470Affinity chromatographyfor protein/peptide purification, 23in recombinant fusion proteinpurification, 58, 59fAFP. See Alpha-fetoproteinAgammaglobulinemia, 595Age, xenobiotic-metabolizing enzymesaffected by, 630Aggrecan, 542, 551t, 553, 553fAggregates, formation of after denaturation,36Aging, glycosaminoglycans and, 549Aglycone, 105, 106AHG. See Antihemophilic factor A/globulinAIB1 coactivator, 472, 472tALA. See AminolevulinateAlanine, 15tpI of, 17in pyruvate formation, 250, 252fsynthesis of, 237, 238fβ-Alanine, 264, 300, 301fAlanine (alanine-pyruvate) aminotransferase(ALT/SGPT)diagnostic significance of, 57tin urea synthesis, 243–244, 244fβ-Alanyl dipeptides, 264, 265fAlbumin, 580, 581f, 583–584, 583tconjugated bilirubin binding to, 283free fatty acids in combination with, 180,206, 206t, 584glomerular membrane permeability to,540–541Albuminuria, 542Alcohol, ethyl. See EthanolAlcohol dehydrogenase, in fatty liver, 212Alcoholismcirrhosis and, 212fatty liver and, 212–214Aldehyde dehydrogenase, 87in fatty liver, 212Aldolase A, deficiency of, 143Aldolase B, 167, 168fdeficiency of, 171Aldolases, in glycolysis, 137, 138fAldose-ketose isomerism, 103f, 104, 104fAldose reductase, 167, 169f, 172Aldoses, 102, 102t, 104tring structure of, 104Aldosteronebinding of, 455synthesis of, 438–440, 441fangiotensin affecting, 451, 452Aldosterone synthase (18-hydroxylase), insteroid synthesis, 440, 441fAlkaline phosphatasein bone mineralization, 550isozymes of, diagnostic significance of,57tin recombinant DNA technology, 400tAlkalosis, metabolic, ammonia in, 245Alkaptonuria, 255Allergic reactions, peptide absorptioncausing, 474Allopurinol, 290, 297Allosteric activators, 157Allosteric effectors/modifiers, 129in gluconeogenesis regulation, 157negative, 74. See also Feedback inhibitionsecond messengers as, 76Allosteric enzymes, 75, 129aspartate transcarbamoylase as model of,75Allosteric properties of hemoglobin, 42–46Allosteric regulation, of enzymatic catalysis,74, 74–76, 75f, 128f, 129gluconeogenesis regulation and, 157Allosteric site, 74, 75Alpha-adrenergic receptors, inglycogenolysis, 148Alpha-amino acids. See also Amino acidsgenetic code specifying, 14, 15–16tin proteins, 14

INDEX / 645Alpha-amino nitrogen. See Amino acidnitrogenAlpha anomers, 104Alpha 1 antiproteinase/antitrypsin. Seeα 1 -AntiproteinaseAlpha- (α) carotene, 482Alpha-fetoprotein, 583tAlpha-globin gene, localization of, 407tAlpha (α) helix, 31–32, 32f, 33famphipathic, 31–32in myoglobin, 40, 41fAlpha (α) ketoglutarate. Seeα-KetoglutarateAlpha-lipoproteins, 205. See also Highdensitylipoproteinsfamilial deficiency of, 228tAlpha-R groups, amino acid propertiesaffected by, 18Alpha (α) thalassemia, 47Alpha-tocopherol. See TocopherolAlport syndrome, 538, 538tALT. See Alanine aminotransferaseAlteplase (tissue plasminogen activator/t-PA),604–605, 605f, 606t, 607tAlternative pathway, of complementactivation, 596Altitude, high, adaptation to, 46Alu family, 321–322Alzheimer disease, amyloid in, 37, 590α-Amanitin, RNA polymerases affected by,343Ambiguity, genetic code and, 359Amino acid carbon skeletons, catabolism of,249–263acetyl-CoA formation and, 254f,255–259, 255f, 256f, 257f, 258f,259fbranched-chain, 259, 260f, 261f, 262fdisorders of, 259–262pyruvate formation and, 250–255, 252f,253ftransamination in initiation of, 249–250,249f, 250f, 251fAmino acid nitrogencatabolism of, 242–248in amino acid carbon skeletoncatabolism, 249, 249fend products of, 242–243urea as, 245–247, 246fL-glutamate dehydrogenase in,244–245, 244f, 245ftransamination of, 243–244, 243fL-Amino acid oxidase, 86–87in nitrogen metabolism, 244, 244fAmino acid sequences. See also Proteinsequencingdetermination of, for glycoproteins, 515tprimary structure determined by, 18–19repeating, in mucins, 519, 520fAmino acids, 2, 14–20, 15–16t. See alsoPeptidesabsorption of, 477analysis/identification of, 20blood glucose and, 159branched chain, catabolism of, 259,260f, 261f, 262fdisorders of, 259–262in catalysis, conservation of, 54, 55tchemical reactions of, functional groupsdictating, 18–20deamination of. See Deaminationdeficiency of, 237, 480excitatory. See also Aspartate; Glutamateglucogenic, 231–232in gluconeogenesis, 133–134, 134finterconvertability of, 231–232keto acid replacement of in diet, 240ketogenic, 232melting point of, 18metabolism of, 122f, 124, 124f, 125f. Seealso Amino acid carbon skeletons,catabolism of; Amino acidnitrogen, catabolism ofpyridoxal phosphate in, 491net charge of, 16–17, 17fnutritionally essential, 124nutritionally nonessential, 124synthesis of, 237–241in peptides, 14, 19, 19fpK/pK a values of, 15–16t, 17, 17fenvironment affecting, 18, 18tproducts derived from, 264–269. See alsospecific productproperties of, 14–18α-R group determining, 18protein degradation and, 242, 243fin proteins, 14removal of ammonia from, 244, 244frequirements for, 480sequence of, primary structuredetermined by, 18–19solubility point of, 18substitutions of, missense mutationscaused by, 362–363, 362fsynthesis of, 237–241in carbohydrate metabolism, 123citric acid cycle in, 133, 134ftransamination of. See Transaminationtransporter/carrier systems for, 99glutathione and, 629–630hormones affecting, 427Aminoacyl residues, 18–19peptide structure and, 19Aminoacyl (A/acceptor) site, aminoacyltRNAbinding to, in proteinsynthesis, 368, 368fAminoacyl-tRNA, in protein synthesis, 368Aminoacyl-tRNA synthetases, 360, 360fγ-Aminobutyrate, 267, 268fβ-Aminoisobutyrate, 300, 301fAminolevulinate (ALA), 270, 273fin porphyria, 278Aminolevulinate dehydratase, 270, 273fin porphyria, 274, 277tAminolevulinate synthase, 270, 272–273,273f, 276fin porphyria, 274, 277f, 278Aminopeptidases, 477Aminophospholipids, membraneasymmetry and, 420Aminoproteinase, procollagen, 537Amino sugars (hexosamines), 106, 106fglucose as precursor of, 169, 171fin glycosaminoglycans, 109, 169, 171fin glycosphingolipids, 169, 171finterrelationships in metabolism of,171fAminotransferases (transaminases),133–134, 134fdiagnostic significance of, 57tin urea biosynthesis, 243–244, 244fAmmoniain acid-base balance, 245detoxification of, 242excess of, 247glutamine synthase fixing, 245, 245fnitrogen removed as, 244, 244fAmmonia intoxication, 244Ammonium ion, pK/pK a value of, 12tAmobarbital, oxidative phosphorylation/respiratory chain affected by, 92,95, 96fAMP, 287f, 288f, 288t, 297fcoenzyme derivatives of, 290tcyclic. See Cyclic AMPfree energy of hydrolysis of, 82tIMP conversion to, 293, 296ffeedback-regulation of, 294, 296fPRPP glutamyl amidotransferaseregulated by, 294structure of, 83f, 288fAmp resistance genes, 402, 403fAmphibolic pathways/processes, 122citric acid cycle and, 133Amphipathic α-helix, 31–32Amphipathic lipids, 119–121, 120fin lipoproteins, 205, 207fin membranes, 119, 120f, 417–418, 417fAmphipathic molecules, folding and, 6Ampicillin resistance genes, 402, 403fAmplification, gene, in gene expressionregulation, 392–393, 393fAmylasesdiagnostic significance of, 57tin hydrolysis of starch, 474β-Amyloid, in Alzheimer disease, 37, 590Amyloid-associated protein, 590Amyloid precursor proteins, 590in Alzheimer disease, 37, 590Amyloidosis, 590–591Amylopectin, 107, 108fAmylopectinosis, 152tAmylose, 107, 108f

646 / INDEXAnabolic pathways/anabolism, 81, 122. Seealso Endergonic reaction;MetabolismAnaerobic glycolysis, 136, 137f, 139as muscle ATP source, 574–576, 575f,575tAnalbuminemia, 584Anaphylaxis, slow-reacting substance of,196Anaplerotic reactions, in citric acid cycle,133Anchorin, in cartilage, 551tAndersen’s disease, 152tAndrogen response element, 459tAndrogensestrogens produced from, 442–445, 444freceptors for, 471synthesis of, 440–442, 441f, 443fAndrostenedioneestrone produced from, 444f, 445synthesis of, 442, 443fAnemias, 47Fanconi’s, 338hemolytic, 136, 143, 609, 619, 620tglucose-6-phosphate dehydrogenasedeficiency causing, 163,169–170, 613, 614f, 619haptoglobin levels in, 584hyperbilirubinemia/jaundice in, 282,284, 284tpentose phosphate pathway/glutathione peroxidase and, 166,167f, 169–170primaquine-sensitive, 613red cell membrane abnormalitiescausing, 619iron deficiency, 478, 497, 586, 610tmegaloblasticfolate deficiency causing, 482t, 492,494vitamin B 12 deficiency causing, 482t,492, 610tpernicious, 482t, 492recombinant erythropoietin for, 526, 610sickle cell. See Sickle cell diseaseAngiotensin II, 437, 451, 452fsynthesis of, 451–452, 452fAngiotensin III, 452, 452fAngiotensin-converting enzyme, 451–452,452fAngiotensin-converting enzyme inhibitors,451–452Angiotensinogen, 451, 452fAnion exchange protein, 615, 615f, 616tAnkyrin, 615f, 616f, 616t, 617Anomeric carbon atom, 104Anomers, α and β, 104Anserine, 264, 265, 265fAntennae (oligosaccharide branches), 521Anterior pituitary gland hormones, bloodglucose affected by, 161Anti conformers, 287, 287fAntibioticsamino sugars in, 106bacterial protein synthesis affected by,371–372folate inhibitors as, 494Antibodies, 580, 581. See alsoImmunoglobulinsmonoclonal, hybridomas in productionof, 595–596, 596fin xenobiotic cell injury, 631, 631fAntibody diversity, 591DNA/gene rearrangement and, 593–594Antichymotrypsin, 583tAnticoagulants, coumarin, 604Anticodon region, of tRNA, 359, 360, 360fAntifolate drugs, purine nucleotidesynthesis affected by, 293Antigenic determinant (epitope), 33, 591Antigenicity, xenobiotics altering, cell injuryand, 631, 631fAntigens, 591Antihemophilic factor A/globulin (factorVIII), 599f, 600, 600tdeficiency of, 604Antihemophilic factor B (factor IX), 599f,600, 600tcoumarin drugs affecting, 604deficiency of, 604Antimalarial drugs, folate inhibitors as,494Antimycin A, respiratory chain affected by,95, 96fAntioxidants, 91, 119, 611–613, 613tretinoids and carotenoids as, 119, 482tvitamin C as, 119vitamin E as, 91, 119, 486, 487fAntiparallel loops, mRNA and tRNA, 360Antiparallel β sheet, 32, 33fAntiparallel strands, DNA, 303Antiport systems, 426, 426ffor nucleotide sugars, 517α 1 -Antiproteinase (α 1 -antitrypsin), 583t,589deficiency of, 589–590, 589f, 590f,623as thrombin inhibitor, 603Antiproteinases, 623, 624tAntithrombin/antithrombin III, 583t,603–604heparin binding to, 547, 603–604α 1 -Antitrypsin (α 1 -antiproteinase), 583t,589deficiency of, 589–590, 589f, 590f,623–624as thrombin inhibitor, 603APC. See Activated protein CApo A-I, 206, 206t, 224deficiencies of, 228tApo A-II, 206tlipoprotein lipase affected by, 207–208Apo A-IV, 206, 206tApo B-48, 206, 206tApo B-100, 206, 206tin LDL metabolism, 209, 210fregulation of, 223Apo C-I, 206, 206tApo C-II, 206, 206tin lipoprotein lipase activity, 207–208Apo C-III, 206, 206tlipoprotein lipase affected by, 208Apo D, 206, 206tApo E, 206, 206treceptors forin chylomicron remnant uptake,208–209, 209fin LDL metabolism, 209, 210fApoferritin, 586Apolipoproteins/apoproteins, 205,205–206distribution of, 205–206, 206themoglobin; oxygenation affecting, 42Apomyoglobin, hindered environment forheme iron and, 41, 41fApoproteins. See Apolipoproteins/apoproteinsApoptosis, 201p53 and, 339Apo-transketolase, activation of, in thiaminnutritional status assessment, 489APP. See Amyloid precursor proteinApurinic endonuclease, in base excisionrepair,337Apyrimidinic endonuclease, in baseexcision-repair, 337Aquaporins, 424–426D-Arabinose, 104f, 105tArabinosyl cytosine (cytarabine), 290Arachidonic acid/arachidonate, 113t, 190,190feicosanoid formation and, 192, 193f,194, 194f, 195ffor essential fatty acid deficiency,191–192ARC, 472t, 473ARE. See Androgen response elementArgentaffinoma (carcinoid), serotonin in,266–267Arginasein periodic hyperlysinemia, 258in urea synthesis, 246f, 247Arginine, 16t, 265, 266fcatabolism of, 250, 251fin urea synthesis, 246f, 247Arginosuccinasedeficiency of, 248in urea synthesis, 246f, 247Arginosuccinate, in urea synthesis, 245,246f, 247Arginosuccinate synthase, 246f, 247deficiency of, 247Arginosuccinicaciduria, 248

INDEX / 647Aromatase enzyme complex, 442, 444f, 445ARS (autonomously replicating sequences),326, 413Arsenate, oxidation and phosphorylationaffected by, 137, 142Arterial wall, intima of, proteoglycans in,548Arthritisgouty, 299proteoglycans in, 548rheumatoid, glycosylation alterations in,533Artificial membranes, 421–422Ascorbate, 167, 168fAscorbic acid (vitamin C), 163, 482t,495–496, 496fas antioxidant, 119in collagen synthesis, 38, 496, 535deficiency of, 482t, 496collagen affected in, 38–39, 496,538–539iron absorption and, 478, 496supplemental, 496Asialoglycoprotein receptorsin cotranslational insertion, 506, 506fin glycoprotein clearance, 517Asn-GlcNAc linkagein glycoproteins, 521in glycosaminoglycans, 543Asparaginase, in amino acid nitrogencatabolism, 245, 245fAsparagine, 15tin amino acid nitrogen catabolism, 245catabolism of, 249, 250fsynthesis of, 237–238, 238fAsparagine synthetase, 238, 238fAspartatecatabolism of, 249, 250fsynthesis of, 237–238, 238fin urea synthesis, 246f, 247Aspartate 102, in covalent catalysis, 53–54,54fAspartate aminotransferase (AST/SGOT),diagnostic significance of, 57tAspartate transcarbamoylase, 75in pyrimidine synthesis, 298f, 299Aspartic acid, 15tpI of, 17Aspartic protease family, in acid-basecatalysis, 52, 53fAspartylglycosaminuria, 532–533, 533tAspirinantiplatelet actions of, 607–608cyclooxygenase affected by, 193prostaglandins affected by, 190Assembly particles, in absorptivepinocytosis, 430AST. See Aspartate aminotransferaseAsthma, leukotrienes in, 112Asymmetric substitution, in porphyrins,270, 271fAsymmetryimportin binding and, 501lipid and protein, membrane assemblyand, 511, 512fin membranes, 416, 419–420Ataxia-telangiectasia, 338ATCase. See Aspartate transcarbamoylaseAtherosclerosis, 205, 607cholesterol and, 117, 219, 227HDL and, 210–211hyperhomocysteinemia and, folic acidsupplements in prevention of,494LDL receptor deficiency in, 209lysophosphatidylcholine (lysolecithin)and, 116Atorvastatin, 229ATP, 82, 82–85, 287f, 289in active transport, 427–428, 428fin coupling, 82, 84fatty acid oxidation producing, 182free energy of hydrolysis of, 82–83, 82tin free energy transfer from exergonic toendergonic processes, 82–83, 82fhydrolysis ofin muscle contraction, 561–562, 561fby NSF, 509, 510finorganic pyrophosphate productionand, 85in mitochondrial protein synthesis andimport, 499in muscle/muscle contraction, 556,561–562, 561fdecrease in availability of, 564sources of, 573–574, 574–576, 575f,575tin purine synthesis, 293–294, 295frespiratory control in maintenance ofsupply of, 94–95, 97, 97t, 98f,134–135structure of, 83fsynthesis ofATP synthase in, 96, 97f, 98fin citric acid cycle, 131f, 133, 142,143tglucose oxidation yielding, 142, 143trespiratory chain in, 93–95, 98fATP/ADP cycle, 83, 84fATPasein active transport, 427–428, 428fchaperones exhibiting activity of, 508copper-binding P-type, mutations ingene forMenkes diseases caused by, 588Wilson disease caused by, 588–589ATP-binding cassette transporter-1, 210,211fATP-chaperone complex, 508. See alsoChaperonesATP-citrate lyase, 134, 135f, 156t, 157acetyl-CoA for lipogenesis and, 177ATP synthase, membrane-located, 96, 97f,98fAtractyloside, respiratory chain affected by,95Attachment proteins, 540, 541fAutoantibodies, in myasthenia gravis, 431Autonomously replicating sequences (ARS),326, 413Auto-oxidation. See PeroxidationAutoradiography, definition of, 413Autotrophic organisms, 82Avidin, biotin deficiency caused by, 494Axial ratios, 30Axonemal dyneins, 5775- or 6-Azacytidine, 2908-Azaguanine, 290, 291fAzathioprine, 2905- or 6-Azauridine, 290, 291fB blood group substance, 618, 619fB (β) cells, pancreatic, insulin produced by,160B cyclins, 333, 334f, 335tB gene, Gal transferase encoded by,618–619B lymphocytes, 591in hybridoma production, 595–596, 596fB vitamins. See Vitamin B complexBAC vector. See Bacterial artificialchromosome (BAC) vectorBacteriaintestinal, in bilirubin deconjugation, 281transcription cycle in, 342–343, 342fBacterial artificial chromosome (BAC)vector, 401–402, 402tfor cloning in gene isolation, 635tin Human Genome project, 634Bacterial gyrase, 306Bacterial promoters, in transcription,345–346, 345fBacteriophage, definition of, 413Bacteriophage lambda (λ), 378–383, 379f,380f, 381f, 382fBAL. See DimercaprolBAL 31 nuclease, in recombinant DNAtechnology, 400tBalanced chemical equations, 60BamHI, 398, 399tBarbiturates, respiratory chain affected by,95, 96fBasal lamina, laminin as component of,540–542Basal metabolic rate, 478Base excision-repair of DNA, 336t, 337,337fBase pairing in DNA, 7, 303, 304, 305fmatching of for renaturation, 305–306recombinant DNA technology and,396–397replication/synthesis and, 328–330, 330f

648 / INDEXBase substitution, mutations occurring by,361, 361f, 362Basement membranes, collagen in, 537Basesconjugate, 10as proton acceptors, 9strong, 9weak, 9Bence Jones protein, 595Bends (protein conformation), 32–33,34fBeriberi, 482t, 489Beta-alanine. See β-AlanineBeta anomers, 104Beta- (β) carotene, 482, 482t, 483, 483f.See also Vitamin Aas antioxidant, 119, 482tBeta-endorphins, 453, 453fBeta-globin genelocalization of, 407trecombinant DNA technology indetection of variations in,407–408, 408f, 409tBeta-lipoproteins, 205. See also low densitylipoproteinsBeta-oxidation of fatty acids, 181–183,181f, 182fketogenesis regulation and, 186–187,187f, 188fmodified, 183, 183fBeta (β) sheet, 32, 33fBeta subunits of hemoglobin, myoglobinand, 42Beta (β) thalassemia, 47Beta (β) turn, 32, 34fBFU-E. See Burst-forming unit-erythroidBgIII, 399tBHA. See Butylated hydroxyanisoleBHT. See Butylated hydroxytolueneBi-Bi reactions, 69–70, 69f, 70fMichaelis-Menten kinetics and, 70, 70fBicarbonate, in extracellular andintracellular fluid, 416tBiglycanin bone, 548tin cartilage, 551tBilayers, lipid, 418–419, 418f, 419fmembrane proteins and, 419Bile, bilirubin secretion into, 280, 281fBile acids (salts), 225–227enterohepatic circulation of, 227in lipid digestion and absorption, 475,476fsecondary, 226f, 227synthesis of, 225–227, 226fregulation of, 226, 226f, 227Bile pigments, 278–284, 282f. See alsoBilirubinBiliary obstruction,hyperbilirubinemia/jaundicecaused by, 283, 284, 284tBilirubinaccumulation of (hyperbilirubinemia),281–284, 284tconjugatedbinding to albumin and, 283reduction of to urobilinogen, 281,282fconjugation of, 280, 280f, 281ffecal, in jaundice, 284theme catabolism producing, 278–280,279fliver uptake of, 280–281, 280f, 281f,282fnormal values for, 284tsecretion of into bile, 280, 281funconjugated, disorders occurring in,282–283urine, in jaundice, 284, 284tBiliverdin, 278, 279fBiliverdin reductase, 278Bimolecular membrane layer, 418–419. Seealso Lipid bilayerBinding constant, Michaelis constant (K m )approximating, 66Binding proteins, 454–455, 454t, 455t, 583tBiochemistryas basis of health/disease, 2–4, 3tdefinition of, 1Human Genome Project and, 3–4methods and preparations used in, 1, 2trelationship of to medicine, 1–4, 3fBiocytin, 494, 495fBioenergetics, 80–85. See also ATPBioinformatics, 412, 638protein function and, 28–29Biologic oxidation. See also OxidationBiomolecules. See also specific typestabilization of, 7water affecting structure of, 6–7, 6tBiotechnology, Human Genome Projectaffecting, 638Biotin, 482t, 494–495, 495fdeficiency of, 482t, 494in malonyl-CoA synthesis, 173, 174fas prosthetic group, 50BiP. See Immunoglobulin heavy chainbinding protein1,3-Bisphosphoglycerate (BPG), free energyof hydrolysis of, 82t2,3-Bisphosphoglycerate (BPG), T structureof hemoglobin stabilized by, 45,45fBisphosphoglycerate mutase, in glycolysis inerythrocytes, 140, 140f2,3-Bisphosphoglycerate phosphatase, inerythrocytes, 140, 140fBlindness, vitamin A deficiency causing, 483Bloodcoagulation of, 598–608. See alsoCoagulation; Coagulation factorsfunctions of, 580, 581tBlood cells, 609–625. See also Erythrocytes;Neutrophils; PlateletsBlood clotting. See CoagulationBlood glucosenormal, 145regulation ofclinical aspects of, 161–162, 161fdiet/gluconeogenesis/glycogenolysis in,158–161, 159f, 160fglucagon in, 160–161glucokinase in, 159–160, 160fglycogen in, 145insulin in, 160limits of, 158metabolic and hormonal mechanismsin, 159, 160t, 161Blood group substances, 618, 619fglycoproteins as, 514, 618Blood group systems, 617–619, 619fBlood plasma. See PlasmaBlood type, 617–618Blood vessels, nitric oxide affecting,571–573, 573f, 574tBlot transfer techniques, 403, 404fBlunt end ligation/blunt-ended DNA, 398,399–400, 400f, 413BMR. See Basal metabolic rateBody mass index, 478Body water. See WaterBohr effect, 44, 45fin hemoglobin M, 46Bone, 549–550, 549f, 550fmetabolic and genetic disordersinvolving, 551–552, 551tproteins in, 548t, 549Bone Gla protein, 548tBone marrow, heme synthesis in, 272Bone matrix Gla protein, 488Bone morphogenetic proteins, 548tBone sialoprotein, 548tBone SPARC protein, 548tBotulinum B toxin, 511Bovine spongiform encephalopathy, 37BPG. See 1,3-Bisphosphoglycerate;2,3-BisphosphoglycerateBradykinin, in inflammation, 621Brain, metabolism in, 235tglucose as necessity for, 232Branched chain amino acids, catabolism of,259, 260f, 261f, 262fdisorders of, 259–262Branched-chain α-keto acid dehydrogenase,259Branched chain ketonuria (maple syrupurine disease), 259Branching enzymesabsence of, 152tin glycogen biosynthesis, 145, 147fBrefeldin A, 510–511Brittle bones (osteogenesis imperfecta), 551tBroad beta disease, 228t

INDEX / 649Bronze diabetes, 587Brown adipose tissue, 217, 217fBrush border enzymes, 475BSE. See Bovine spongiform encephalopathyBuffersHenderson-Hasselbalch equationdescribing behavior of, 11, 12fweak acids and their salts as, 11–12, 12f“Bulk flow,” of membrane proteins, 507Burst-forming unit-erythroid, 610, 611fButylated hydroxyanisole (BHA), asantioxidant/food preservative, 119Butylated hydroxytoluene (BHT), asantioxidant/food preservative, 119Butyric acid, 112tC1–9 (complement proteins), 596C-peptide, 449, 450fC 20 polyunsaturated acids, eicosanoidsformed from, 190, 192, 193f,194fC-reactive protein, 583, 583tC regions/segments. See Constantregions/segmentsCa 2+ ATPase, 463Ca 2+ -Na + exchanger, 463, 567–568Cachexia, cancer, 136, 479Caffeine, 289, 289fhormonal regulation of lipolysis and, 215Calbindin, 477Calcidiol (25-hydroxycholecalciferol), invitamin D metabolism, 484, 485fCalciferol. See Vitamin DCalcineurin, 566tCalcinosis, 486Calcitonin, 437Calcitriol (1,25(OH) 2 -D 3 ), 437, 439f, 485calcium concentration regulated by, 485storage/secretion of, 453, 454tsynthesis of, 445, 446f, 484, 485fCalcium, 496tabsorption of, 477vitamin D metabolism and, 477, 484,484–485in blood coagulation, 599f, 600, 600t,601in bone, 549in extracellular fluid, 416, 416tin intracellular fluid, 416, 416tiron absorption affected by, 478in malignant hyperthermia, 564–565,565fmetabolism of, 463vitamin D metabolism and, 484–485in muscle contraction, 562in cardiac muscle, 566–568phosphorylase activation and, 148sarcoplasmic reticulum and, 563–564,563f, 564fin smooth muscle, 570, 571in platelet activation, 606f, 607as second messenger, 436–437, 437t,457, 463–465, 463tphosphatidylinositide metabolismaffecting, 464–465, 464f, 465fvitamin D metabolism affected by,485–486Calcium ATPase, 463, 568Calcium-binding proteins, vitamin K andglutamate carboxylation andpostsynthetic modificationand, 487–488, 488fsynthesis and, 488, 604Calcium/calmodulin, 463Calcium/calmodulin-sensitive phosphorylasekinase, in glycogenolysis, 148Calcium channels, 463. See also Calciumrelease channelin cardiac muscle, 566–567Calcium-induced calcium release, in cardiacmuscle, 567Calcium pump, 463, 568Calcium release channeldihydropyridine receptor and, 563–564,563fmutations in gene for, malignanthyperthermia caused by,564–565, 565f, 630tCalcium release channel, 563, 564fCalcium-sodium exchanger, 463Caldesmon, 571Calmodulin, 463, 463t, 562muscle phosphorylase and, 148, 149fCalmodulin-4Ca 2+ , in smooth musclecontraction, 570–571, 571fCalnexin, 508, 526Calreticulin, 508, 526Calsequestrin, 563, 563fcAMP. See Cyclic AMPCancer/cancer cells. See alsoCarcinogenesis/carcinogenscyclins and, 334glycoproteins and, 514, 526, 530t, 531hormone-dependent, vitamin B 6deficiency and, 491membrane abnormalities and, 432tmucins produced by, 520Cancer cachexia, 136, 479Cancer chemotherapyfolate inhibitors in, 494neutropenia caused by, 610synthetic nucleotide analogs in,290–291, 291fCancer phototherapy, porphyrins in,273CAP. See Catabolite gene activator proteinCaproic acid, 112tCarbamates, hemoglobin, 44Carbamoyl phosphateexcess, 301free energy of hydrolysis of, 82tin urea synthesis, 245, 246–247, 246f,247Carbamoyl phosphate synthase Ideficiency of, 247in urea synthesis, 245–246, 246fCarbamoyl phosphate synthase II, in pyrimidinesynthesis, 296, 298f, 299Carbohydrates, 102–110. See also specifictype and Glucose; Sugarsin cell membranes, 110cell surface, glycolipids and, 116classification of, 102, 102tcomplex (glycoconjugate), glycoproteinsas, 514digestion and absorption of, 474–475,475finterconvertibility of, 231isomerism of, 102–104, 103fin lipoproteins, 110metabolism of, 122–123, 122f, 123f,124–125, 125fdiseases associated with, 102vitamin B 1 (thiamin) in, 488–489,489fin proteoglycans, 542, 543, 543fCarbon dioxidecitric acid cycle in production of,130–133, 132ftransport of, by hemoglobin, 44, 45fCarbon monoxideheme catabolism producing, 278oxidative phosphorylation/respiratorychain affected by, 92, 95, 96fCarbon skeleton, amino acid. See Aminoacid carbon skeletonsCarbonic acid, pK/pK a value of, 12tCarbonic anhydrase, in osteopetrosis, 552Carboxybiotin, 494, 495fγ-Carboxyglutamate, vitamin K in synthesisof, 487, 488fCarboxyl terminal repeat domain, 350Carboxylase enzymes, biotin as coenzymeof, 494–495Carboxypeptidases, 477Carboxyproteinase, procollagen, 537Carcinogenesis/carcinogens, 631chemical, 631cytochrome P450 induction and, 628indirect, 631Carcinoid (argentaffinoma), serotonin in,266–267Carcinoid syndrome, 490Cardiac developmental defects, 570Cardiac glycosides, 106Cardiac muscle, 556, 566–570, 568t, 569tCardiolipin, 115, 115fsynthesis of, 197, 197f, 199, 199fCardiomyopathies, 556, 569–570, 569tCargo proteins/molecules, 510in export, 503in import, 501, 502f

650 / INDEXCarnitinedeficiency of, 180, 187in fatty acid transport, 180–181, 181fCarnitine-acylcarnitine translocase, 180,181fCarnitine palmitoyltransferase, deficiencyof, 180Carnitine palmitoyltransferase-I, 180,181fdeficiency of, 187in ketogenesis regulation, 186–187, 187f,188fCarnitine palmitoyltransferase-II, 181, 181fdeficiency of, 187–188Carnosinase deficiency, 264Carnosine, 264, 265, 265fCarnosinuria, 264β-Carotene, 482, 482t, 483, 483f. See alsoVitamin Aas antioxidant, 119, 482tCarotene dioxygenase, 482–483, 483fCarotenoids, 482–484, 483f, 484f. See alsoVitamin ACarrier proteins/systems, 426, 426ffor nucleotide sugars, 517Cartilage, 543, 551t, 552–553, 553fchondrodysplasia affecting, 553–554Catabolic pathways/catabolism, 81, 122.See also Exergonic reaction;MetabolismCatabolite gene activator protein (cyclicAMP regulatory protein), 376,378Catabolite regulatory protein, 460Catalase, 88–89as antioxidant, 119, 611–613, 613tin nitrogen metabolism, 244, 244fCatalysis/catalytic reactions (enzymatic). Seealso Metabolismacid-base, 51–52HIV protease in, 52, 53fat active site, 51, 51fBi-Bi, 69–70, 69f, 70fMichaelis-Menten kinetics and, 70,70fcoenzymes/cofactors in, 50–51, 51fconservation of residues and, 54, 55tcovalent, 52, 52f, 63chymotrypsin in, 52–54, 54f, 63fructose-2,6-bisphosphatase in, 54, 55fdouble displacement, 69–70, 69fenzyme detection facilitated by, 55–56,56fequilibrium constant and, 63isozymes and, 54–55kinetics of, 63–70activation energy and, 61, 63balanced equations and, 60competitive versus noncompetitiveinhibition and, 67–69, 67f,68f, 69ffactors affecting rates of, 61–63, 62f,63–64, 64ffree energy changes and, 60–61initial velocity and, 64multiple substrates and, 69–70, 69f,70fsubstrate concentration and, 64, 64f,65fmodels of, 65–67, 66f, 67ftransition states and, 61mechanisms of, 51–52, 52fprosthetic groups/cofactors/coenzymesin, 50–51, 51fsite-directed mutagenesis in study of,58oxaloacetate and, 130ping-pong, 69–70, 69fprosthetic groups in, 50–51, 51fby proximity, 51regulation of, 72–79, 128f, 129active and passive processes in, 72, 73fallosteric, 74, 74–76, 75f, 128f, 129compartmentation in, 72–73covalent, 74, 76, 77–78, 78fenzyme quantity and, 73–74feedback inhibition and, 74–76, 75f,76, 129feedback regulation and, 76, 129metabolite flow and, 72, 73fMichaelis constant (K m ) in, 72, 73fphosphorylation-dephosphorylationin, 78–79, 78tproteolysis in, 76–77, 77fsecond messengers in, 76RNA and, 356sequential (single) displacement, 69, 69fspecificity of, 49, 50fby strain, 52substrate concentration affecting rate of,64, 64f, 65fHill model of, 66–67, 67fMichaelis-Menten model of, 65–66,66fCatalytic residues, conserved, 54, 55tCatalytic site, 75. See also Active siteCataracts, diabetic, 172Catecholamines. See also specific typereceptors for, 436storage/secretion of, 453, 454tsynthesis of, 445–447, 447fCathepsins, in acid-base catalysis, 52Caveolae, 422Caveolin-1, 422CBG. See Corticosteroid-binding globulinCBP/CBP/p300 (CREB-binding protein),461, 468, 469, 469f, 471–472,472tCD11a-c/CD18, in neutrophils, 621, 621tCD18, 620–621CD49a/e/f, 622tCD59, 531CDK-cyclin inhibitor/CDKI, DNA/-chromosome integrity and, 339CDKs. See Cyclin-dependent proteinkinasescDNA, 413sequencing, in glycoprotein analysis, 515tcDNA library, 402, 413CDRs. See Complementarity-determiningregionsCeliac disease, 474Cell, 1injury tooxygen species causing, 611–613,613txenobiotics causing, 631, 631flysis of, complement in, 596in macromolecule transport, 428–431,429f, 430fCell adhesionfibronectin in, 540, 541fglycosphingolipids in, 202integrins in, 620–621, 622tselectins in, 528–529, 529t, 530fCell biology, 1Cell-cell interactions, 415mucins in, 520Cell cycle, S phase of, DNA synthesisduring, 333–335, 334f, 335tCell death, 201Cell-free systems, vesicles studied in, 509Cell fusion, 595Cell-mediated immunity, 591Cell membrane. See Plasma membraneCell migration, fibronectin in, 540Cell recognition, glycosphingolipids in, 202Cell sap. See CytosolCell surface carbohydrates, glycolipids and,116Cell surfaces, heparan sulfate on, 545Cellulose, 109Cellulose acetate zone electrophoresis, 580,582fCentral core disease, 565, 569tCentral nervous system, glucose asmetabolic necessity for, 232Centromere, 318, 319fCephalin (phosphatidylethanolamine), 115,115fmembrane asymmetry and, 420synthesis of, 197, 197fCeramide, 116, 116f, 201–202, 202f, 203fsynthesis of, 201–202, 202fCerebrohepatorenal (Zellweger) syndrome,188, 503, 503tCerebrosides, 201Ceruloplasmin, 583t, 587, 588deficiency of, 589diagnostic significance of, 57t, 587Cervonic acid, 113tCFTR. See Cystic fibrosis transmembraneregulator

INDEX / 651CFU-E. See Colony-forming unit-erythroidChain elongation. See also Elongationby DNA polymerase, 328in glycosaminoglycan synthesis, 543in transcription cycle, 342, 342fChain initiation. See also Initiationin transcription cycle, 342, 342fChain termination. See also Terminationin glycosaminoglycan synthesis, 543in transcription cycle, 342, 342fChanneling, in citric acid cycle, 130Channelopathies, 568, 569tChaperones, 36–37, 507–508, 508tATPase activity of, 508ATP-dependent protein binding to, 499,508histone, 315in protein sorting, 499, 508tChaperonins, 36–37Charging, in protein synthesis, 360, 360fCheckpoint controls, 339Chédiak-Higashi syndrome, 512tChemical carcinogenesis/carcinogens, 631Chemiosmotic theory, 92, 95–97, 97fexperimental findings in support of, 96respiratory control and uncouplers and,97Chemotactic factors, 620Chemotherapy, cancerfolate inhibitors in, 494neutropenia caused by, 610synthetic nucleotide analogs in,290–291, 291fChenodeoxycholic acid, 225, 226fChenodeoxycholyl CoA, 226, 226fChimeric gene approach, 385–386, 387f,388fChimeric molecules, 397–406, 413restriction enzymes and DNA ligase inpreparation of, 399–400, 401fChips, gene array, protein expression and,28Chitin, 109, 109fChloridein extracellular and intracellular fluid,416, 416tpermeability coefficient of, 419fChlorophyll, 270Cholecalciferol (vitamin D 3 )synthesis of in skin, 445, 446f, 484, 485fin vitamin D metabolism, 484, 485fCholestatic jaundice, 283Cholesterol, 117, 118, 119f, 205, 219–230in bile acid synthesis, 225–227, 226fin calcitriol (1,25(OH) 2 -D 3 ) synthesis,445, 446fdietary, 219excess of. See Hypercholesterolemiaexcretion of, 225–227, 226fin hormone synthesis, 438, 438–445,439t, 440fin lipoprotein, 205, 207fin membranes, 417fluid mosaic model and, 422metabolism of, 123–124, 123fclinical aspects of, 227–229, 228tdiurnal variations in, 220high-density lipoproteins in,209–211, 211fplasma levels ofatherosclerosis and coronary heartdisease and, 227dietary changes affecting, 227drug therapy affecting, 229lifestyle changes affecting, 227–229normal, 223synthesis of, 219–220, 220f, 221f, 222facetyl-CoA in, 123f, 124, 219–220,220f, 221f, 222fcarbohydrate metabolism and, 123HMG-CoA reductase in regulation of,220, 223fin tissues, 118, 119ffactors affecting balance of, 220–223,224ftransport of, 223–224, 225freverse, 210, 211f, 219, 224Cholesteryl ester hydrolase, 223Cholesteryl ester transfer protein, 224, 225fCholesteryl esters, 118, 205, 224in lipoprotein core, 205, 207fCholestyramine resins, forhypercholesterolemia, 229Cholic acid, 225Choline, 114–115, 115fdeficiency of, fatty liver and, 212in glycine synthesis, 238, 239fmembrane asymmetry and, 420Cholinesterase. See AcetylcholinesteraseCholuric jaundice, 282Cholyl CoA, in bile acid synthesis, 226,226fChondrodysplasias, 551t, 553–554, 554fChondroitin sulfates, 109, 109f, 538,543–545, 544f, 544tfunctions of, 547Chondronectin, 551t, 553Chorionic gonadotropin, human (hCG),438Christmas factor (factor IX), 599f, 600, 600tcoumarin drugs affecting, 604deficiency of, 604Chromatidsnucleoprotein packing in, 318, 319t,320fsister, 318, 319fexchanges between, 325, 325fChromatin, 314–316, 315f, 315tactive vs. inactive regions of, 316–318,318fhigher order structure/compaction of,316, 317freconstitution of, in DNA replication,333remodeling of in gene expression,383–384Chromatography. See also specific typeaffinityfor protein/peptide purification, 23for recombinant fusion proteinpurification, 58, 59ffor protein/peptide purification, 21–24Sepharose-lectin column, forglycoprotein analysis, 515tChromium, 496tChromosomal integration, 324, 324fChromosomal recombination, 323–324,323f, 324fChromosomal transposition, 324–325Chromosome jumping, 635tChromosome walking, 411, 411f, 635tChromosomes, 318–319, 319f, 319t, 320f,321fintegrity of, monitoring, 339interphase, chromatin fibers in, 316metaphase, 317f, 318, 319tpolytene, 318, 318fvariations in, 636Chronic granulomatous disease, 623, 623fChyle, 207Chylomicron remnants, 206t, 208, 209fliver uptake of, 208–209Chylomicrons, 125, 205, 206tapolipoproteins of, 206, 206tmetabolism of, 125, 126f, 207–209,209fin triacylglycerol transport, 207, 208f,209fChymotrypsin, 477conserved residues and, 55tin covalent catalysis, 52–54, 54fin digestion, 477for polypeptide cleavage, 26tChymotrypsinogen, 477cI repressor protein/cI repressor gene,379–383, 380f, 381f, 382fCICR. See Calcium-induced calcium releaseCirrhosis of liver, 130, 212in α 1 -antitrypsin deficiency, 590Cistron, 375–376Citratein citric acid cycle, 130, 131fin lipogenesis regulation, 178Citrate synthase, 130, 132fCitric acid, pK/pK a value of, 12tCitric acid cycle (Krebs/tricarboxylic acidcycle), 83, 130–135, 131f, 132fATP generated by, 131f, 133, 142, 143tcarbon dioxide liberated by, 130–133,132fdeamination and, 133–134gluconeogenesis and, 133, 134f,153–155, 154f

652 / INDEXCitric acid cycle (cont.)in metabolism, 122, 122f, 123f, 124f,126, 127f, 130, 133–135, 134famino acid, 122f, 124fcarbohydrate, 122–123, 122f, 123f,133–134, 134flipid/fatty acid, 122f, 123, 123f, 134,135fat subcellular level, 126, 127fin mitochondria, 126, 127freducing equivalents liberated by,130–133, 132fregulation of, 134–135respiratory chain substrates provided by,130, 131ftransamination and, 133–134, 134fvitamins in, 133Citrulline, in urea synthesis, 245, 246–247,246f, 247Citrullinemia, 247CJD. See Creutzfeldt-Jakob diseaseCl. See ChlorideClass B scavenger receptor B1, 210, 211fClass (isotype) switching, 594Classic pathway, of complement activation,596Clathrin, 429f, 430Clathrin-coated vesicles, 510Cleavage, in protein sequencing, 25, 26tCLIP, 453, 453fClofibrate, 229Clonesdefinition of, 413library of, 402, 413in monoclonal antibody production, 596Cloning, 400–402, 401f, 402t, 403fin gene isolation, 635tCloning vectors, 400–402, 401f, 402t,403f, 414Closed complex, 345Clotting factors, 600t. See also specific typeunder Factorvitamin K in synthesis of, 486–488, 488fCMP, 288tCMP-NeuAc, 516t, 517CNBr. See Cyanogen bromideCO. See Carbon monoxideCO 2 . See Carbon dioxideCoA. See Coenzyme ACoactivators, transcription, 351, 351tCoagulation (blood), 598–608endothelial cell products in, 607, 607textrinsic pathway of, 598, 599f, 601fibrin formation in, 598–601, 599ffinal common pathway in, 598, 599,601, 602fintrinsic pathway of, 598, 599f, 600–601laboratory tests in evaluation of, 608prostaglandins in, 190proteins involved in, 599–600, 600t. Seealso Coagulation factorsvitamin K in, 486–488, 488fcoumarin anticoagulants affecting, 604Coagulation factors, 600t. See also specifictype under Factorvitamin K in synthesis of, 486–488, 488fCoat proteins, recruitment of, 509, 510fCoated pits, in absorptive pinocytosis, 429f,430Coating, vesicle, 509, 510fbrefeldin A affecting, 510–511Cobalamin (vitamin B 12 ), 482t, 491–492,492fabsorption of, 491–492intrinsic factor in, 477, 491–492deficiency of, 482t, 492functional folate deficiency and, 492,494in methylmalonic aciduria, 155Cobalophilin, 492Cobalt, 496tin vitamin B 12Cobamide, coenzymes derived from, 51Coding regions, 319, 321f, 637in recombinant DNA technology, 397,398fCoding strand, 304in RNA synthesis, 341Codon usage tables, 359–360Codons, 358, 359tamino acid sequence of encoded proteinspecified by, 358nonsense, 359Coenzyme A, synthesis of from pantothenicacid, 495, 495fCoenzyme Q (Q/ubiquinone), 92, 95fCoenzymes, 50in catalysis, 50–51, 51fnucleotide derivatives, 290, 290tCofactors, 50in blood coagulation, 600, 600t, 603in catalysis, 50–51, 51fin citric acid cycle regulation, 134–135Colipase, 475Collagen, 37–39, 371, 535–539, 536tin bone, 548t, 549in cartilage, 551t, 552, 553fclassification of, 535, 536telastin differentiated from, 539tfibril formation by, 535–539, 536f, 537tgenes for, 535, 536tdiseases caused by mutations in, 39,538–539, 538tchondrodysplasias, 551t, 553osteogenesis imperfecta, 551maturation/synthesis of, 38–39ascorbic acid (vitamin C) in, 38, 496disorders of, 38–39O-glycosidic linkage in, 518in platelet activation, 605, 606f, 607posttranslational modification of,537–538, 537tsecretion of, 537triple helix structure of, 38, 38f,535–539, 536ftypes of, 535, 536tCollision-induced dissociation, in massspectrometry, 27Collision (kinetic) theory, 61Colon cancer. See Colorectal cancerColony-forming unit-erythroid, 610, 611fColony hybridization, 403–404. See alsoHybridizationColorectal cancer, mismatch repair genes in,336Coltranslational glycosylation, 504Column chromatography, for protein/peptide purification, 21, 22fCombinatorial diversity, 592Compartmentation, 72–73Competitive inhibition, noncompetitiveinhibition differentiated from,67–69, 67f, 68f, 69fComplement, 583t, 596–597in inflammation, 596, 621tComplementarityof DNA, 306, 307frecombinant DNA technology and,396–397of RNA, 306, 309fComplementarity-determining regions,591–592, 594fComplementary DNA (cDNA), 413Complementary DNA (cDNA) library,402, 413Complex (glycoconjugate) carbohydrates,glycoproteins as, 514Complex oligosaccharide chains, 521, 522fformation of, 521, 524Concanavalin A (ConA), 110, 518tConformational diseases, 590Congenital disorders of glycosylation(CDG), 530t, 531Congenital long QT syndrome, 432tCongenital nonhemolytic jaundice (type ICrigler-Najjar syndrome), 283Conjugate acid, 10Conjugate base, 10Conjugationof bilirubin, 280, 280f, 281fof xenobiotics, 626, 628–630Connective tissue, 535bone as, 549–550keratan sulfate I in, 545Connexin, 431Consensus sequences, 353, 353fKozak, 365Conservation of energy, 83Conserved residues, 54, 55tConstant regions/segments, 593gene for, 593DNA rearrangement and, 325–326,393, 593–594

INDEX / 653immunoglobulin heavy chain, 591, 592fimmunoglobulin light chain, 325–326,393, 591, 592fConstitutive enzymes, 75Constitutive gene expression, 376, 378Constitutive heterochromatin, 316Constitutive mutation, 376Constitutive secretion, 498Contig map, 634fContractility/contraction. See MusclecontractionCooperative bindinghemoglobin, 42Bohr effect and, 44, 45fHill equation describing, 66–67, 67fCOPI vesicles, 510COPII vesicles, 510Coplanar atoms, partial double-bondcharacter and, 19, 20fCopper, 496tceruloplasmin in binding of, 587, 588as cofactor, 588, 588tenzymes containing, 588tin Menkes disease, 588metallothioneins in regulation of, 588in oxidases, 86tests for disorders of metabolism of, 588,589tin Wilson disease, 587, 588–589Copper-binding P-type ATPase, mutationsin gene forMenkes diseases caused by, 588Wilson disease caused by, 588–589Copper toxicosis, 588. See also WilsondiseaseCoproporphyrinogen I, 271, 275fCoproporphyrinogen III, 271, 275fCoproporphyrinogen oxidase, 271, 275f,276fin porphyria, 277tCoproporphyrins, 272fspectrophotometry in detection of,273–274Coprostanol (coprosterol), 225Core proteins, 542, 543fin glycosaminoglycan synthesis, 542–543Coregulator proteins, 469, 471–473, 472fCorepressors, 472t, 473Cori cycle, 159, 159fCori’s disease, 152tCornea, keratan sulfate I in, 545, 546, 547Coronary (ischemic) heart disease. See alsoAtherosclerosischolesterol and, 227Corrinoids, 491. See also CobalaminCorticosteroid-binding globulin (CBG/transcortin), 454–455, 455t, 583tcyclooxygenases affected by, 193Corticosteronebinding of, 454, 455tsynthesis of, 438, 440, 441fCorticotropin. See AdrenocorticotropichormoneCortisol, 439f, 440fbinding of, 454, 455, 455tsynthesis of, 440, 441fCos sites, 401Cosmids, 401, 402t, 413for cloning in gene isolation, 635tCothromboplastin (factor VII), 599f, 600t,601coumarin drugs affecting, 604Cotranslational insertion, 504, 505–506Cotransport systems, 426, 426fCoulomb’s law, 5Coumarin, 604Coupling, 81–82, 81f, 82fATP in, 82, 84hormone receptor-effector, 435–436Coupling domains, on hormone receptors,435–436Covalent bondsbiologic molecules stabilized by, 6, 6tmembrane lipid-protein interaction and,419xenobiotic cell injury and, 631, 631fCovalent catalysis, 52, 52f, 63chymotrypsin in, 52–54, 54f, 63fructose-2,6-bisphosphatase in, 54, 55fCovalent cross-links, collagen, 537Covalent modificationmass spectrometry in detection of, 27,27f, 27tin protein maturation, 37–39in regulation of enzymatic catalysis, 74,76, 77–78, 78f. See alsoPhosphorylation; Proteolysisgluconeogenesis regulation and, 157irreversible, 76–77, 77fmetabolite flow and, 79reversible, 77–79, 78f, 78tCPT-I. See Carnitine palmitoyltransferase-ICRE. See Cyclic AMP response elementCreatine, 267, 268fCreatine kinase, diagnostic significance of,57tCreatine phosphate, 267, 268ffree energy of hydrolysis of, 82tin muscle, 573–574, 574–576, 575f,575tCreatine phosphate shuttle, 100, 101fCreatinine, 267, 268fCREB, 461CREB-binding protein, 461, 469, 469f, 471Creutzfeldt-Jakob disease, 37Crigler-Najjar syndrometype I (congenital nonhemolyticjaundice), 283type II, 283Cro protein/cro gene, 379–383, 380f, 381f,382fbinding of to DNA, by helix-turn-helixmotif, 389–390, 389fCross-bridges, 557, 557–559, 558f, 562fCross-links, covalent in collagen, 537Crossing-over, in chromosomal recombination,323–324, 323f, 324fCrouzon syndrome, 551tCRP. See C-reactive protein; Cataboliteregulatory protein; Cyclic AMPregulatory proteinCryoprecipitates, recombinant DNA technologyin production of, 604Cryptoxanthin, 482Crystallography, x-ray, protein structuredemonstrated by, 35CS-PG I/II/III, in bone, 548tCT. See CalcitoninCTD. See Carboxyl terminal repeat domainCTP, 290in phosphorylation, 85CY282Y mutation, in hemochromatosis, 587Cyanide, oxidative phosphorylation/respiratory chain affected by, 92,95, 96fCyanogen bromide, for polypeptide cleavage,25, 26tCyclic AMP, 147, 148f, 289, 289f, 290t,458–462, 460t, 462fadenylyl cyclase affecting, 147, 458–459,460tin cardiac muscle regulation, 566in gluconeogenesis, 158, 158fin glycogen metabolism regulation,147–150, 148f, 149f, 150fas second messenger, 147, 436, 437t,457, 458–462, 460t, 462fsmooth muscle contraction affected by,571Cyclic AMP-dependent protein kinase. SeeProtein kinasesCyclic AMP regulatory protein (catabolitegene activator protein), 376, 378Cyclic AMP response element, 459t, 461Cyclic AMP response element bindingprotein, 461Cyclic GMP, 289f, 290as second messenger, 290, 436, 437t,457, 462–463role in smooth muscle, 573fCyclic 3′,5′-nucleotide phosphodiesterase,in lipolysis, 215Cyclin-dependent protein kinases, 333,334f, 335tinhibition of, DNA/chromosomeintegrity and, 339Cyclins, 333–335, 334f, 335tCycloheximide, 372Cyclooxygenase, 192as “suicide enzyme,” 194Cyclooxygenase pathway, 192, 192–194,193f, 194f

654 / INDEXCYP nomenclature, for cytochrome P450isoforms, 627CYP2A6, polymorphism of, 628, 630tCYP2C9, in warfarin–phenobarbitalinteraction, 628CYP2D6, polymorphism of, 628, 630tCYP2E1, enzyme induction and, 628Cysteine, 15t, 265metabolism of, 250, 252f, 253fabnormalities of, 250–255, 253fin pyruvate formation, 250, 252frequirements for, 480synthesis of, 238–239, 239fCystic fibrosis, 431–432, 432t, 474, 569tCystic fibrosis transmembrane regulator(CFTR), 431, 431f, 432tCystine reductase, 250, 252fCystinosis (cystine storage disease),250–255Cystinuria (cystine-lysinuria), 250Cytarabine (arabinosyl cytosine), 290Cytidine, 287f, 288tCytidine monophosphate (CMP), 288tCytidine monophosphateN-acetylneuraminic acid(CMP-NeuAc), 516t, 517Cytidine triphosphate (CTP), 290in phosphorylation, 85Cytochrome b 5 , 89, 627Cytochrome b 558 , 622Cytochrome c, 93Cytochrome oxidase/cytochrome aa 3 , 86,93Cytochrome P450-dependent microsomalethanol oxidizing system,212–214Cytochrome P450 side chain cleavage enzyme(P450scc), 438, 440f, 442Cytochrome P450 system, 86, 89–90, 90f,626ALA synthase affected by, 272, 278enzyme induction and, 272–273,627–628genes encoding, nomenclature for, 627isoforms of, 627–628in metabolism of xenobiotics, 626–628,629tmembrane insertion, 504mitochondrial, 89–90nomenclature system for, 627in xenobiotic cell injury, 631, 631fCytochromes, as dehydrogenases, 88Cytogenetic abnormalities, detection of,635tCytogenetic map, 633, 634fCytokines, α 2 -macroglobulin binding of,590Cytosine, 288tbase pairing of in DNA, 303, 304, 305fdeoxyribonucleosides of, in pyrimidinesynthesis, 296–297, 298fCytoskeleton/cytoskeletal proteins, 556,576–578red cell, 615f, 616–617, 616f, 616tCytosolALA synthesis in, 270, 273fglycolysis in, 126, 127f, 136lipogenesis in, 173–177, 174f, 175fpentose phosphate pathway reactions in,163pyrimidine synthesis in, 296, 298fCytosolic branch, for protein sorting, 498,499fCytosolic dynein, 577Cytosolic proteins, O-glycosidic linkages in,518Cytotoxicity, xenobiotic, 631, 631fD-amino acids, free, 14D arm, of tRNA, 310, 312f, 360, 361fD cyclins, 333, 334f, 335tcancer and, 334D isomerism, 102–104, 103fDAF. See Decay accelerating factordAMP, 288fDantrolene, for malignant hyperthermia,564DBD. See DNA-binding domainDBH. See Dopamine-β-hydroxylaseDeamination, 124, 124fcitric acid cycle in, 133–134liver in, 125Debranching enzymesabsence of, 152tin glycogenolysis, 146–147, 148fDebrisoquin, CYP2D6 in metabolism of,628Decay accelerating factor, 531Decorinin bone, 548tin cartilage, 551tDefensins, 621tDegeneracy, of genetic code, 359Degradation, rate of (k deg ), 74Dehydrocholesterol, in vitamin Dmetabolism, 484, 485fDehydroepiandrosterone (DHEA),synthesis of, 440, 441fDehydroepiandrosterone (∆ 5 ) pathway, 442,443fDehydrogenases, 86, 87–88, 88fin enzyme detection, 56, 56fnicotinamide coenzyme-dependent, 87,89fin respiratory chain, 87riboflavin-dependent, 87Deletions, DNA, recombinant DNA technologyin detection of, 409,409tDelta 5 , 4 (∆ 5,4 ) isomerase, 438, 441f, 442,443fDelta 4 (∆ 4 ) (progesterone) pathway, 442,443fDelta 5 (∆ 5 ) (dehydroepiandrosterone)pathway, 442, 443fDelta 9 (∆ 9 ) desaturasein monounsaturated fatty acid synthesis,191, 191fin polyunsaturated fatty acid synthesis,191, 191fDenaturationDNA structure analysis and, 304–305protein refolding and, 36temperature and, 63Deoxoynojirimycin, 527, 527tDeoxyadenylate, 303Deoxycholic acid, synthesis of, 226Deoxycorticosteronebinding of, 454–455synthesis of, 438, 441f11-Deoxycortisol, synthesis of, 440, 441fDeoxycytidine residues, methylation of,gene expression affected by, 383Deoxycytidylate, 303Deoxyguanylate, 303Deoxyhemoglobin, proton binding by, 44,45fDeoxyhemoglobin A, “sticky patch” receptoron, 46Deoxyhemoglobin S, “sticky patch”receptor on, 46Deoxynucleotides, 303–304, 304f, 305fDeoxyribonucleases (DNase)/DNase I, 312active chromatin and, 316Deoxyribonucleic acid. See DNADeoxyribonucleoside diphosphates(dNDPs), reduction of NDPs to,294, 297fDeoxyribonucleosides, 286in pyrimidine synthesis, 296Deoxyribose, 102, 106, 106fDeoxy sugars, 106, 106f3-Deoxyuridine, 290Dephosphorylation. See alsoPhosphorylationin covalent modification, 78–79, 78tDepolarization, in nerve impulsetransmission, 428Depurination, DNA, base excision-repairand, 337Dermatan sulfate, 544f, 544t, 545functions of, 547∆ 9 Desaturasein monounsaturated fatty acid synthesis,191, 191fin polyunsaturated fatty acid synthesis,191, 191fDesmin, 566t, 577tDesmosines, 539Desmosterol, in cholesterol synthesis, 220,222fDetergents, 417–418

INDEX / 655Detoxification, 626cytochrome P450 system in, 89–90, 90f,626–628, 629tDextrinosis, limit, 152tDextrins, 109Dextrose, 104DHA. See Docosahexaenoic acidDHEA. See DehydroepiandrosteroneDHPR. See Dihydropyridine receptorDHT. See DihydrotestosteroneDiabetes mellitus, 102, 161–162fatty liver and, 212free fatty acid levels in, 206hemochromatosis and, 587insulin resistance and, 611ketosis/ketoacidosis in, 188lipid transport and storage disorders and,205lipogenesis in, 173as metabolic disease, 122, 231starvation and, 236Diabetic cataract, 172Diacylglycerol, 115, 475, 476fin calcium-dependent signaltransduction, 464, 465fformation of, 197f, 198fin platelet activation, 606, 606fin respiratory burst, 623Diacylglycerol acyltransferase, 198f, 199Diagnostic enzymology, 57, 57tDicarboxylate anions, transporter systemsfor, 98–99Dicarboxylic aciduria, 188Dicumarol (4-hydroxydicoumarin), 486Dielectric constant, of water, 5Diet. See also Nutritionblood glucose regulation and, 159–161cholesterol levels affected by, 227hepatic VLDL secretion and, 211–212,213fhigh-fat, fatty liver and, 212Diet-induced thermogenesis, 217, 478Diethylenetriaminepentaacetate (DTPA),as preventive antioxidant, 119Diffusionfacilitated, 423, 423t, 424f, 426–427,427, 427fof bilirubin, 280of glucose. See also Glucosetransportersinsulin affecting, 427in red cell membrane, 611hormones in regulation of, 427“Ping-Pong” model of, 427, 427fnet, 423, 424fpassive, 423, 423t, 424fsimple, 423, 423t, 424fDigestion, 474–480DigitalisCa 2+ -Na + exchanger in action of, 568Na + -K + ATPase affected by, 428, 568Dihydrobiopterin, defect in synthesis of, 255Dihydrobiopterin reductase, defect in, 255Dihydrofolate/dihydrofolate reductase,methotrexate affecting, 296–297,494Dihydrolipoyl dehydrogenase, 140, 141fDihydrolipoyl transacetylase, 140, 141fDihydropyridine receptor, 563–564, 563f,564fDihydrotestosterone, 442, 444fbinding of, 455tDihydroxyacetone, 106fDihydroxyacetone phosphate, in glycolysis,197, 197f, 198f1,25-Dihydroxyvitamin D 3 . See Calcitriol24,25-Dihydroxyvitamin D 3 (24-hydroxycalcidiol),in vitamin Dmetabolism, 484, 485fDiiodotyrosine (DIT), 447, 448f, 449Dilated cardiomyopathy, 570Dimercaprol (BAL), respiratory chainaffected by, 95, 96fDimeric proteins, 34DimersCro protein, 380, 381fhistone, 315lambda repressor (cI) protein, 380, 381fDimethylallyl diphosphate, in cholesterolsynthesis, 219, 221fDimethylaminoadenine, 289fDinitrophenol, respiratory chain affectedby, 95, 96fDinucleotide, 291Dioxygenases, 89Dipalmitoyl lecithin, 115Dipeptidases, 477Diphosphates, nucleoside, 287, 287fDiphosphatidylglycerol. See CardiolipinDiphtheria toxin, 372Dipoles, water forming, 5, 6fDisaccharidases, 102, 475Disaccharides, 106–107, 107f, 107t. Seealso specific typeDiseasebiochemical basis of, 2, 3tHuman Genome Project and, 3–4major causes of, 3tDisplacement reactionsdouble, 69–70, 69fsequential (single), 69, 69fDissociation, of water, 8–9Dissociation constant, 8–9Michaelis constant (K m ) and, 66in pH calculation, 10of weak acids, 10–11, 12Distal histidine (histidine E7), in oxygenbinding, 40, 41fDisulfide bonds, protein folding and, 37DIT. See DiiodotyrosineDiurnal rhythm, in cholesterol synthesis,220Diversityantibody, 592, 593–594combinatorial, 592in gene expression, 387, 388fjunctional, 593–594Diversity segment, DNA rearrangementand, 593–594DNA, 303, 303–306, 314–340base excision-repair of, 336t, 337, 337fbase pairing in, 7, 303, 304, 305fmatching of for renaturation, 305–306recombinant DNA technology and,396–397replication/synthesis and, 328–330,330fbinding of to regulatory proteins, motifsfor, 387–390, 388t, 389f, 390f,391fblunt-ended, 398, 399–400, 400f, 413in chromatin, 314–318, 315f, 315t,317f, 318fchromosomal, 318–319, 319f, 319t,320f, 321frelationship of to mRNA, 321fcoding regions of, 319, 321f, 637complementarity of, 306, 307frecombinant DNA technology and,396–397damage to, 335, 335trepair of, 335–339, 335t, 336tADP-ribosylation for, 490deletions in, recombinant DNA technologyin detection of, 409, 409tdepurination of, base excision-repair and,337double-strand break repair of, 336t,337–338, 338fdouble-stranded, 304flanking sequence, 397genetic information contained in,303–306grooves in, 305f, 306insertions in, recombinant DNAtechnology in detection of, 409integrity of, monitoring, 339“jumping,” 325mismatch repair of, 36f, 336, 336f,336tmitochondrial, 322–323, 322f, 323tmutations in, 314, 323–326, 323f, 324f,325f. See also Mutationsin nucleosomes, 315–316, 316fnucleotide excision-repair of, 336, 337,338frearrangements ofin antibody diversity, 325–326, 393,593–594recombinant DNA technology indetection of, 409, 409trecombinant. See Recombinant DNA/recombinant DNA technology

656 / INDEXDNA (cont.)relaxed form of, 306renaturation of, base pair matching and,305–306repair of, 335–339, 335t, 336trepetitive-sequence, 320–322replication/synthesis of, 306, 307f,326–339, 326t, 327f, 328tDNA polymerase complex in, 328,328tDNA primer in, 328, 329f, 330finitiation of, 328–330, 329f, 330f,331forigin of, 326polarity of, 330–331proteins involved in, 328treconstitution of chromatin structureand, 333repair during, 335–339, 335t, 336treplication bubble formation and,331–333, 331f, 332f, 333freplication fork formation and,327–328, 327fribonucleoside diphosphate reductionand, 294, 297fin S phase of cell cycle, 333–335,334f, 335tsemiconservative nature of, 306, 307fsemidiscontinuous, 327f, 331, 331funwinding and, 326, 326–327in RNA synthesis, 341–343, 342f, 343tstabilization of, 7structure of, 303–306, 304f, 305fdenaturation in analysis of, 304–305double-helical, 7, 303, 304, 305frecombinant DNA technology and,396, 397supercoiled, 306, 332, 333ftranscription of, 306transposition of, 325unique-sequence (nonrepetitive), 320,320–321unwinding of, 326, 326–327RNA synthesis and, 344xenobiotic cell injury and, 631DNA binding domains, 390–391, 392f, 470DNA binding motifs, 387–390, 388t, 389f,390f, 391fDNA-dependent protein kinase, indouble-strand break repair, 338DNA-dependent RNA polymerases,342–343, 342f, 343tDNA elements, gene expression affected by,384–385, 384f, 385t, 386fdiversity and, 387, 388fDNA fingerprinting, 413DNA footprinting, 413DNA helicase, 326–327, 327f, 328, 328tDNA ligase, 328t, 330in recombinant DNA technology,399–400, 400t, 401fDNA-PK. See DNA-dependent proteinkinaseDNA polymerases, 326, 327–328, 327f,328, 328tin recombinant DNA technology, 400tDNA primase, 327, 327f, 328tDNA probes, 402, 414for gene isolation, 635tlibrary searched with, 402in porphyria diagnosis, 274DNA-protein interactions, bacteriophagelambda as paradigm for,378–383, 379f, 380f, 381f, 382fDNA sequencesamplification of by PCR, 405–406,406fdetermination of, 404, 405fin gene isolation, 635tprotein sequencing and, 25–26DNA topoisomerases, 306, 328t, 332,332fDNA transfection, identification ofenhancers/regulatory elementsand, 386DNA unwinding element, 326DNase (deoxyribonuclease)/DNase I, 312active chromatin and, 316in recombinant DNA technology, 400tdNDPs. See DeoxyribonucleosidediphosphatesDOC. See 11-DeoxycorticosteroneDocking, in nuclear import, 501, 502fDocking protein, 504Docosahexaenoic acid, 191–192Dolichol, 118, 119f, 522, 523fin cholesterol synthesis, 220, 221fin N-glycosylation, 522Dolichol kinase, 522Dolichol-P-P-GlcNAc, 522–523Dolichol-P-P-oligosaccharide (dolicholpyrophosphate-oligosaccharide),521, 524fin N-glycosylation, 521–524, 523fDolichol phosphate, 522Domains. See also specific typealbumin, 584carboxyl terminal repeat, 350chromatin, 316, 318, 319fcoupling, on hormone receptors, 435–436DNA binding, 390–391, 392f, 470fibronectin, 540, 541fprotein, 33–34Src homology 2 (SH2)in insulin signal transmission, 465,466f, 467in Jak/STAT pathway, 467, 467ftrans-activation, of regulatory proteins,390–391, 392ftranscription, 387L-Dopa, 446, 447fDopa decarboxylase, 267, 267f, 446, 447fDopamine, 446, 447f. See also Catecholaminessynthesis of, 267, 267f, 445–447, 447fDopamine-β-hydroxylase, 447vitamin C as coenzyme for, 495–496Dopamine β-oxidase, 267, 267fDouble displacement reactions, 69–70, 69fDouble helix, of DNA structure, 7, 303,304, 305frecombinant DNA technology and, 396,397Double reciprocal plotinhibitor evaluation and, 68, 68f, 69fK m and V max estimated from, 66, 66fDouble-strand break repair of DNA, 336t,337–338, 338fDouble-stranded DNA, 304, 314unwindingfor replication, 326, 326–327RNA synthesis and, 344Downstream promoter element, 346–348,347fDPE. See Downstream promoter elementDRIPs, 472t, 473Drug detoxification/interactions,cytochromes P450 and,89–90, 90f, 628Drug development, pharmacogenetics and,631–632Drug resistance, gene amplification in, 393DS-PG I/DS-PG II, in cartilage, 551tdsDNA. See Double-stranded DNADTPA, as preventive antioxidant, 119Dubin-Johnson syndrome, 283Duchenne muscular dystrophy, 556,565–566, 566fDUE. See DNA unwinding elementDwarfism, 551t, 553–554Dynamin, in absorptive pinocytosis, 430,577Dyneins, 577Dysbetalipoproteinemia, familial, 228tDyslipoproteinemias, 228t, 229Dystrophin, 556, 565–566, 566t, 567fmutation in gene for, in musculardystrophy, 565–566, 566fE 0 . See Redox (oxidation-reduction)potentialE act . See Activation energyE coli, lactose metabolism in, operonhypothesis and, 376–378,376f, 377fE coli bacteriophage P1-based (PAC) vector,401–402, 402t, 413E cyclins, 333, 334f, 335tE-selectin, 529tE (exit) site, in protein synthesis, 38f, 368ECF. See Extracellular fluidECM. See Extracellular matrix

INDEX / 657EcoRI, 398, 399t, 401fEcoRII, 399tEdemain kwashiorkor, 479plasma protein concentration and, 580in thiamin deficiency, 489Edman reaction, for peptide/proteinsequencing, 25, 26fEdman reagent (phenylisothiocyanate), inprotein sequencing, 25, 26fEDRF. See Endothelium-derived relaxingfactorEDTA, as preventive antioxidant, 119EFA. See Essential fatty acidsEFs. See Elongation factorsEGF. See Epidermal growth factorEgg white, uncooked, biotin deficiencycaused by, 494Ehlers-Danlos syndrome, 538, 538tEicosanoids, 112, 190, 192, 193f, 194f,621tEicosapentaenoic acid, 190feIF-4E complex, in protein synthesis, 367,367feIFs, in protein synthesis, 36580S initiation complex, in protein synthesis,366f, 367Elaidic acid, 112, 113, 113t, 114fElastase, in digestion, 477Elastin, 539, 539tElectrochemical potential difference, inoxidative phosphorylation, 96Electron carriers, flavin coenzymes as, 490Electron movement, in active transport, 427Electron-transferring flavoprotein, 87, 181Electron transport chain system, 622. Seealso Respiratory chainElectrophiles, 7Electrophoresisfor plasma protein analysis, 580polyacrylamide, for protein/peptidepurification, 24, 24f, 25fpulsed-field gel, for gene isolation, 635ttwo-dimensional, protein expression and,28Electrospray dispersion, in massspectrometry, 27Electrostatic bonds/interactions, 7. See alsoSalt (electrostatic) bondsoxygen binding rupturing, Bohr effectprotons and, 44–45, 45fELISAs. See Enzyme-linked immunoassaysElliptocytosis, hereditary, 617Elongase, 177, 177fin polyunsaturated fatty acid synthesis,191, 191f, 192fElongationchainin fatty acid synthesis, 177, 177fin transcription cycle, 342, 342fin DNA synthesis, 328in glycosaminoglycan synthesis, 543in protein synthesis, 367–370, 368fin RNA synthesis, 342, 342f, 344Elongation arrest, 504Elongation factor 2, in protein synthesis,368, 368fElongation factor EF1A, in proteinsynthesis, 368, 368fElongation factors, in protein synthesis,367, 368, 368fEmelin, 539Emphysema, α 1 -antiproteinase deficiencyand, 589, 589f, 623–624Emulsions, amphipathic lipids forming,120f, 121Encephalopathieshyperbilirubinemia causing (kernicterus),282, 283mitochondrial, with lactic acidosis andstroke (MELAS), 100–101spongiform (prion diseases), 37Wernicke’s, 489Endergonic reaction, 80, 81coupling and, 81–82, 81f, 82fATP in, 82, 84Endocrine system, 434–455. See alsoHormonesdiversity of, 437–438Endocytosis, 428, 429–430, 429freceptor-mediated, 429fEndoglycosidase F, 517Endoglycosidase H, 517Endoglycosidases, in glycoprotein analysis,515t, 517, 517tEndonucleases, 312, 413apurinic and apyrimidinic, in baseexcision-repair, 337restriction, 312, 397–399, 399t, 400f,414in recombinant DNA technology,399–400, 399t, 400f, 400t, 401fEndopeptidases, 477Endoplasmic reticulum, 370acylglycerol synthesis and, 126, 127fcore protein synthesis in, 543fatty acid chain elongation in, 177, 177froughglycosylation in, 524–525, 525fin protein sorting, 498, 499f, 500fprotein synthesis and, 370routes of protein insertion into,505–507, 506fsignal hypothesis of polyribosomebinding to, 503–505, 504t, 505fsmooth, cytochrome P450 isoforms in,627Endoproteinase, for polypeptide cleavage,26tEndorphins, 453, 453fEndothelial cellsin clotting and thrombosis, 607, 607tneutrophil interaction andintegrins in, 529t, 620–621, 622tselectins in, 528–529, 529t, 530fEndothelium-derived relaxing factor, 572,607t. See also Nitric oxideEnergyactivation, 61, 63conservation of, 83free. See Free energyin muscle, creatine phosphate as reservefor, 573–574, 575fnutritional requirement for, 478transduction ofmembranes in, 415in muscle, 556–559Energy balance, 478–479Energy capture, 82–83, 82f, 83Energy expenditure, 478Energy transfer, 82–83, 82fEnhanceosome, 385, 386fEnhancers/enhancer elements, 348in gene expression, 384–385, 384f, 385ttissue-specific expression and, 385recombinant DNA technology and, 397reporter genes in definition of, 385–386,387f, 388fEnolase, in glycolysis, 137, 138f∆ 2 Enoyl-CoA hydratase, 181, 182f∆ 3 cis-Enoyl-CoA isomerase, 183∆ 2 trans-Enoyl-CoA isomerase, 183Entactin, in basal lamina, 540Enterohepatic circulation, 227lipid absorption and, 475Enterohepatic urobilinogen cycle, 281Enteropeptidase, 477Enthalpy, 80Entropy, 80Enzyme induction, 630cytochrome P450 and, 272–273,627–628in gluconeogenesis regulation, 155–157,156tEnzyme-linked immunoassays (ELISAs), 55Enzymes, 7–8active sites of, 51, 51fassay of, 55–56, 56fbranching, in glycogen biosynthesis, 145,147fcatalytic activity of, 49, 50f. See alsoCatalysisdetection facilitated by, 55–56, 56fkinetics of, 63–70. See also Kinetics(enzyme)regulation of, 72–79, 128f, 129RNA and, 356specificity of, 49, 50fclassification of, 49–50constitutive, 75debranchingabsence of, 152tin glycogenolysis, 146–147, 148f

658 / INDEXEnzymes (cont.)degradation of, control of, 74in disease diagnosis/prognosis, 56–57,57, 57t, 58f, 580in DNA repair, 335, 336t, 338–339hydrolysis rate affected by, 7–8irreversible inhibition (“poisoning”) of,69isozymes and, 54–55kinetics of, 60–71. See also Kinetics(enzyme)mechanisms of action of, 49–59membranes in localization of, 415metal-activated, 50as mitochondrial compartment markers,92plasma, diagnostic significance of, 57, 57tquantity of, catalytic capacity affected by,73–74rate of degradation of (k deg ), control of,74rate of synthesis of (k s ), control of, 74recombinant DNA technology in studyof, 58, 59fregulatory, 126–129, 128frestriction. See Restriction endonucleasesspecificity of, 49, 50fsubstrates affecting conformation of, 52,53fEnzymopathies, 619Epidermal growth factor (EGF), receptorfor, 436Epidermolysis bullosa, 538, 538tEpimerasesin galactose metabolism, 167, 170fin glycosaminoglycan synthesis, 543in pentose phosphate pathway, 163, 165fEpimers, 104, 104fEpinephrine, 439f, 447, 447f. See alsoCatecholaminesblood glucose affected by, 161in gluconeogenesis regulation, 157in lipogenesis regulation, 178synthesis of, 267, 267f, 445–447, 447fEpitope (antigenic determinant), 33, 591Epoxide hydrolase, 631Epoxides, 631Equilibrium constant (K eq ), 62–63in enzymatic catalysis, 63free energy changes and, 60–61ER. See Estrogens, receptors forErcalcitriol, 484ERE. See Estrogen response elementeRF. See Releasing factorsErgocalciferol (vitamin D 2 ), 484Ergosterol, 118, 119fErythrocyte aminotransferases, in vitaminB 6 status assessment, 491Erythrocyte transketolase activation, inthiamin nutritional statusassessment, 489Erythrocytes, 609–610, 610–6192,3-bisphosphoglycerate pathway in,140, 140fdisorders affecting, 609, 610terythropoietin in regulation of, 609–610,611fglucose-6-phosphate dehydrogenase deficiencyaffecting, 613, 614f, 619glucose as metabolic necessity for, 232glycolysis in, 140, 140fhemoglobin S “sticky patch” affecting, 46hemolysis of, pentose phosphatepathway/glutathione peroxidaseand, 166, 167f, 169–170life span of, 609membranes of, 614–617, 615f, 615t,616f, 616tglucose transporter of, 611, 612themolytic anemias and, 619, 620tmetabolism of, 235t, 610–614, 612toxidants produced during, 611–613,613trecombinant DNA technology in studyof, 624structure and function of, 609–610Erythroid ALA synthase (ALAS2), 272, 273in porphyria, 274, 277tErythropoiesis, 609–610, 611fErythropoietin/recombinant erythropoietin(epoitin alfa/EPO), 526, 583t,609–610, 611fD-Erythrose, 104fEscherichia coli, lactose metabolism in,operon hypothesis and, 376–378,376f, 377fEscherichia coli bacteriophage P1-based(PAC) vector, 401–402, 402t,413Essential amino acids. See Nutritionallyessential amino acidsEssential fatty acids, 190, 190f, 193abnormal metabolism of, 195–196deficiency of, 191–192, 194–195prostaglandin production and, 190Essential fructosuria, 163, 171–172Essential pentosuria, 163, 170Estradiol/17β-Estradiol, 439f, 440fbinding of, 455, 455tsynthesis of, 442–445, 444fEstriol, synthesis of, 442, 444fEstrogen response element, 459tEstrogensbinding of, 455, 455treceptors for, 471synthesis of, 442–445, 444f, 445fEstronebinding of, 455tsynthesis of, 442, 444fEthanolCYP2E1 induction and, 628fatty liver and, 212–214iron absorption and, 478thiamin deficiency and, 489Ethylenediaminetetraacetate (EDTA), aspreventive antioxidant, 119Euchromatin, 316Eukaryotic gene expression, 383–387,391–395, 392t. See also Geneexpressionchromatic remodeling in, 383–384diversity of, 387, 388fDNA elements affecting, 384–385, 384f,385t, 386fDNA-protein interactions in, bacteriophagelambda as paradigm for,378–383, 379f, 380f, 381f, 382flocus control regions and insulators in,387prokaryotic gene expression comparedwith, 391–395, 392treporter genes and, 385–386, 387f, 388ftissue-specific, 385Eukaryotic promoters, in transcription,346–349, 347f, 348f, 349fEukaryotic transcription complex,350–352, 351tExchange transporters, 98–100, 98f, 99fExcitation-response coupling, membranesin, 415Exergonic reaction, 80, 81coupling and, 81–82, 81f, 82fATP in, 82, 84Exinuclease, in DNA repair, 337, 337fExit (E) site, in protein synthesis, 368,368fExocytosis, 429, 430–431, 430fin insulin synthesis, 430–431Exocytotic (secretory) pathway, 498Exoglycosidases, in glycoprotein analysis,515t, 517, 517tExons, 319, 358, 413interruptions in. See Intronsin recombinant DNA technology, 397,398fsplicing, 352–354, 414alternative, in regulation of geneexpression, 354, 354f,393–394, 636recombinant DNA technology and,397, 398fExonucleases, 312, 413in recombinant DNA technology, 400tExopeptidases, 477Exportins, 503Expression vector, 402Extra arm, of tRNA, 310, 312fExtracellular environment, membranes inmaintenance of, 415–416, 416tExtracellular fluid (ECF), 415–416, 416,416tExtracellular matrix, 535–555. See also specificcomponent and under Matrix

INDEX / 659Extrinsic pathway of blood coagulation,598, 599f, 601Eye, fructose and sorbitol in, diabeticcataract and, 172F 0 , in ATP synthesis, 96, 97f, 98fF 1 , in ATP synthesis, 96, 97f, 98fFab region, 591, 592fFabry’s disease, 203tFacilitated diffusion/transport system, 423,423t, 424f, 426–427, 427, 427ffor bilirubin, 280for glucose. See also Glucose transportersinsulin affecting, 427in red cell membrane, 611hormones in regulation of, 427“Ping-Pong” model of, 427, 427fFactor I (fibrinogen), 580, 600, 600tconversion of to fibrin, 601–602Factor II (prothrombin), 600t, 601coumarin drugs affecting, 487, 604vitamin K in synthesis of, 487Factor III (tissue factor), 599f, 600t, 601Factor IV. See CalciumFactor V (proaccelerin/labile factor/acceleratorglobulin), 600t, 601, 602fFactor V Leiden, 603–604Factor VII (proconvertin/serumprothrombin conversion accelerator/cothromboplastin),599f,600t, 601coumarin drugs affecting, 604Factor VIII (antihemophilic factorA/globulin), 599f, 600, 600tdeficiency of, 604Factor VIII concentrates, recombinantDNA technology in productionof, 604Factor IX (antihemophilic factor B/Christmasfactor/plasma thromboplastincomponent), 599f, 600, 600tcoumarin drugs affecting, 604deficiency of, 604Factor X (Stuart-Prower factor), 599f, 600,600tactivation of, 599f, 600–601coumarin drugs affecting, 604Factor XI (plasma thromboplastinantecedent), 599f, 600, 600tdeficiency of, 601Factor XII (Hageman factor), 599f, 600,600tFactor XIII (fibrin stabilizing factor/fibrinoligase), 600tFacultative heterochromatin, 316FAD. See Flavin adenine dinucleotideFADH 2 , fatty acid oxidation yielding, 181Familial hypertrophic cardiomyopathy,569–570, 570fFanconi’s anemia, 338Farber’s disease, 203tFarnesoid X receptor, in bile acid synthesisregulation, 227Farnesyl diphosphate, in cholesterol/polyisoprenoid synthesis,219, 220, 221fFast acetylators, 630Fast (white) twitch fibers, 574–576, 575tFat tissue. See Adipose tissueFatal infantile mitochondrial myopathy andrenal dysfunction, oxidoreductasedeficiency causing, 100Fatigue (muscle), 136Fats, 111. See also Lipidsdiets high in, fatty liver and, 212metabolism of, 122f, 123–124, 123f,125–126, 126fFatty acid-binding protein, 180, 207Fatty acid chains, elongation of, 177, 177fFatty acid elongase system, 177, 177fin polyunsaturated fatty acid synthesis,191, 191f, 192fFatty acid oxidase, 181, 182fFatty acid synthase, 156t, 173Fatty acid synthase complex, 173–176,174f, 175f, 179Fatty acid-transport protein, membrane,207Fatty acids, 2, 111–114activation of, 180–181, 181fcalcium absorption affected by, 477eicosanoids formed from, 190, 192,193f, 194fessential, 190, 190f, 193abnormal metabolism of, 195–196deficiency of, 191–192, 194–195prostaglandin production and, 190free. See Free fatty acidsinterconvertibility of, 231in membranes, 417, 418fmetabolism of, 123–124, 123fnomenclature of, 111–112, 112foxidation of, 180–189. See alsoKetogenesisacetyl-CoA release and, 123–124,123f, 181–183, 181f, 182fβ, 181–183, 181f, 182fketogenesis regulation and,186–187, 187f, 188fmodified, 183,183fclinical aspects of, 187–189hypoglycemia caused by impairmentof, 187–188in mitochondria, 180–181, 181fphysical/physiologic properties of, 114saturated, 111, 112, 112tsynthesis of, 173–179, 174f, 175f. Seealso Lipogenesiscarbohydrate metabolism and, 123citric acid cycle in, 133, 134, 135fextramitochondrial, 173trans, 113–114, 192transport of, carnitine in, 180–181, 181ftriacylglycerols (triglycerides) as storageform of, 114, 115funesterified (free). See Free fatty acidsunsaturated. See Unsaturated fatty acidsFatty liveralcoholism and, 212–214of pregnancy, 188triacylglycerol metabolism imbalanceand, 212Favism, 170Fc fragment, 591, 592freceptors for, in neutrophils, 621tFe. See IronFed state, metabolic fuel reserves and, 232,234tFeedback inhibition, in allosteric regulation,74–76, 75f, 76, 129Feedback regulationin allosteric regulation, 76, 129thrombin levels controlled by, 602Fenton reaction, 612Ferric iron, 278in methemoglobinemia, 46Ferrireductase, 585Ferritin, 478, 585, 586protein synthesis affected by, 370Ferritin receptor, 586Ferrochelatase (heme synthase), 271, 272fin porphyria, 277tFerrous ironincorporation of into protoporphyrin,271–272, 272fin oxygen transport, 40–41Fertilization, glycoproteins in, 528FeS. See Iron sulfur protein complexFetal hemoglobin, P 50 of, 42Fetal warfarin syndrome, 488α-Fetoprotein, 583tFFA. See Free fatty acidsFGFs. See Fibroblast growth factorsFibrillin, 535, 539Marfan syndrome caused by mutations ingene for, 539–540, 540fFibrils, collagen, 535–539, 536f, 537tFibrindissolution of by plasmin, 604–605,604fformation of, 598–601, 599fthrombin in, 601–602, 603fin thrombi, 598Fibrin deposit, 598Fibrin mesh, formation of, 598Fibrin split products, in inflammation, 621tFibrin stabilizing factor (factor XIII), 600tFibrinogen (factor I), 580, 600, 600tconversion of to fibrin, 601–602Fibrinoligase (factor XIII), 600tFibrinolysis, 604–605Fibrinopeptides A and B, 602, 603f

660 / INDEXFibroblast growth factor receptor 3, achondroplasiacaused by mutationin gene for, 551t, 554, 554fFibroblast growth factor receptors, chondrodysplasiascaused by mutationin gene for, 551t, 554, 554fFibroblast growth factors (FGFs), 554Fibronectin, 535, 537–538, 540, 541fFibrous proteins, 30collagen as, 38Figlu. See FormiminoglutamateFinal common pathway of bloodcoagulation, 599, 601, 602fFingerprinting, DNA, 413FISH. See Fluorescence in situ hybridizationFish-eye disease, 228t5′ cap, mRNA modification and, 355Flanking-sequence DNA, 397Flavin adenine dinucleotide (FAD), 86–87,290t, 489in citric acid cycle, 133Flavin mononucleotide (FMN), 50, 86–87,489Flavoproteinselectron-transferring, 87as oxidases, 86–87, 88fFlip-flop rate, phospholipid, membraneasymmetry and, 420Flippases, membrane asymmetry and, 420Fluid mosaic model, 421f, 422Fluid-phase pinocytosis, 429–430, 429fFluidity, membrane, 422Fluorescence, of porphyrins, 273–274,277fFluorescence in situ hybridization, in genemapping, 406–407, 407t,635tFluoride, 496tFluoroacetate, 130, 132f1-Fluoro-2,4-dinitrobenzene (Sanger’sreagent), for polypeptidesequencing, 255-Fluorouracil, 290, 291f, 297Flux-generating reaction, 129FMN. See Flavin mononucleotideFolate. See Folic acidFolate trap, 492f, 494Foldingpolar and charged group positioning and,6protein, 36–37, 37fafter denaturation, 36Folic acid (folate/pteroylglutamic acid),482t, 492–494, 493fcoenzymes derived from, 51deficiency of, 250, 482t, 494functional, 492, 494forms of in diet, 492, 493finhibitors of metabolism of, 494supplemental, 494Folinic acid, 493Follicle-stimulating hormone (FSH), 437,438, 439fFootprinting, DNA, 413Forbes’ disease, 152tForensic medicinepolymerase chain reaction (PCR) in,405restriction fragment lengthpolymorphisms (RFLPs) in, 411variable numbers of tandemly repeatedunits (VNTRs) in, 411Formic acid, pK/pK a value of, 12tFormiminoglutamate, in histidinecatabolism, 250, 251fFormyl-tetrahydrofolate, 493, 493f, 49443S initiation complex, in protein synthesis,365, 366f43S preinitiation complex, in proteinsynthesis, 365, 366fFPA/FPB. See Fibrinopeptides A and BFrameshift mutations, 363, 364fABO blood group and, 619Framework regions, 592Free amino acids, absorption of, 477Free energychanges in, 80–81chemical reaction direction and,60–61coupling and, 81–82, 81f, 82fenzymes affecting, 63equilibrium state and, 60–61redox potential and, 86, 87ttransition states and, 61of hydrolysis of ATP, 82–83, 82tliberation of as heat, 95Free fatty acids, 111, 180, 205, 206tin fatty liver, 212glucose metabolism affecting, 215insulin affecting, 215ketogenesis regulation and, 186–187,187f, 188flipogenesis affected by, 177–178, 177fmetabolism of, 206–207starvation and, 232–234, 234f, 234tFree polyribosomes, protein synthesis on,498, 506. See alsoPolyribosomesFree radicals (reactive oxygen species). Seealso Antioxidantshydroperoxidases in protection against,88in kwashiorkor, 479lipid peroxidation producing, 118–119,120fin oxygen toxicity, 90–91, 611–613,613txenobiotic cell injury and, 631, 631fD-Fructofuranose, 103fFructokinase, 167, 169fdeficiency of, 171–172D-Fructopyranose, 103fFructoseabsorption of, 475, 475fin diabetic cataract, 172glycemic index of, 474hepatichyperlipidemia/hyperuricemia and,170–171metabolism affected by, 167, 169firon absorption affected by, 478metabolism of, 167, 169fdefects in, 171–172pyranose and furanose forms of, 103fD-Fructose, 105t, 106fFructose-1,6-bisphosphatase, 156t, 166deficiency of, 171–172Fructose-2,6-bisphosphatase, 157, 158fin covalent catalysis, 54, 55fFructose-1,6-bisphosphatein gluconeogenesis, 153, 154fin glycolysis, 137, 138fFructose-2,6-bisphosphate, 157–158,158fFructose intolerance, hereditary, 171Fructose 6-phosphatefree energy of hydrolysis of, 82tin gluconeogenesis, 153, 154fin glycolysis, 137, 138fFructosuria, essential, 163, 171–172FSF. See Fibrin stabilizing factorFSH. See Follicle-stimulating hormoneFucose, in glycoproteins, 516tFucosidosis, 532–533, 533tFucosylated oligosaccharides, selectinsbinding, 530Fucosyltransferase/fucosyl (Fuc) transferase,618Fumarase (fumarate hydratase), 132f, 133Fumarate, 132f, 133in tyrosine catabolism, 254, 255fin urea synthesis, 246f, 247Fumarylacetoacetate, in tyrosine catabolism,254f, 255Fumarylacetoacetate hydrolase, defect at, intyrosinemia, 255Functional groupsamino acid chemical reactions affectedby, 18–20amino acid properties affected by, 18physiologic significance of, 10–11pK of, medium affecting, 13Functional plasma enzymes, 57. See alsoEnzymesFuranose ring structures, 103f, 104Fusion proteins, recombinant, in enzymestudy, 58, 59fFXR. See Farnesoid X receptor∆G. See Free energy∆G 0 , 60, 61, 81∆G D , 61

INDEX / 661∆G F , 61enzymes affecting, 63G-CSF. See Granulocyte colony-stimulatingfactorG protein-coupled receptors (GPCRs), 458,459, 460fG proteins, 459, 461tin calcium-dependent signaltransduction, 464f, 465in cAMP-dependent signal transduction,436, 459, 461tin Jak/STAT pathway, 467in respiratory burst, 623GABA. See γ-AminobutyrateGAGs. See GlycosaminoglycansGal-Gal-Xyl-Ser trisaccharide, 518Gal-hydroxylysine (Hyl) linkage, 518Gal transferase, 618–619, 619fGalactokinase, 167, 170finherited defects in, 172Galactosamine, 169, 171fD-Galactosamine (chondrosamine), 106Galactose, 102, 167–169, 170fabsorption of, 475, 475fglycemic index of, 474in glycoproteins, 516tmetabolism of, 167–169, 170fenzyme deficiencies and, 172D-Galactose, 104f, 105tα-D-Galactose, 104fGalactose 1-phosphate uridyl transferase,167, 170fGalactosemia, 102, 163, 172Galactosidases, in glycoprotein analysis,517Galactoside, 106Galactosylceramide, 116, 117f, 201, 203fGalCer. See GalactosylceramideGallstones, 474cholesterol, 219GalNAc, in glycoproteins, 515, 516tGalNAc-Ser(Thr) linkagein glycoproteins, 518, 519fin glycosaminoglycans, 543GalNAc transferase, in ABO system,618–619, 619fGamma- (γ) aminobutyrate. Seeγ-AminobutyrateGamma- (γ) carotene, 482Gamma-globulin, 581fGamma- (γ) glutamyltransferase, 630Gamma- (γ) glutamyl transpeptidase(γ-glutamyltransferase),diagnostic significance of, 57tGangliosides, 116amino sugars in, 106, 169, 171fsialic acids in, 110synthesis of, 201–202, 203fGap junctions, 431GAPs. See Guanine activating proteinsGastric lipase, 475Gastroenteropathy, protein-losing, 582Gated ion channels, 424Gaucher’s disease, 203tGDH. See Glutamate dehydrogenase/L-Glutamate dehydrogenaseGDP-Fuc, 516tGDP-Man, 516t, 517GEFs. See Guanine nucleotide exchangefactorsGel electrophoresispolyacrylamide, for protein/peptidepurification, 24, 24f, 25fpulsed-field, for gene isolation, 635tGel filtration, for protein/peptidepurification, 21–22, 23fGemfibrozil, 229Gender, xenobiotic-metabolizing enzymesaffected by, 630Gene. See Genes; GenomeGene array chips, protein expression and,28Gene conversion, 325Gene disruption/knockout, targeted, 412Gene expressionconstitutive, 376, 378in pyrimidine nucleotide synthesis,regulation of, 297–299regulation of, 374–395alternative splicing and, 354, 354f,393–394, 636eukaryotic transcription and, 383–387hormones in, 458fnegative versus positive, 374, 375t,378, 380in prokaryotes versus eukaryotes,391–395, 392tregulatory protein–DNA bindingmotifs and, 387–390, 388t,389f, 390f, 391fregulatory protein DNA binding andtrans-activation domains and,390–391, 392fretinoic acid in, 483temporal responses and, 374–375,375fGene mapping, 319, 406–407, 407t, 633,634f, 635tGene products, diseases associated withdeficiency of, 407tgene therapy for, 411Gene therapy, 411for α 1 -antitrypsin deficiency emphysema,589for urea biosynthesis defects, 248Gene transcription. See TranscriptionGeneral acid/base catalysis, 52Genesalteration of, 323–326, 324f, 325famplification of, in gene expressionregulation, 323–326, 324f,325fdisease causing, recombinant DNAtechnology in detection of,407–408, 408f, 409thuman genome information and, 638heterogeneous nuclear RNA processingin regulation of, 354housekeeping, 376immunoglobulin, DNA rearrangementand, 325–326, 393, 593–594double-strand break repair and,337–338inducible, 376knockout, 412processed, 325reporter, 385–386, 387f, 388ftargeted disruption of, 412variations inmethods for isolation of, 635tnormal, recombinant DNA techniquesfor identification of, 407size/complexity and, 397, 399tGenetic code, 303, 358–363, 359tfeatures of, 358–359, 360tL-α-amino acids encoded by, 14, 15–16tGenetic diseasesdiagnosis ofenzymes in, 57recombinant DNA technology in,407–412, 408f, 409t, 410f, 411fgene therapy for, 411Genetic linkage. See Linkage analysisGenetic mapping, 633, 634f. See alsoHuman Genome ProjectGenetics. See also Human Genome Projectmolecular, 1xenobiotic-metabolizing enzymes affectedby, 630Genevan system, for fatty acidnomenclature, 111Genomeredundancy in, 320–322removal of gene from (targeted genedisruption/knockout), 412sequencing. See also Human GenomeProjectapproaches used in, 634, 635tresults of, 636–637, 636t, 637tworking draft of, 633Genomic library, 402, 413Genomic technology, 396. See alsoRecombinant DNA/recombinantDNA technologyGenomics, protein sequencing and, 28Geometric isomerism, of unsaturated fattyacids, 112–114, 114fGeranyl diphosphate, in cholesterolsynthesis, 219, 221fGGT. See γ-GlutamyltransferaseGhosts, red cell membrane analysis and, 614Gibbs free energy/Gibbs energy. See Freeenergy

662 / INDEXGilbert syndrome, 283GK (glucokinase) gene, regulation of, 355,355fGla. See γ-CarboxyglutamateGlcCer. See GlucosylceramideGlcNAc. See N-AcetylglucosamineGlial fibrillary acid protein, 577tGlibenclamide. See GlyburideGlobin, 278α-Globin gene, localization of, 407tβ-Globin genelocalization of, 407trecombinant DNA technology indetection of variations in,407–408, 408f, 409tGlobular proteins, 30β-turns in, 32, 34fβ 1 -Globulin, 581ftransferrin as, 586γ-Globulin, 581fGlobulins, 580Glomerular filtration, basal lamina in, 540Glomerular membrane, laminin in, 540–542Glomerulonephritis, 541Glomerulus, renal, laminin in, 540–542Glucagon, 148, 160–161in gluconeogenesis regulation, 157in lipogenesis regulation, 178, 178fGlucagon/insulin ratio, in ketogenesisregulation, 187Glucagon-like peptide, 437Glucan (glucosan), 107Glucan transferase, in glycogenolysis, 146,146f, 148fGlucocorticoid receptor-interacting protein(GRIP1 coactivator), 472, 472tGlucocorticoid response element (GRE),456, 458f, 459tGlucocorticoids, 437. See also specific typein amino acid transport, 427blood glucose affected by, 161in lipolysis, 215, 216fNF-κB pathway regulated by, 468, 468freceptors for, 471synthesis of, 440, 441ftransport of, 454–455, 455tD-Glucofuranose, 103fGlucogenic amino acids, 231–232Glucokinase, 156tin blood glucose regulation, 159–160,160fin glycogen biosynthesis, 145, 146f, 156tin glycolysis, 137, 138f, 156tGlucokinase gene, regulation of, 355, 355fGluconeogenesis, 123, 125, 153–162, 154fblood glucose regulation and, 158–161,159f, 160fcitric acid cycle in, 133–134, 134f,153–155, 154fglycolysis and, 136–140, 138f, 139f,153–155, 154fregulation of, 155–158, 156t, 158fallosteric modification in, 157covalent modification in, 157enzyme induction/repression in,155–157, 156tfructose 2,6-bisphosphate in,157–158, 158fsubstrate (futile) cycles in, 158thermodynamic barriers to glycolysisand, 153–155, 154fGluconolactone hydrolase, 163, 165fD-Glucopyranose, 103fαD-Glucopyranose (α-anomer), 103f, 104βD-Glucopyranose (β-anomer), 103f, 104Glucosamine, 106f, 169, 171fin heparin, 545Glucosan (glucan), 107Glucose, 102, 102–106absorption of, 474, 475, 475fas amino sugar precursor, 169, 171fblood levels of. See Blood glucoseepimers of, 104, 104fin extracellular and intracellular fluid,416, 416tfuranose forms of, 103f, 104galactose conversion to, 167–169,170fglycemic index of, 474in glycogen biosynthesis, 145, 146fin glycoproteins, 516tinsulin secretion and, 160, 161–162interconvertibility of, 231isomers of, 102–104, 103fas metabolic necessity, 232metabolism of, 122–123, 122f, 123f,124–125, 125f, 136–140, 138f,139f, 140f, 159, 159f. See alsoGluconeogenesis; GlycolysisATP generated by, 142, 143tin fed state, 232free fatty acids and, 215insulin affecting, 160, 161–162by pentose phosphate pathway, 123,163–166, 164f, 165f, 167fstarvation and, 232–234, 234f, 234t,236permeability coefficient of, 419fpyranose forms of, 103f, 104renal threshold for, 161structure of, 102, 103ftransport of, 159, 160t, 428, 429f, 475,475finsulin affecting, 427D-Glucose, 103f, 104f, 105tα-D-Glucose, 104fL-Glucose, 103fGlucose-alanine cycle, 159Glucose-6-phosphatasedeficiency of, 152t, 300in gluconeogenesis, 156tin glycogenolysis, 147Glucose 1-phosphatefree energy of hydrolysis of, 82tin gluconeogenesis, 154f, 155Glucose 6-phosphatefree energy of hydrolysis of, 82tin gluconeogenesis, 153, 154fin glycogen biosynthesis, 145, 146fin glycolysis, 137, 138fGlucose-6-phosphate dehydrogenasedeficiency of, 163, 169–170, 613, 614f,619, 630tin pentose phosphate pathway, 156t,163, 164f, 165fGlucose tolerance, 161–162, 161fGlucose transporters, 159, 160tin blood glucose regulation, 159, 160insulin affecting, 427red cell membrane, 611, 612tGlucoside, 106Glucosuria, 161Glucosylceramide, 116, 201, 203fGlucuronate/glucuronic acid, 166–167,168fbilirubin conjugation with, 280, 280f,281fD-Glucuronate, 105, 106fβ-Glucuronidases, 281Glucuronidationof bilirubin, 280, 280f, 281fof xenobiotics, 628–629Glucuronides, 163GLUT 1–4. See Glucose transportersGlutamatecarboxylation of, vitamin K as cofactorfor, 487–488, 488fcatabolism of, 249, 250fin proline synthesis, 238, 239fsynthesis of, 237, 238ftransamination and, 243–244, 243f,244fin urea biosynthesis, 243–244, 243f,244fL-Glutamate decarboxylase, 267, 268fGlutamate dehydrogenase/L-glutamatedehydrogenase, 237, 238fin nitrogen metabolism, 244–245, 244f,245fGlutamate-α-ketoglutarate transaminase(glutamate transaminase), in ureabiosynthesis, 243–244, 244fGlutamate-γ-semialdehyde dehydrogenase,block at in hyperprolinemia,250Glutamic acid, 15tGlutaminase, in amino acid nitrogencatabolism, 245, 245fGlutamine, 15tin amino acid nitrogen catabolism, 245,245fcatabolism of, 249, 250fsynthesis of, 237, 238f

INDEX / 663Glutamine analogs, purine nucleotidesynthesis affected by, 293Glutamine synthetase/synthase, 237, 238f,245, 245fGlutamyl amidotransferase, PRPP,regulation of, 294, 295fγ-Glutamyltransferase, 630γ-Glutamyl transpeptidase(γ-glutamyltransferase),diagnostic significance of, 57tGlutaric acid, pK/pK a value of, 12tGlutathioneas antioxidant, 611–613, 613tin conjugation of xenobiotics,629–630as defense mechanism, 629functions of, 629–630Glutathione peroxidase, 88, 166, 167f, 170,612, 613tGlutathione reductase, erythrocytepentose phosphate pathway and, 166,167friboflavin status and, 490Glutathione S-transferases, 629in enzyme study, 58, 59fGlyburide (glibenclamide), 188Glycan intermediates, formation of duringN-glycosylation, 526Glycemic index, 474Glyceraldehyde (glycerose), D and L isomersof, 103f, 104fGlyceraldehyde 3-phosphatein glycolysis, 137, 138foxidation of, 137, 139fGlyceraldehyde 3-phosphate dehydrogenasein glycolysis, 137, 138fin red cell membranes, 615f, 616tGlycerol, 114in lactic acid cycle, 159permeability coefficient of, 419fsynthesis of, 155Glycerol ether phospholipids, synthesis of,199, 200fGlycerol kinase, 155, 197, 198f, 214Glycerol moiety, of triacylglycerols, 123Glycerol phosphate pathway, 198fGlycerol-3-phosphateacylglycerol biosynthesis and, 197, 197f,198ffree energy of hydrolysis of, 82ttriacylglycerol esterification and,214–215, 214fGlycerol-3-phosphate acyltransferase, 198f,199Glycerol-3-phosphate dehydrogenase, 155,198f, 199mitochondrial, 87Glycerophosphate shuttle, 99, 100fGlycerophospholipids, 111Glycerose (glyceraldehyde), D and L isomersof, 103f, 104fGlycine, 15t, 264catabolism of, pyruvate formation and,250, 252fin collagen, 535in heme synthesis, 264, 270–273, 273f,274f, 275f, 276fsynthesis of, 238, 239fGlycine synthase complex, 250Glycinuria, 250Glycocalyx, 110Glycochenodeoxycholic acid, synthesis of,226fGlycocholic acid, synthesis of, 226fGlycoconjugate (complex) carbohydrates,glycoproteins as, 514Glycoforms, 514Glycogen, 102, 107, 108fin carbohydrate metabolism, 123, 123f,155carbohydrate storage and, 145, 146tmetabolism of, 145–152. See alsoGlycogenesis; Glycogenolysisbranching in, 145, 147fclinical aspects of, 151–152, 152fregulation ofcyclic AMP in, 147–150, 148f,149f, 150fglycogen synthase andphosphorylase in, 150–151, 151fin starvation, 234muscle, 145, 146t, 573, 575fGlycogen phosphorylase, 145–146, 146fpyridoxal phosphate as cofactor for,491regulation of, 148–150, 150–151, 150f,151fGlycogen storage diseases, 102, 145,151–152, 152tGlycogen synthase, in glycogen metabolism,145, 146f, 155, 156tregulation of, 148–150, 150–151, 150f,151fGlycogen synthase a, 148–150, 150fGlycogen synthase b, 150, 150fGlycogenesis, 124–125, 145, 146fregulation ofcyclic AMP in, 147–150, 148f, 149f,150fenzymes in, 156tglycogen synthase and phosphorylasein, 148–150, 150–151, 150f,151fGlycogenin, 145, 146fGlycogenolysis, 125, 145–147, 146fblood glucose regulation and, 158–161,159f, 160fcyclic AMP-independent, 148cyclic AMP in regulation of, 147–150,148f, 149f, 150fdebranching enzymes in, 146–147,148fglycogen synthase and phosphorylase inregulation of, 148–150,150–151, 150f, 151fGlycolipid storage diseases, 197Glycolipids (glycosphingolipids), 111, 116,117fABO blood group and, 618amino sugars in, 169, 171fgalactose in synthesis of, 167–169, 170fGlycolysis, 83, 122, 123f, 136–144, 137faerobic, 139anaerobic, 136, 137f, 139as muscle ATP source, 574–576, 575f,575tATP generated by, 142, 143tclinical aspects of, 142–143in erythrocytes, 140, 140fglucose utilization/gluconeogenesis and,136–140, 138f, 139f, 153–155,154f. See also Gluconeogenesispathway of, 136–140, 138f, 139f, 140fpyruvate oxidation and, 134, 135f,140–142, 141f, 142f, 143tregulation of, 140enzymes in, 156tfructose 2,6-bisphosphate in,157–158, 158fgluconeogenesis and, 140, 155–158,156t, 158fat subcellular level, 126, 127fthermodynamic barriers to reversal of,153–155Glycolytic enzymes, in muscle, 556Glycomics, 533Glycophorins, 110, 518, 615–616, 615f,616f, 616tGlycoprotein IIb-IIIa, in platelet activation,607, 622tGlycoprotein glycosyltransferases, 520Glycoproteins, 30, 109–110, 109t, 439f,514–534, 580, 581–582. See alsospecific type and Plasma proteinsamino sugars in, 106, 169, 171fasialoglycoprotein receptor in clearanceof, 517as blood group substances, 514, 618carbohydrates in, 109tclasses of, 518, 519fcomplex, 521, 522fformation of, 521, 524diseases associated with abnormalities of,530, 530t, 531f, 531t, 532f, 533textracellular, absorptive pinocytosis of,430in fertilization, 528functions of, 514, 515t, 528–533, 529tgalactose in synthesis of, 167–169, 170fglycosylphosphatidylinositol-anchored,518, 519f, 527–528, 528thigh-mannose, 521, 522fformation of, 521, 524

664 / INDEXGlycoproteins (cont.)hybrid, 521, 522fformation of, 521immunoglobulins as, 593membrane asymmetry and, 420N-linked, 518, 519f, 521–527nucleotide sugars, 516–517, 516tO-linked, 518, 518–520, 519f, 520f,520t, 521toligosaccharide chains of, 514red cell membrane, 615, 615fsugars in, 515–517, 516ttechniques for study of, 514, 515tasialoglycoprotein receptor in, 517glycosidases in, 517, 517tlectins in, 517–518, 518tin zona pellucida, 528Glycosaminoglycans, 109, 109f, 542,542–547. See also specific typeamino sugars in, 106deficiencies of enzymes in degradation of,545–547, 546t, 547fdisease associations of, 548–549distributions of, 543–545, 544f, 544tfunctions of, 547–549, 548tstructural differences among, 543–545,544f, 544t, 545fsynthesis of, 542–543Glycosidases, in glycoprotein analysis, 517Glycosides, 105–106β-N-Glycosidic bond, 286, 287fGlycosphingolipids (glycolipids), 111, 116,117f, 201–202, 203fABO blood group and, 618amino sugars in, 169, 171fgalactose in synthesis of, 167–169, 170fin membranes, 417membrane asymmetry and, 420Glycosuria, 161N-Glycosylases, in base excision-repair, 337,337fGlycosylated hemoglobin (HbA 1c ), 47Glycosylationof collagen, 537congenital disorders of, 531, 531tin covalent modification, mass increasesand, 27tinhibitors of, 527, 527tnucleotide sugars in, 516–517, 516tN-Glycosylation, 521–527, 523f, 524f,525f, 526tdolichol-P-P-oligosaccharide in,521–524, 523fin endoplasmic reticulum, 524–525,525fglycan intermediates formed during, 526in Golgi apparatus, 524–525, 525finhibition of, 527, 527tregulation of, 526–527, 527ttunicamycin affecting, 527, 527tO-Glycosylation, 520, 521fGlycosylphosphatidylinositol-anchored(GPI-anchored/GPI-linked)glycoproteins, 518, 519f,527–528, 528tin paroxysmal nocturnal hemoglobinuria,531, 531fGlycosyltransferases, glycoprotein, 520,526–527Glypiation, 528GM-CSF. See Granulocyte-macrophagecolony-stimulating factorG M1 ganglioside, 116, 117fG M3 ganglioside, 116GMP, 288t, 297fcyclic, 289f, 290as second messenger, 290, 436, 437t,457, 462–463IMP conversion to, 293, 296ffeedback-regulation of, 294, 296fPRPP glutamyl amidotransferaseregulated by, 294Golgi apparatuscore protein synthesis in, 543glycosylation and, 509, 524–525, 525fin membrane synthesis, 509in protein sorting, 498, 500f, 509in VLDL formation. 213fretrograde transport from, 507Gout/gouty arthritis, 299GPIIb-IIIa, in platelet activation, 607, 622tGPCRs. See G protein-coupled receptorsGPI-anchored/linked glycoproteins, 518,519f, 527–528, 528tin paroxysmal nocturnal hemoglobinuria,531, 531fGranulocyte-colony stimulating factor, 610Granulocyte-macrophage colonystimulatingfactor, 610Granulomatous disease, chronic, 623, 623fGranulosa cells, hormones produced by,442Gratuitous inducers, 378GRE. See Glucocorticoid response elementGRIP1 coactivator, 472, 472tGroup transfer potential, 83, 83fof nucleoside triphosphates, 289–290,289f, 290f, 290tGroup transfer reactions, 8Growth factors, hematopoietic, 610Growth hormone, 437, 438amino acid transport affected by, 427localization of gene for, 407treceptor for, 436GSH. See GlutathioneGSLs. See GlycosphingolipidsGST (glutathione S-transferase) tag, inenzyme study, 58, 59fGTP, 290cyclic GMP formed from, 462in phosphorylation, 85GTPases, 459GTP-γ-S, vesicle coating and, 510Guanine, 288tGuanine activating proteins, 501, 502fGuanine nucleotide exchange factors, 501,502fGuanosine, 287f, 288tbase pairing of in DNA, 303, 304, 305fin uric acid formation, 299, 299fGuanosine diphosphate fucose (GDP-Fuc),516tGuanosine diphosphate mannose(GDP-Man), 516t, 517Guanosine monophosphate. See GMPGuanylyl cyclase, 462, 463Gyrase, bacterial, 306Gyrate atrophy of retina, 250H bands, 556, 557f, 558fH blood group substance, 618, 619fH chains. See Heavy chainsH1 histones, 314, 315fH2A histones, 314, 315H2B histones, 314, 315H3 histones, 314, 314–315H4 histones, 314, 314–315H 2 S. See Hydrogen sulfideH substance, ABO blood group and, 618Haber-Weiss reaction, 612Hageman factor (factor XII), 599f, 600,600tHairpin, 306, 309f, 413Half lifeenzyme, 242protein, 242plasma protein, 582Halt-transfer signal, 506Hapten, in xenobiotic cell injury, 631,631fHaptoglobin, 583t, 584, 584fHartnup disease, 258, 490HAT activity. See Histone acetyltransferaseactivityHbA (hemoglobin A), P 50 of, 42HbA 1c (glycosylated hemoglobin), 47HbF (fetal hemoglobin), P 50 of, 42HbM (hemoglobin M), 46, 363, 614HbS (hemoglobin S), 46, 46f, 363hCG. See Human chorionic gonadotropinHDL. See High-density lipoproteinsHealth, normal biochemical processes asbasis of, 2–4, 3tHeartdevelopmental defects of, 570metabolism in, 235tthiamin deficiency affecting, 489Heart disease, coronary (ischemic). See alsoAtherosclerosischolesterol and, 227Heart failure, 556in thiamin deficiency, 489

INDEX / 665Heat, free energy liberated as, 95Heat-shock proteins, as chaperones, 36–37Heavy chainsimmunoglobulin, 591, 592fgenes producing, 593myosin, 560familial hypertrophic cardiomyopathycaused by mutations in gene for,569–570, 570fHeavy meromyosin, 560f, 561, 561fHeinz bodies, 613Helicases, DNA, 326–327, 327f, 328, 328tHelicobacter pylori, ulcers associated with,474HelixDouble, of DNA structure, 7, 303, 304,305frecombinant DNA technology and,396, 397triple, of collagen structure, 38, 38f,535–539, 536fα-Helix, 31–32, 32f, 33famphipathic, 31–32in myoglobin, 40, 41fHelix-loop-helix motifs, 33Helix-turn-helix motif, 387, 388t,389–390, 389fHemagglutinin, influenza virus, calnexinbinding to, 526Hematology, recombinant DNAtechnology affecting, 624Hematopoietic growth factors, 610Heme, 40, 41f, 270catabolism of, bilirubin produced by,278–280, 279fin proteins, 270. See also Heme proteinssynthesis of, 270–273, 273f, 274f, 275f,276fdisorders of (porphyrias), 274–278,277f, 277tHeme iron, 278, 585absorption of, 478, 585, 585fhindered environment for, 41, 41fHeme oxygenase system, 278, 279fHeme proteins (hemoproteins), 270, 271t.See also Hemoglobin; Myoglobincytochrome P450 isoforms as, 627Heme synthase (ferrochelatase), 271, 272fin porphyria, 277tHemin, 278, 279fHemochromatosis, 478, 586–587, 587fHemoglobin, 40–48, 581fallosteric properties of, 42–46β subunits of, myoglobin and, 42bilirubin synthesis and, 278–280, 279fin carbon dioxide transport, 44, 45fextracorpuscular, haptoglobin binding of,583f, 584glycosylated (HbA 1c ), 47mutant, 46, 362–363oxygen affinities (P 50 ) and, 42–43, 43foxygen dissociation curve for, 41–42,42fin oxygen transport, 40–41oxygenation ofconformational changes and, 43, 43f,44fapoprotein, 422,3-bisphosphoglycerate stabilizing,45, 45fhigh altitude adaptation and, 46mutant hemoglobins and, 46in proton transport, 44tetrameric structure of, 42changes in during development,42–43, 43fHemoglobin A (HbA), P 50 of, 42Hemoglobin A 1c (glycosylated hemoglobin),47Hemoglobin A 2Hemoglobin Bristol, 362Hemoglobin Chesapeake, 46Hemoglobin F(fetal hemoglobin), P 50 of, 42Hemoglobin Hikari, 362–363Hemoglobin M, 46, 363, 614Hemoglobin Milwaukee, 362Hemoglobin S, 46, 46f, 363Hemoglobin Sydney, 362Hemoglobinopathies, 46, 619Hemoglobinuria, paroxysmal nocturnal,432t, 528, 530t, 531, 531fHemolytic anemias, 136, 143, 609, 619,620tglucose-6-phosphate dehydrogenasedeficiency causing, 163,169–170, 613, 614f, 619haptoglobin levels in, 584hyperbilirubinemia/jaundice in, 282,284, 284tpentose phosphate pathway/glutathioneperoxidase and, 166, 167f,169–170primaquine-sensitive, 613red cell membrane abnormalities causing,619Hemopexin, 583tHemophilia A, 604Hemophilia B, 604Hemoproteins. See Heme proteinsHemosiderin, 586Hemostasis, 598–608. See also Coagulationlaboratory tests in evaluation of, 608phases of, 598HEMPAS. See Hereditary erythroblasticmultinuclearity with a positiveacidified lysis testHenderson-Hasselbalch equation, 11Heparan sulfate, 538, 544f, 544t, 545,547–548in basal lamina, 540clotting/thrombosis affected by, 607,607tHeparin, 109, 109f, 544f, 544t, 545, 545f,603–604antithrombin III activity affected by,547, 603–604in basal lamina, 540binding of, fibronectin in, 540, 541ffunctions of, 547lipoprotein and hepatic lipases affectedby, 207Heparin cofactor II, as thrombin inhibitor,603Hepatic ALA synthase (ALAS1), 272in porphyria, 277t, 278Hepatic lipase, 207in chylomicron remnant uptake, 209,209fdeficiency of, 228tHepatic portal system, 158in metabolite circulation, 124, 125fHepatitis, 130in α 1 -antitrypsin deficiency, 590jaundice in, 284tHepatocytesglycoprotein clearance from, asialoglycoproteinreceptor in, 517heme synthesis in, 272ALA synthase in regulation of,272–273, 276fHepatolenticular degeneration (Wilsondisease), 432t, 587, 588–589ceruloplasmin levels in, 587gene mutations in, 432t, 588–589Heptoses, 102, 102tHereditary elliptocytosis, 617Hereditary erythroblastic multinuclearitywith a positive acidified lysis test(HEMPAS), 530t, 531Hereditary hemochromatosis, 586–587,587fHereditary nonpolyposis colon cancer,mismatch repair genes in, 336Hereditary spherocytosis, 432t, 617, 617fHermansky-Pudlak syndrome, 512tHers’ disease, 152tHeterochromatin, 316Heterogeneous nuclear RNA (hnRNA), 310processing of, gene regulation and, 354Heterotrophic organisms, 82Hexapeptide, in albumin synthesis, 583Hexokinase, 156tin blood glucose regulation, 159, 160fin fructose metabolism, 167, 169fin glycogen biosynthesis, 145, 156tin glycolysis, 136–137, 138f, 156tas flux-generating reaction, 129regulation and, 140Hexosamines (amino sugars), 106, 106fglucose as precursor of, 169, 171fin glycosaminoglycans, 109, 169, 171fin glycosphingolipids, 169, 171finterrelationships in metabolism of, 171f

666 / INDEXHexose monophosphate shunt. See Pentosephosphate pathwayHexoses, 102, 102tin glycoproteins, 109tmetabolism of, 163–166, 164f, 165f,167f. See also Pentose phosphatepathwayclinical aspects of, 169–172physiologic importance of, 105, 105tHFE mutations, in hemochromatosis,586–587HGP. See Human Genome ProjectHhaI, 399tHierarchical shotgun sequencing, 634High altitude, adaptation to, 46High-density lipoproteins, 205, 206tapolipoproteins of, 205–206, 206tatherosclerosis and, 210–211, 227metabolism of, 209–211, 211fratio of to low-density lipoproteins, 227receptor for, 210, 211fHigh-density microarray technology, 412High-energy phosphates, 83. See also ATPin energy capture and transfer, 82–83,82f, 82t, 83fas “energy currency” of cell, 83–85, 84f,85fsymbol designating, 83transport of, creatine phosphate shuttlein, 100, 101fHigh-mannose oligosaccharides, 521,522fformation of, 521, 524High-molecular-weight kininogen, 599f, 600High-performance liquid chromatography,reversed phase, for protein/peptide purification, 23–24Hill coefficient, 67Hill equation, 66–67, 67fHindered environment, for heme iron, 41,41fHindIII, 399tHinge regionimmunoglobulin, 591, 592fnuclear receptor protein, 460Hippuric acid/hippurate, synthesis of, 264,265fHistamine, 621tformation of, 265Histidase, impaired, 250Histidine, 16t, 265, 265fβ-alanyl dipeptides and, 264, 265fcatabolism of, 250, 251fconserved residues and, 55tdecarboxylation of, 265in oxygen binding, 40, 41frequirements for, 480Histidine 57, in covalent catalysis, 53–54,54fHistidine E7, in oxygen binding,40, 41fHistidine F8in oxygen binding, 40, 41freplacement of in hemoglobin M, 46Histidinemia, 250Histone acetyltransferase activity, ofcoactivators, 383, 472, 473Histone chaperones, 315Histone dimer, 315Histone octamer, 315, 315fHistone tetramer, 314–315, 315Histones, 314, 314–315, 315f, 315tacetylation and deacetylation of, geneexpression affected by, 383HIV-I, glycoproteins in attachment of, 533HIV protease, in acid-base catalysis, 52, 53fHMG-CoA. See 3-Hydroxy-3-methylglutaryl-CoAHMM. See Heavy meromyosinhMSH1/hMSH2, in colon cancer, 336HNCC. See Hereditary nonpolyposis coloncancerhnRNA. See Heterogeneous nuclear RNAHolocarboxylase synthetase, biotin ascoenzyme of, 494Homeostasisblood in maintenance of, 580hormone signal transduction inregulation of, 456, 457fHomocarnosine, 264, 265fHomocarnosinosis, 264Homocysteinein cysteine and homoserine synthesis,238–239, 239ffunctional folate deficiency and, 494Homocystinurias, 250vitamin B 12 deficiency/functional folatedeficiency and, 492f, 494Homodimers, 34Homogentisate, in tyrosine catabolism,254f, 255Homogentisate dioxygenase/oxidase, 89deficiency of, in alkaptonuria, 255Homologyconserved residues and, 54, 55tin protein classification, 30Homopolymer tailing, 399Homoserine, synthesis of, 239, 239fHormone-dependent cancer, vitamin B 6deficiency and, 491Hormone response elements, 349, 386,388f, 456–457, 459t, 469, 470fHormone-sensitive lipase, 214–215, 214finsulin affecting, 215Hormones. See also specific typein blood glucose regulation, 159classification of, 436–437, 437tfacilitated diffusion regulated by, 427glycoproteins as, 514lipid metabolism regulated by, 215–217,216fin metabolic control, 128f, 129receptors for, 435–436, 436f, 471proteins as, 436recognition and coupling domains on,435–436specificity/selectivity of, 435, 436fsignal transduction and, 456–473intracellular messengers and,457–468, 461t, 463tresponse to stimulus and, 456, 457fsignal generation and, 456–457, 458f,459f, 459ttranscription modulation and,468–473, 470f, 471f, 472tstimulus recognition by, 456, 457fstorage/secretion of, 453, 454tsynthesis ofchemical diversity of, 438, 439fcholesterol in, 438, 438–445, 439t,440fpeptide precursors and, 449–453specialization of, 437tyrosine in, 438, 439–449, 439ttarget cells for, 434–435, 435ttransport of, 454–455, 454t, 455tvitamin D as, 484–486Housekeeping genes, 376Hp. See HaptoglobinHpaI, 399tHPETE. See HydroperoxidesHPLC. See High-performance liquidchromatographyHREs. See Hormone response elementsHRPT. See Hypoxanthine-guaninephosphoribosyl transferasehsp60/hsp70, as chaperones, 36–375HT (5-hydroxytryptamine). See SerotoninHuman chorionic gonadotropin (hCG), 438Human Genome Project, 3–4, 633–641approaches used in elucidation of, 634,635tfuture work and, 637goals of, 633–635implications of, 637–638major findings of, 636–637, 636t, 637tprotein sequencing and, 28Human immunodeficiency virus (HIV-I),glycoproteins in attachment of,533Hunter syndrome, 546tHurler syndrome, 546tHurler-Scheie syndrome, 546tHyaluronic acid, 109, 109f, 543, 544f, 544tdisease associations and, 548functions of, 547Hyaluronidase, 547Hybrid glycoproteins, 521, 522fformation of, 521Hybrid mapping, radiation, 635tHybridization, 397, 403–404, 413in situ, in gene mapping, 406–407, 407t,635t

INDEX / 667Hybridomas, 595–596, 596fHydrocortisone. See CortisolHydrogen bonds, 5, 6fin DNA, 303, 304, 305fHydrogen ion concentration. See also pHenzyme-catalyzed reaction rate affectedby, 64, 64fHydrogen peroxideglutathione in decomposition of, 629as hydroperoxidase substrate, 88–89production of in respiratory burst, 622Hydrogen sulfide, respiratory chain affectedby, 95, 96fHydrolases, 50cholesteryl ester, 223fumarylacetoacetate, defect at, intyrosinemia, 255gluconolactone, 163, 165flysosomal, deficiencies of, 532–533, 533tHydrolysis (hydrolytic reactions), 7–8. Seealso specific reactionfree energy of, 82–83, 82tin glycogenolysis, 146, 146f, 148fof triacylglycerols, 197Hydropathy plot, 419Hydroperoxidases, 86, 88–89Hydroperoxides, formation of, 194, 195fHydrophilic compounds, hydroxylationproducing, 627Hydrophilic portion of lipid molecule, 119,120fHydrophobic effect, in lipid bilayer selfassembly,418Hydrophobic interaction chromatography,for protein/peptide purification,23Hydrophobic interactions, 6–7Hydrophobic portion of lipid molecule,119, 120fHydrostatic pressure, 580L(+)-3-Hydroxyacyl-CoA dehydrogenase,181, 182f3-Hydroxyanthranilate dioxygenase/oxygenase, 89Hydroxyapatite, 549D(–)-3-Hydroxybutyrate (β-hydroxybutyrate),183–184, 184fD(–)-3-Hydroxybutyrate dehydrogenase,184, 184f24-Hydroxycalcidiol (24,25-dihydroxyvitaminD 3 ), in vitamin Dmetabolism, 484, 485f25-Hydroxycholecalciferol (calcidiol), invitamin D metabolism, 484, 485f4-Hydroxydicoumarin (dicumarol), 486Hydroxylamine, for polypeptide cleavage,26t7α-Hydroxylase, bile acid synthesisregulated by, 226, 226f, 22711β-Hydroxylase, in steroid synthesis, 440,441f17α-Hydroxylase, in steroid synthesis, 440,441f, 442, 443f18-Hydroxylase, in steroid synthesis, 440,441f21-Hydroxylase, in steroid synthesis, 440,441f27-Hydroxylase, sterol, 226Hydroxylase cycle, 89, 90fHydroxylases, 89–90in steroid synthesis, 438, 440, 441fHydroxylationin collagen processing, 537in covalent modification, mass increasesand, 27tof xenobiotics, 626, 626–628, 629tHydroxylysine, synthesis of, 2405-Hydroxymethylcytosine, 287, 289f3-Hydroxy-3-methylglutaryl-CoA(HMG-CoA)in ketogenesis, 184–185, 185fin mevalonate synthesis, 219, 220f3-Hydroxy-3-methylglutaryl-CoA(HMG-CoA) lyasedeficiency of, 188in ketogenesis, 185, 185f3-Hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductasecholesterol synthesis controlled by, 220,223fin mevalonate synthesis, 219, 220f3-Hydroxy-3-methylglutaryl-CoA(HMG-CoA) synthasein ketogenesis, 185, 185fin mevalonate synthesis, 219, 220fp-Hydroxyphenylpyruvate, in tyrosinecatabolism, 254f, 25517-Hydroxypregnenolone, 440, 441f17-Hydroxyprogesterone, 440, 441fHydroxyprolinesynthesis of, 240, 240f, 535–537tropoelastin hydroxylation and, 5394-Hydroxyproline, catabolism of, 253f,2554-Hydroxyproline dehydrogenase, defect in,in hyperhydroxyprolinemia,25515-Hydroxyprostaglandin dehydrogenase,1943β-Hydroxysteroid dehydrogenase, 438,441f, 442, 443f17β-Hydroxysteroid dehydrogenase, 442,443f5-Hydroxytryptamine. See SerotoninHyperalphalipoproteinemia, familial, 228tHyperammonemia, types 1 and 2, 247Hyperargininemia, 248Hyperbilirubinemia, 281–284, 284tHypercholesterolemia, 205familial, 1, 228t, 432tLDL receptor deficiency in, 209, 432tHyperchromicity of denaturation, 304–305Hyperglycemia. See also Diabetes mellitusglucagon causing, 161insulin release in response to, 466fHyperhomocysteinemia, folic acid supplementsin prevention of, 494Hyperhydroxyprolinemia, 255Hyperkalemic periodic paralysis, 569tHyperlacticacidemia, 212Hyperlipidemia, 170–171, 490Hyperlipoproteinemias, 205, 228t, 229familial, 228tHyperlysinemia, periodic, 258Hypermetabolism, 136, 479Hyperornithinemia-hyperammonemiasyndrome, 250Hyperoxaluria, primary, 250Hyperparathyroidism, bone and cartilageaffected in, 551tHyperphenylalaninemias, 255Hyperprolinemias, types I and II, 249–250Hypersensitive sites, chromatin, 316Hypersplenism, in hemolytic anemia, 619Hypertension, hyperhomocysteinemia and,folic acid supplements inprevention of, 494Hyperthermia, malignant, 556, 564–565,565f, 569tHypertriacylglycerolemiain diabetes mellitus, 205familial, 228tHypertrophic cardiomyopathy, familial,569–570, 570fHyperuricemia, 170–171, 300Hypervariable regions, 591–592, 594fHypoglycemia, 153fatty acid oxidation and, 180, 187–188fructose-induced, 171–172insulin excess causing, 162during pregnancy and in neonate, 161Hypoglycin, 180, 188Hypokalemic periodic paralysis, 569tHypolipidemic drugs, 229Hypolipoproteinemia, 205, 228t, 229Hypouricemia, 300Hypoxanthine, 289Hypoxanthine-guanine phosphoribosyltransferase (HRPT)defect of in Lesch-Nyhan syndrome, 300localization of gene for, 407tHypoxia, lactate production and, 136, 137f,139–140I. See Iodine/iodideI bands, 556, 557f, 558fI-cell disease, 431, 432t, 512t, 524, 530t,531–532, 532t, 546–547, 546tIbuprofen, cyclooxygenases affected by, 193ICAM-1, 529, 529tICAM-2, 529, 529tICF. See Intracellular fluid

668 / INDEXIcterus (jaundice), 270, 281–284, 284tIDDM. See Insulin-dependent diabetesmellitusIdiotypes, 594IDL. See Intermediate-density lipoproteinsL-Iduronate, 105, 106fIEF. See Isoelectric focusingIgA, 591, 594t, 595fIgD, 591, 594tIgE, 591, 594tIGF-I. See Insulin-like growth factor-IIgG, 591, 592f, 594tdeficiency of, 595hypervariable regions of, 591–592,594fIgM, 591, 594t, 595fImmune response, class/isotype switchingand, 594Immunoglobulin genes, 593DNA rearrangement and, 325–326, 393,593–594double-strand break repair and,337–338Immunoglobulin heavy chain bindingprotein, 508Immunoglobulin heavy chains, 591, 592fgenes producing, 593Immunoglobulin light chains, 591, 592fin amyloidosis, 590genes producing, 593DNA rearrangement and, 325–326,393, 593–594Immunoglobulins, 583t, 591–597, 593t.See also specific type under Igclass switching and, 594classes of, 591, 593t, 594tdiseases caused by over- andunderproduction of, 594–595functions of, 593, 594tgenes for. See Immunoglobulin geneshybridomas as sources of, 595–596, 596fstructure of, 591, 592, 592f, 594f, 595fIMP (inosine monophosphate)conversion of to AMP and GMP, 293,296ffeedback regulation of, 294, 296fsynthesis of, 293–294, 295f, 296f, 297fImportins, 501, 502fIn situ hybridization/fluorescence in situhybridization, in gene mapping,406–407, 407t, 635tInactive chromatin, 316–318, 383Inborn errors of metabolism, 1, 249, 545Inclusion cell (I-cell) disease, 431, 432t,512t, 524, 530t, 531–532, 532tIndole, permeability coefficient of, 419fIndomethacin, cyclooxygenases affected by,193Induced fit model, 52, 53fInducersenzyme synthesis affected by, 74in gluconeogenesis regulation,155–157gratuitous, 378in regulation of gene expression, 376Inducible gene, 376Infantile Refsum disease, 188, 503, 503tInfectionglycoprotein hydrolase deficiencies and,533neutrophils in, 620, 621tprotein loss and, 480respiratory burst in, 622–623Inflammation, 190acute phase proteins in, 583, 583tcomplement in, 596neutrophils in, 620, 621tintegrins and, 529t, 620–621, 622tselectins and, 528–529, 529t, 530fNF-κB in, 468prostaglandins in, 190selectins in, 528–530, 529f, 529t, 530fInfluenza virushemagglutinin in, calnexin binding to,526neuraminidase in, 533Information pathway, 457, 459fInhibitioncompetitive versus noncompetitive,67–69, 67f, 68f, 69ffeedback, in allosteric regulation, 74–76,75firreversible, 69Inhibitor-1, 148, 149f, 151, 151fInitial velocity, 64inhibitors affecting, 68, 68f, 69fInitiationin DNA synthesis, 328–330, 329f, 330f,331fin protein synthesis, 365–367, 366fin RNA synthesis, 342, 342f, 343–344Initiation complexes, in protein synthesis,365, 366f, 367Initiator sequence, 346–348, 347fInner mitochondrial membrane, 92, 93fimpermeability of, exchange transportersand, 98–100, 98f, 99fprotein insertion in, 501Inorganic pyrophosphatase, in fatty acidactivation, 85Inosine monophosphate (IMP)conversion of to AMP and GMP, 293,296ffeedback-regulation of, 294, 296fsynthesis of, 293–294, 295f, 296f, 297fInositol hexaphosphate (phytic acid), calciumabsorption affected by, 477Inositol trisphosphate, 464–465, 464f, 465fin platelet activation, 606, 606f, 607in respiratory burst, 623Inotropic effects, 566Inr. See Initiator sequenceInsert/insertions, DNA, 413recombinant DNA technology indetection of, 409Inside-outside asymmetry, membrane,419–420Insulators, 387nonpolar lipids as, 111Insulin, 107–109, 438, 449, 450fadipose tissue metabolism affected by,216–217in blood glucose regulation, 160–162deficiency of, 161. See also Diabetesmellitusfree fatty acids affected by, 215gene for, localization of, 407tglucagon opposing actions of, 160–161in glucose transport, 427in glycolysis, 137, 155–157initiation of protein synthesis affected by,367, 367fin lipogenesis regulation, 178–179in lipolysis regulation, 178–179, 215,216fphosphorylase b affected by, 148receptor for, 436, 465, 466fsignal transmission by, 465–467, 466fstorage/secretion of, 453, 454tsynthesis of, 449, 450fInsulin-dependent diabetes mellitus(IDDM/type 1), 161–162. Seealso Diabetes mellitusInsulin/glucagon ratio, in ketogenesisregulation, 187Insulin-like growth factor I, receptor for, 436Insulin resistance, 611Integral proteins, 30, 420, 421fas receptors, 431red cell membrane, 615–616, 615f, 616f,616tIntegration, chromosomal, 324, 324fIntegrins, neutrophil interactions and, 529t,620–621, 622tIntercellular junctions, 431Intermediate-density lipoproteins, 206tIntermediate filaments, 577–578, 577tIntermembrane space, proteins in, 501Intermittent branched-chain ketonuria, 259Internal presequences, 501Internal ribosomal entry site, 371, 371fInterphase chromosomes, chromatin fibersin, 316Intervening sequences. See IntronsIntestinal bacteria, in bilirubin conjugation,281Intracellular environment, membranes inmaintenance of, 415–416, 416tIntracellular fluid (ICF), 415, 416, 416tIntracellular membranes, 415Intracellular messengers, 457–468, 461t,463t. See also specific type andSecond messengers

INDEX / 669Intracellular signals, 457–458Intracellular traffic, 498–513. See alsoProtein sortingdisorders due to mutations in genesencoding, 512t, 513Intrinsic factor, 477, 491–492in pernicious anemia, 492Intrinsic pathway of blood coagulation,598, 599f, 600–601Introns (intervening sequences), 319,352–354, 353f, 358, 413in recombinant DNA technology, 397,398fremoval of from primary transcript,352–354, 353fInulin, glomerular membrane permeabilityto, 540“Invert sugar,” 107Iodine/iodide, 496tdeficiency of, 447–449in thyroid hormone synthesis, 447, 448f,4495-Iodo-2′-deoxyuridine, 291fIodopsin, 483o-Iodosobenzene, for polypeptide cleavage,26tIodothyronyl residues, 447. See alsoThyroxine; Triiodothyronine5-Iodouracil, 290Ion channels, 415, 423–424, 425f, 426t,568tin cardiac muscle, 566–567, 568, 568tdiseases associated with disorders of, 568,569tIon exchange chromatography, for protein/peptide purification, 22–23Ion product, 8–9Ion transport, in mitochondria, 99Ionizing radiation, nucleotide excisionrepairof DNA damage causedby, 337Ionophores, 99, 424IP 3 . See Inositol trisphosphateIPTG. See IsopropylthiogalactosideIREG1. See Iron regulatory proteinIRES. See Internal ribosomal entry siteIron, 496tabsorption of, 478, 584–586, 585f,585tin hemochromatosis, 478vitamin C and ethanol affecting, 478,496deficiency of, 497distribution of, 585tferrous, in oxygen transport, 40–41heme, 278, 585absorption of, 478, 585, 585fhindered environment for, 41, 41fin methemoglobinemia, 46incorporation of into protoporphyrin,271–272, 272fmetabolism of, 585, 585fdisorders of, 586, 587tnonheme, 92, 95f, 585transferrin in transport of, 584–586,585f, 585tIron-binding capacity, total, 586Iron deficiency/iron deficiency anemia, 478,497, 586Iron porphyrins, 270Iron regulatory protein, 585Iron response elements, 586Iron-sulfur protein complex, 92, 95fIrreversible covalent modifications, 76–77,77fIrreversible inhibition, enzyme, 69IRS 1–4, in insulin signal transmission,465, 466fIschemia, 136, 431Islets of Langerhans, insulin produced by,160Isocitrate dehydrogenase, 130–131, 132fin NADPH production, 176, 176fIsoelectric focusing, for protein/peptidepurification, 24, 25fIsoelectric pH (pI), amino acid net chargeand, 17Isoenzymes. See IsozymesIsoleucine, 15tcatabolism of, 259, 260f, 261finterconversion of, 240requirements for, 480∆ 5,4 -Isomerase, 438, 441f, 442, 443fIsomerases, 50in steroid synthesis, 438, 441f, 442,443fIsomerismgeometric, of unsaturated fatty acids,112–114, 114fof steroids, 117, 118fof sugars, 102–104, 103fIsoniazid, acetylation of, 630Isopentenyl diphosphate, in cholesterolsynthesis, 219, 221fIsoprene units, polyprenoids synthesizedfrom, 118, 119fIsoprenoids, synthesis of, in cholesterolsynthesis, 219, 221f, 222fIsopropylthiogalactoside, 378Isoprostanes (prostanoids), 112, 119cyclooxygenase pathway in synthesis of,192, 192–194, 193f, 194fIsothermic systems, biologic systems as,80Isotopes. See also specific typein plasma protein analysis, 581Isotype (class) switching, 594Isotypes, 594Isovaleric acidemia, 259, 259–262Isovaleryl-CoA dehydrogenase, in isovalericacidemia, 259–262Isozymes, 54–55J chain, 595fJackson-Weiss syndrome, 551tJAK kinases, 436, 467, 467fJak-STAT pathway, 436, 467, 467fJamaican vomiting sickness, 188Jaundice (icterus), 270, 281–284, 284tJoining region, gene for, 593DNA rearrangement and, 393, 593–594“Jumping DNA,” 325Junctional diversity, 593–594Juxtaglomerular cells, in renin-angiotensinsystem, 451K. See Dissociation constantK. See Potassiumk. See Rate constantK d . See Dissociation constantk deg . See Rate of degradationK eq . See Equilibrium constantK m . See Michaelis constantk s . See Rate of synthesisK w . See Ion productKappa (κ) chains, 591Kartagener syndrome, 577Karyotype, 320fKayser-Fleischer ring, 588KDEL-containing proteins, 506–507,508tKeratan sulfates, 544f, 544t, 545functions of, 547Keratins, 577t, 578Kernicterus, 282, 283α-Keto acid decarboxylase, defect/absenceof, in maple syrup urine disease(branched-chain ketonuria), 259α-Keto acid dehydrogenase,branched-chain, 259thiamin diphosphate as coenzyme for,488–489α-Keto acidsamino acids in diet replaced by, 240oxidative carboxylation of, 259, 260f,261f, 262fKetoacidosis, 180, 188–1893-Ketoacyl-CoA thiolase deficiency, 1883-Ketoacyl synthase, 173, 175fKetogenesis, 125–126, 126f, 183–187high rates of fatty acid oxidation and,183–186, 184fHMG-CoA in, 184–185, 185fregulation of, 186–187, 187f, 188fKetogenic amino acids, 232α-Ketoglutarate, 131in amino acid carbon skeletoncatabolism, 249, 250, 250f,251fin glutamate synthesis, 237, 238f, 243,243f, 244ftransporter systems for, 99in urea synthesis, 244, 244f

670 / INDEXα-Ketoglutarate dehydrogenase complex,131, 132fregulation of, 135thiamin diphosphate as coenzyme for,488–489Ketone bodies, 124, 125–126, 126f, 180,183–184, 184ffree fatty acids as precursors of, 186as fuel for extrahepatic tissues, 185–186,186fin starvation, 232–234, 234f, 234tKetonemia, 186, 188Ketonuria, 188branched chain (maple syrup urinedisease), 259Ketoses (sugars), 102, 102tKetosis, 180, 186, 188in cattlefatty liver and, 212lactation and, 188in diabetes mellitus, 188ketoacidosis caused by, 188–189nonpathologic, 188–189in starvation, 188Kidneyglycogenolysis in, 147metabolism in, 235tin renin-angiotensin system, 451vitamin D 3 synthesis in, 445, 446f, 484Kinases, protein. See Protein kinasesKinesin, 577Kinetic (collision) theory, 61Kinetics (enzyme), 60–71. See also Catalysisactivation energy affecting, 61, 63balanced equations and, 60competitive versus noncompetitive inhibitionand, 67–69, 67f, 68f, 69ffactors affecting reaction rate and,61–63, 62f, 63–64, 64ffree energy changes affecting, 60–61initial velocity and, 64multisubstrate enzymes and, 69–70, 69f,70fsaturation, 64f, 66sigmoid (Hill equation), 66–67, 67fsubstrate concentration and, 64, 64f, 65fmodels of effects of, 65–67, 66f, 67ftransition states and, 61Kinetochore, 318Kininogen, high-molecular-weight, 599f,600Kinky hair disease (Menkes disease), 588Knockout genes, 412Korsakoff’s psychosis, 489Kozak consensus sequences, 365Krabbe’s disease, 203tKrebs cycle. See Citric acid cycleKu, in double-strand break repair, 338,338fKwashiorkor, 237, 478, 478–479Kynureinase, 257f, 258Kynurenine-anthranilate pathway, fortryptophan catabolism, 257f, 258Kynurenine formylase, 257f, 258L-α-amino acids, 14. See also Amino acidsgenetic code specifying, 14, 15–16tin proteins, 14L chains. See Light chainsL-Dopa, 446, 447fL isomerism, 102–104, 103fL-selectin, 529f, 529tL-type calcium channel, 567Labile factor (factor V), 600t, 601, 602flacA gene, 376, 376f, 377f, 378lacI gene, 377, 377f, 378lac operon, 375, 376–378, 376f, 377flac repressor, 377, 377flacY gene, 376, 376f, 377f, 378lacZ gene, 376, 376f, 377f, 378Lactase, 475deficiency of (lactose/milk intolerance),102, 474, 475Lactateanaerobic glycolysis and, 136, 137f,139–140hypoxia and, 137f, 139–140Lactate dehydrogenase, in anaerobicglycolysis, 139Lactate dehydrogenase isozymes, 57, 139diagnostic significance of, 57, 57t, 58fLactic acid, pK/pK a value of, 12tLactic acid cycle, 159, 159fLactic acidosis, 92, 136with mitochondrial encephalopathy andstroke-like episodes (MELAS),100–101pyruvate metabolism and, 142–143thiamin deficiency and, 489Lactoferrin, 621tLactogenic hormone. See ProlactinLactose, 106–107, 107f, 107tgalactose in synthesis of, 167–169, 170fmetabolism of, operon hypothesis and,376–378, 376f, 377fLactose (milk) intolerance, 102, 474, 475Lactose synthase, 167, 170fLagging (retrograde) strand, in DNAreplication, 327f, 328, 330–331Lambda (λ) chains, 591Lambda (λ) phage, 378–383, 379f, 380f,381f, 382fLambda repressor (cI) protein/gene,379–383, 380f, 381f, 382fLaminin, 535, 540–542, 541fLamins, 577t, 578Langerhans, islets of, insulin produced by,160Lanosterol, in cholesterol synthesis, 219,220, 222fLatch state, 571Lauric acid, 112tLaws of thermodynamics, 80–81hydrophobic interactions and, 7LBD. See Ligand-binding domainLCAT. See Lecithin:cholesterolacyltransferaseLCRs. See Locus control regionsLDH. See Lactate dehydrogenase isozymesLDL. See Low-density lipoproteinsLDL:HDL cholesterol ratio, 227Lead poisoning, ALA dehydratase inhibitionand, 270, 278Leader sequence. See Signal peptideLeading (forward) strand, in DNAreplication, 327f, 328, 330Lecithin:cholesterol acyltransferase (LCAT),200–201, 209–210, 211f, 223,224familial deficiency of, 228tLecithins (phosphatidylcholines), 114–115,115fin cytochrome P450 system, 617membrane asymmetry and, 420synthesis of, 197, 197f, 198fLectins, 110, 517–518, 518tin glycoprotein analysis, 515t, 517–518,518tLeiden factor V, 603Lens of eye, fructose and sorbitol in,diabetic cataract and, 172Leptin, 215–216Lesch-Nyhan syndrome, 300Leucine, 15tcatabolism of, 259, 260f, 261finterconversion of, 240requirements for, 480Leucine aminomutase, 492Leucine zipper motif, 387–388, 388t, 390,391fLeucovorin, 493Leukocyte adhesion deficiencytype I, 621type II, 530t, 531Leukocytes, 620–624growth factors regulating production of,610recombinant DNA technology in studyof, 624Leukodystrophy, metachromatic, 203tLeukotriene A 4 , 114fLeukotrienes, 112, 114f, 190, 192clinical significance of, 196lipoxygenase pathway in formation of,192, 193f, 194, 195fLFA-1, 529, 529t, 620, 622tLH. See Luteinizing hormoneLibrary, 402, 413Lifestyle changes, cholesterol levels affectedby, 227–228Ligand-binding domain, 470Ligand-gated channels, 424, 568t

INDEX / 671Ligand-receptor complex, in signalgeneration, 456–457Ligases, 50DNA, 328t, 330, 332, 332fLigation, 413in RNA processing, 352Light, in active transport, 427Light chainsimmunoglobulin, 591, 592fin amyloidosis, 590genes producing, 593DNA rearrangement and, 325–326,393, 593–594myosin, 560in smooth muscle contraction, 570Light meromyosin, 560–561, 560fLimit dextrinosis, 152tLines, definition of, 413LINEs. See Long interspersed repeatsequencesLineweaver-Burk plotinhibitor evaluation and, 68, 68f, 69fK m and V max estimated from, 66, 66fLingual lipase, 475Link trisaccharide, in glycosaminoglycansynthesis, 543Linkage analysis, 635tin glycoprotein study, 515tLinoleic acid/linoleate, 113t, 190, 190f,192in essential fatty acid deficiency, 191synthesis of, 191fα-Linolenic acid/α-linolenate, 113t, 190,190f, 192in essential fatty acid deficiency, 191synthesis of, 191, 191fγ-Linolenic acid/γ-linolenate, 113tin essential fatty acid deficiency, 191in polyunsaturated fatty acid synthesis,191, 192fLipasesdiagnostic significance of, 57tin digestion, 475, 476fin triacylglycerol metabolism, 197,214–215, 214f, 475, 476fLipid bilayer, 418–419, 418f, 419fmembrane proteins and, 419Lipid core, of lipoprotein, 205Lipid rafts, 422Lipid storage disorders (lipidoses),202–203, 203tLipids, 111–121. See also specific typeamphipathic, 119–121, 120fasymmetry of, membrane assembly and,511, 512fclassification of, 111complex, 111in cytochrome P450 system, 627derived, 111digestion and absorption of, 475–477,476fdisorders associated with abnormalitiesof, 431fatty acids, 111–114glycolipids, 111, 116, 117finterconvertibility of, 231in membranes, 416–418ratio of to protein, 416, 416fmetabolism of, 122f, 123–124, 123f,125–126, 126f. See also Lipolysisin fed state, 232in liver, 211–212, 213fneutral, 111peroxidation of, 118–119, 120fphospholipids, 111, 114–116, 115fprecursor, 111simple, 111steroids, 117–118, 117f, 118f, 119ftransport and storage of, 205–218adipose tissue and, 214–215, 214fbrown adipose tissue and, 217, 217fclinical aspects of, 212–214fatty acid deficiency and, 194–195as lipoproteins, 205–206, 206t, 207fliver in, 211–212, 213ftriacylglycerols (triglycerides), 114, 115fturnover of, membranes and, 511–512Lipogenesis, 125, 173–177, 174f, 175facetyl-CoA for, 176–177fatty acid synthase complex in, 173–176,174f, 175fmalonyl-CoA production in, 173, 174fNADPH for, 175f, 176, 176fregulation of, 178–179, 178fenzymes in, 156t, 173, 174f, 178,178fnutritional state in, 177–178Lipolysis, 125, 126f, 216–217, 216f. Seealso Lipids, metabolism ofhormone-sensitive lipase in, 214–215,214fhormones affecting, 215–216, 216finsulin affecting, 178–179triacylglycerol, 197Lipophilic compounds, cytochrome P450isoforms in hydroxylation of, 627Lipoprotein lipase, 125, 126f, 207–208,209f, 210ffamilial deficiency of, 228tinvolvement in remnant uptake, 208,209fα 1 -Lipoprotein, 581fβ 1 -Lipoprotein, 581fLipoprotein(a) excess, familial, 228tLipoproteins, 30, 111, 125, 205–206, 206t,207f, 580, 583t. See also specifictypecarbohydrates in, 110in cholesterol transport, 223–224, 225fclassification of, 205, 206tdeficiency of, fatty liver and, 212disorders of, 228t, 229remnant, 206t, 208, 209fliver uptake of, 208–209Liposomes, 421amphipathic lipids forming, 119–121,120fartificial membranes and, 421Lipotropic factor, 212β-Lipotropin, 453, 453fLipoxins, 112, 114f, 190, 192clinical significance of, 196lipoxygenase pathway in formation of,192, 193f, 194, 195fLipoxygenase, 119, 194, 195freactive species produced by, 1195-Lipoxygenase, 194, 195fLipoxygenase pathway, 192, 193f, 194,195fLiquid chromatography, high-performancereversed-phase, for peptideseparation, 23–24Lithium, 496tLithocholic acid, synthesis of, 226fLiverangiotensinogen made in, 451bilirubin uptake by, 280–281, 280f,281f, 282fcirrhosis of, 130, 212in α 1 -antitrypsin deficiency, 590cytochrome P450 isoforms in, 627disorders of, in α 1 -antitrypsin deficiency,589–590, 590ffattyalcoholism and, 212–214of pregnancy, 188triacylglycerol metabolism imbalanceand, 212fructose overload and, 170–171glycogen in, 145, 146theme synthesis in, 272ALA synthase in regulation of,272–273, 276fketone bodies produced by, 183–184,184f, 186metabolism in, 124–125, 125f, 126f,130, 235tfatty acid oxidation, ketogenesis and,183–186, 184ffructose, 167, 169fglucose, 154f, 159, 159–160,159ffructose 2,6-bisphosphate inregulation of, 157–158, 158fglycogen, 145–147, 145, 146f, 148lipid, 211–212, 213fplasma protein synthesis in, 125, 581vitamin D 3 synthesis in, 445, 446f, 484,485fLiver phosphorylase, 147deficiency of, 152tLMM. See Light meromyosinLock and key model, 52

672 / INDEXLocus control regions, 387Long interspersed repeat sequences(LINEs), 321–322Looped domains, chromatin, 316, 318,319fLoops (protein conformation), 32–33Loose connective tissue, keratan sulfate I in,545Low-density lipoprotein receptor-relatedprotein, 206in chylomicron remnant uptake,208–209, 209fLow-density lipoproteins, 205, 206tapolipoproteins of, 206, 206tmetabolism of, 209, 210fratio of to high-density lipoproteins,atherosclerosis and, 227receptors for, 209in chylomicron remnant uptake,208–209, 209fin cotranslational insertion, 505–506,506fregulation of, 223Low-energy phosphates, 83β-LPH. See β-LipotropinLRP. See Low-density lipoproteinreceptor-related proteinL-tryptophan dioxygenase (tryptophanpyrrolase), 89LTs. See LeukotrienesLung surfactant, 115, 197deficiency of, 115, 202Luteinizing hormone (LH), 437, 438, 439fLXs. See LipoxinsLXXLL motifs, nuclear receptorcoregulators, 473Lyases, 50in steroid synthesis, 440–442, 441f,443fLymphocyte homing, selectins in, 528–530,529f, 529t, 530fLymphocytes. See also B lymphocytes;T lymphocytesrecombinant DNA technology in studyof, 624Lysine, 16tcatabolism of, 256f, 258pI of, 17requirements for, 480Lysine hydroxylase, vitamin C as coenzymefor, 496Lysis, cell, complement in, 596Lysogenic pathway, 379, 379fLysolecithin (lysophosphatidylcholine),116, 116fmetabolism of, 200–201, 201fLysophosphatidylcholine. See LysolecithinLysophospholipase, 200, 201fLysophospholipids, 116, 116fLysosomal degradation pathway, defect inin lipidoses, 203Lysosomal enzymes, 623in I-cell disease, 431, 432t, 531–532,532fLysosomal hydrolases, deficiencies of,532–533, 533tLysosomesin oligosaccharide processing, 524protein entry into, 507, 507f, 508tdisorders associated with defects in,512t, 513Lysozyme, 621tLysyl hydroxylasediseases caused by deficiency of, 538tin hydroxylysine synthesis, 240, 537Lysyl oxidase, 537, 539Lytic pathway, 379, 379fD-Lyxose, 104f, 105tMac-1, 529, 529tα 2 -Macroglobulin, 583t, 590, 624antithrombin activity of, 603Macromolecules, cellular transport of,428–431, 429f, 430fMad cow disease (bovine spongiformencephalopathy), 37Magnesium, 496tin chlorophyll, 270in extracellular and intracellular fluid,416, 416tMajor groove, in DNA, 305f, 306operon model and, 378Malate, 132f, 133Malate dehydrogenase, 132f, 133Malate shuttle, 99, 100fMALDI. See Matrix-assisted laser-desorptionMaleylacetoacetate, in tyrosine catabolism,254f, 255Malic enzyme, 156t, 157in NADPH production, 176, 176fMalignancy/malignant cells. SeeCancer/cancer cellsMalignant hyperthermia, 556, 564–565,565f, 569tMalonaterespiratory chain affected by, 95, 96fsuccinate dehydrogenase inhibition by,67–68, 67fMalonyl-CoA, in fatty acid synthesis, 173,174fMalonyl transacylase, 173, 174f, 175fMaltase, 475Maltose, 106–107, 107f, 107tMammalian target of rapamycin (mTOR),in insulin signal transmission,466f, 467Mammotropin. See ProlactinManganese, 496tMannosamine, 169, 171fD-Mannosamine, 106Mannose, in glycoproteins, 516tD-Mannose, 104f, 105tα-D-Mannose, 104fMannose-binding protein, deficiency of, 533Mannose 6-phosphate/mannose 6-P signal,526in I-cell disease, 531, 532, 532fin protein flow, 507, 508tMannosidosis, 532–533, 533tMAP (mitogen-activated protein) kinasein insulin signal transmission, 466f, 467in Jak/STAT pathway, 467Maple syrup urine disease (branched-chainketonuria), 259Marasmus, 80, 237, 478, 478–479Marble bone disease (osteopetrosis), 552Marfan syndrome, fibrillin mutationscausing, 539–540, 540fMaroteaux-Lamy syndrome, 546tMass spectrometry, 27, 27fcovalent modifications detected by, 27,27f, 27tfor glycoprotein analysis, 514, 515ttandem, 27transcript-protein profiling and, 412Mast cells, heparin in, 545Matrixextracellular, 535–555. See also specificcomponentmitochondrial, 92, 93f, 130Matrix-assisted laser-desorption (MALDI),in mass spectrometry, 27Matrix-processing peptidase, 499Matrix proteins, 499diseases caused by defects in import of,503Maxam and Gilbert’s method, for DNAsequencing, 404–405Maximal velocity (V max )allosteric effects on, 75–76inhibitors affecting, 68, 68f, 69fMichaelis-Menten equation indetermination of, 65–66, 66fsubstrate concentration and, 64, 64fMcArdle’s disease/syndrome, 152t, 573Mechanically gated ion channels, 568tMediator-related proteins, 472t, 473Medicinepreventive, biochemical researchaffecting, 2relationship of to biochemistry, 1–4, 3fMedium-chain acyl-CoA dehydrogenase,deficiency of, 188Megaloblastic anemiafolate deficiency causing, 482t, 492, 610tvitamin B 12 deficiency causing, 482t,492, 494, 610tMelanocyte-stimulating hormone (MSH),453, 453fMELAS (mitochondrial encephalomyopathywith lactic acidosis andstroke-like episodes), 100–101

INDEX / 673Melting point, of amino acids, 18Melting temperature/transitiontemperature, 305, 422Membrane attack complex, 596Membrane fatty acid-transport protein, 207Membrane proteins, 419, 420t, 514. Seealso Glycoproteinsassociation of with lipid bilayer, 419flow of, 507, 507f, 508tintegral, 30, 420, 421fmutations affecting, diseases caused by,431–432, 432f, 432tperipheral, 420–421, 421fred cell, 614–617, 615f, 616f, 616tstructure of, dynamic, 419Membrane transport, 423, 423t, 424f,426–431, 426f. See also specificmechanismMembranes, 415–433artificial, 421–422assembly of, 511–513, 512f, 512tasymmetry of, 416, 419–420bilayers of, 418–419, 418f, 419fmembrane protein association and, 419biogenesis of, 511–513, 512f, 512tcholesterol in, 417fluid mosaic model and, 422depolarization of, in nerve impulsetransmission, 428function of, 415–416, 421–422fluidity affecting, 422glycosphingolipids in, 417Golgi apparatus in synthesis of, 509intracellular, 415lipids in, 416–418amphipathic, 119, 120f, 417–418, 417fmutations affecting, diseases caused by,431–432, 432f, 432tphospholipids in, 114–116, 115f,416–417, 417fplasma. See Plasma membraneprotein:lipid ratio in, 416, 416fproteins in, 419, 420t. See alsoMembrane proteinsred cell, 614–617, 615f, 615t, 616f, 616themolytic anemias and, 619, 620tselectivity of, 415, 423–426, 423t, 424f,425f, 426tsterols in, 417structure of, 416–421, 416fasymmetry and, 416, 419–420fluid mosaic model of, 421f, 422Menadiol, 486, 487fMenadiol diacetate, 486, 488fMenadione, 486. See also Vitamin KMenaquinone, 482t, 486, 488f. See alsoVitamin KMenkes disease, 588MEOS. See Cytochrome P450-dependentmicrosomal ethanol oxidizingsystem6-Mercaptopurine, 290, 291fMercapturic acid, 629Mercuric ions, pyruvate metabolismaffected by, 142Meromyosinheavy, 560f, 561, 561flight, 560–561, 560fMessenger RNA (mRNA), 307, 309–310,310f, 311f, 341, 342t, 359. Seealso RNAalternative splicing and, 354, 354f,393–394, 636codon assignments in, 358, 359tediting of, 356expression of, detection of in geneisolation, 635tmodification of, 355–356nucleotide sequence of, 358mutations caused by changes in,361–363, 361f, 362f, 364fpolycistronic, 376recombinant DNA technology and, 397relationship of to chromosomal DNA,321fstability of, regulation of gene expressionand, 394–395, 394ftranscription starting point and, 342variations in size/complexity of, 397,399tMetabolic acidosis, ammonia in, 245Metabolic alkalosis, ammonia in, 245Metabolic fuels, 231–236. See also Digestionclinical aspects of, 236diet providing, 474, 478in fed and starving states, 232–234,233f, 234f, 234tinterconvertability of, 231–232Metabolic pathway/metabolite flow, 122,122–124. See also specific typeand Metabolismflux-generating reactions in, 129nonequilibrium reactions in, 128–129regulation of, 72, 73f, 126–129, 128fcovalent modification in, 79unidirectional nature of, 72, 73fMetabolism, 81, 122–129, 235t. See alsospecific type and Catalysis;Metabolic pathwayblood circulation and, 124–126, 125f,126fgroup transfer reactions in, 8inborn errors of, 1, 249integration of, metabolic fuels and,231–236regulation of, 72, 73f, 126–129, 128fallosteric and hormonal mechanismsin, 74, 74–76, 75f, 128f, 129enzymes in, 126–129, 128fallosteric regulation and, 74,74–76, 75f, 128f, 129compartmentation and, 72–73control of quantity and, 73–74covalent modification and, 74, 76,77–78, 78frate-limiting reactions and, 73at subcellular level, 126, 127fat tissue and organ levels, 124–126, 125f,126f, 235tof xenobiotics, 626–632Metachromatic leukodystrophy, 203tMetal-activated enzymes, 50Metal ions, in enzymatic reactions, 50Metalloenzymes, 50Metalloflavoproteins, 86–87Metalloproteins, 30Metallothioneins, 588Metaphase chromosomes, 317f, 318, 319tMetastasisglycoproteins and, 514, 526, 530t, 531membrane abnormalities and, 432tMethacrylyl-CoA, catabolism of, 262fMethemoglobin, 46, 363, 613–614Methemoglobinemia, 46, 614Methionine, 15t, 264, 266factive (S-adenosylmethionine), 258f,259, 264, 266f, 289, 290f, 290tcatabolism of, 258f, 259, 259frequirements for, 480Methionine synthase, 492, 494Methotrexate, 296–297, 494dihydrofolate/dihydrofolate reductaseaffected by, 296–297, 494Methylationin covalent modification, mass increasesand, 27tof deoxycytidine residues, geneexpression affected by, 383in glycoprotein analysis, 515tof xenobiotics, 626, 630β-Methylcrotonyl-CoA, catabolism of, 261f5-Methylcytosine, 287, 289fα-Methyldopa, 446Methylene tetrahydrofolate, 493, 493fin folate trap, 493f, 4947-Methylguanine, 289fMethylhistidine, 576in Wilson’s disease, 265Methylmalonic aciduria, 155Methylmalonyl-CoA, accumulation of invitamin B 12 deficiency, 492Methylmalonyl-CoA isomerase (mutase), inpropionate metabolism, 155,155f, 492Methylmalonyl-CoA mutase (isomerase),155, 155f, 492Methylmalonyl-CoA racemase, in propionatemetabolism, 155, 155fMethyl pentose, in glycoproteins, 109tMethyl-tetrahydrofolate, in folate trap,493f, 494Mevalonate, synthesis of, in cholesterolsynthesis, 219, 220f, 221f, 222f

674 / INDEXMg. See MagnesiumMicelles, 418, 418famphipathic lipids forming, 119, 120f,418, 418fin lipid absorption, 475Michaelis constant (K m ), 65allosteric effects on, 75–76binding constant approximated by, 66enzymatic catalysis rate and, 65–66, 66f,72, 73finhibitors affecting, 68, 69fMichaelis-Menten equation indetermination of, 65–66, 66fMichaelis-Menten equation, 65Bi-Bi reactions and, 70, 70fregulation of metabolite flow and, 72,73fMicrofilaments, 576–577α 2 -Microglobulin, 583tMicrosatellite instability, 322Microsatellite polymorphism, 322, 411,413Microsatellite repeat sequences, 322, 413Microsomal elongase system, 177, 177fMicrosomal fraction, cytochrome P450isoforms in, 627Microtubules, 577Migration, cell, fibronectin in, 540Milk (lactose) intolerance, 102, 474, 475Mineralocorticoid response element, 459tMineralocorticoids, 437receptor for, 471synthesis of, 438–440, 441fMinerals, 2, 496–497, 496tdigestion and absorption of, 477–478Minor groove, in DNA, 305f, 306Mismatch repair of DNA, 336, 336f, 336tcolon cancer and, 336Missense mutations, 361, 362–363, 362ffamilial hypertrophic cardiomyopathycaused by, 569–570, 570fMIT. See MonoiodotyrosineMitchell’s chemiosmotic theory. SeeChemiosmotic theoryMitochondriaALA synthesis in, 270, 273fcitric acid cycle in, 122, 122f, 123f, 124f,126, 127f, 130, 133–135, 134ffatty acid oxidation in, 180–181, 181fion transport in, 99protein synthesis and import by,499–501, 501trespiration rate of, ADP in control of,94–95, 97t, 98frespiratory chain in, 92. See alsoRespiratory chainMitochondrial cytochrome P450, 89–90,627. See also Cytochrome P450systemMitochondrial DNA, 322–323, 322f,323tMitochondrial encephalomyopathies, withlactic acidosis and stroke-likeepisodes (MELAS), 100Mitochondrial genome, 499Mitochondrial glycerol-3-phosphatedehydrogenase, 87Mitochondrial membrane proteins, mutationsof, 431Mitochondrial membranes, 92, 93fenzymes as markers and, 92exchange transporters and, 98–100, 98f,99fprotein insertion in, 501Mitochondrial myopathies, fatal infantile,and renal dysfunction, oxidoreductasedeficiency causing, 100Mitogen-activated protein (MAP) kinasein insulin signal transmission, 466f,467in Jak/STAT pathway, 467Mitotic spindle, microtubules in formationof, 577Mixed-function oxidases, 89–90, 627. Seealso Cytochrome P450 systemML. See MucolipidosesMOAT. See Multispecific organic aniontransporterModeling, molecular, in protein structureanalysis, 36Molecular biology, 1. See also RecombinantDNA/recombinant DNAtechnologyin primary structure determination,25–26Molecular chaperones. See ChaperonesMolecular genetics, 1, 396. See alsoRecombinant DNA/recombinantDNA technologyMolecular modeling, in protein structureanalysis, 36Molecular motors, 577Molybdenum, 496tMonoacylglycerol acyltransferase, 198f, 199Monoacylglycerol pathway, 198f, 199,475–477, 476f2-Monoacylglycerols, 198f, 199Monoclonal antibodies, hybridomas inproduction of, 595–596, 596fMonoglycosylated core structure, calnexinbinding and, 526Monoiodotyrosine (MIT), 447, 448f, 449Monomeric proteins, 34Mononucleotides, 287“salvage” reactions and, 294, 295f, 297fMonooxygenases, 89–90. See alsoCytochrome P450 systemin metabolism of xenobiotics, 626Monosaccharides, 102. See also specific typeand Glucoseabsorption of, 475, 475fphysiologic importance of, 104–105, 105tMonounsaturated fatty acids, 112, 113t. Seealso Fatty acids; Unsaturated fattyacidsdietary, cholesterol levels affected by, 227synthesis of, 191, 191fMorquio syndrome, 546tMPP. See Matrix-processing peptidaseMPS. See MucopolysaccharidosesMRE. See Mineralocorticoid responseelementmRNA. See Messenger RNAMRP2. See Multidrug resistance-likeprotein 2MSH. See Melanocyte-stimulating hormoneMstII, 399tin sickle cell disease, 409, 410fmtDNA. See Mitochondrial DNAmTOR, in insulin signal transmission,466f, 467Mucins, 519–520, 520tgenes for, 520O-glycosidic linkages in, 518, 519–520,519frepeating amino acid sequences in, 519,520fMucolipidoses, 546–547, 546tMucopolysaccharides, 109, 109fMucopolysaccharidoses, 545–547, 546t,547fMucoproteins. See GlycoproteinsMucus, 519–520Multidrug resistance-like protein 2, inbilirubin secretion, 280Multipass membrane protein, anionexchange protein as, 615, 615f,616tMultiple myeloma, 595Multiple sclerosis, 202Multiple sulfatase deficiency, 203Multisite phosphorylation, in glycogenmetabolism, 151Multispecific organic anion transporter, inbilirubin secretion, 280Muscle, 556–576, 557f. See also Cardiacmuscle; Skeletal muscleATP in, 556, 561–562, 573–574, 575fcontraction of. See Muscle contractionin energy transduction, 556–559, 557f,558f, 559ffibers in, 556glycogen in, 145, 146tmetabolism in, 125, 125f, 235t, 576tglycogen, 145lactate production and, 139as protein reserve, 576proteins of, 566t. See also Actin; Myosin;TitinMuscle contraction, 556, 558f, 561–565,564tATP hydrolysis in, 561–562, 561fin cardiac muscle, 566–568

INDEX / 675regulation ofactin-based, 562–563calcium in, 562in cardiac muscle, 566–568sarcoplasmic reticulum and,563–564, 563f, 564fin smooth muscle, 570–571, 571fmyosin-based, 570myosin light chain kinase in,570–571, 571frelaxation phase of, 561, 564, 564tin smooth musclecalcium in, 571nitric oxide in, 571–573, 573fsliding filament cross-bridge model of,557–559, 558fin smooth muscle, 570–573tropomyosin and troponin in, 562Muscle fatigue, 136Muscle phosphorylase, 147absence of, 152tactivation ofcalcium/muscle contraction and,148cAMP and, 147–148, 149fMuscular dystrophy, Duchenne, 556,565–566, 566fMutagenesis, site-directed, in enzyme study,58Mutations, 314, 323–326, 323f, 324f,325fbase substitution, 361, 361f, 362constitutive, 376frameshift, 363, 364fABO blood group and, 619gene conversion and, 325integration and, 324, 324fof membrane proteins, diseases causedby, 431–432, 432f, 432tmissense, 361, 362–363, 362ffamilial hypertrophic cardiomyopathycaused by, 569–570, 570fmRNA nucleotide sequence changes causing,361–363, 361f, 362f, 364fnonsense, 362point, 361recombinant DNA technology indetection of, 408–409, 408frecombination and, 323–324, 323f, 324fsilent, 361sister chromatid exchanges and, 325, 325fsuppressor, 363transition, 361, 361ftransposition and, 324–325transversion, 361, 361fMyasthenia gravis, 431Myelin sheets, 428Myeloma, 595Myeloma cells, hybridomas grown from,596, 596fMyeloperoxidase, 612, 621t, 623Myocardial infarction, lactatedehydrogenase isoenzymes indiagnosis of, 57, 57t, 58fMyofibrils, 556, 557f, 558fMyoglobin, 40–48α-helical regions of, 40, 41fβ subunits of hemoglobin and, 42oxygen dissociation curve for, 41–42, 42foxygen stored by, 40, 41–42, 42f, 573Myoglobinuria, 47Myokinase (adenylyl kinase), 84deficiencies of, 151–152in gluconeogenesis regulation, 157as source of ATP in muscle, 573, 575fMyopathies, 92mitochondrial, 100–101fatal infantile, and renal dysfunction,oxidoreductase deficiencycausing, 100Myophosphorylase deficiency, 152tMyosin, 557, 559, 560fin muscle contraction, 557–559, 558f,561–562, 561f, 562fregulation of smooth musclecontraction and, 570in striated versus smooth muscle, 572tstructure and function of, 560–561, 560fMyosin-binding protein C, 566tMyosin (thick) filaments, 557, 558fMyosin head, 560, 560fconformational changes in, in musclecontraction, 561Myosin heavy chains, 560familial hypertrophic cardiomyopathycaused by mutations in gene for,569–570, 570fMyosin light chain kinase, 570–571, 571fMyosin light chains, 560in smooth muscle contraction, 570Myotonia congenita, 569tMyristic acid, 112t, 510Myristylation, 510in covalent modification, mass increasesand, 27tN-acetyl neuraminic acid, 169, 171fin gangliosides, 201, 203fin glycoproteins, 169, 171f, 515, 516tin mucins, 519f, 520N-linked glycoproteins, 518, 519f,521–527classes of, 521, 522fsynthesis of, 521–527, 523f, 524f, 525f,526tdolichol-P-P-oligosaccharide in,521–524, 523fin endoplasmic reticulum and Golgiapparatus, 524–525, 526tglycan intermediates formed during,526regulation of, 526–527, 527ttunicamycin affecting, 527, 527tNa. See SodiumNa + -Ca 2+ exchanger, 463Na + -K + ATPase, 427–428, 428fin glucose transport, 428, 429fNAD + (nicotinamide adenine dinucleotide),87, 490, 490fabsorption spectrum of, 56, 56fin citric acid cycle, 133as coenzyme, 87, 89f, 290tNADHabsorption spectrum of, 56, 56fextramitochondrial, oxidation of, 99,100ffatty acid oxidation yielding, 181in pyruvate dehydrogenase regulation,141–142, 142fNADH dehydrogenase, 87, 93NADP + (nicotinamide adenine dinucleotidephosphate), 87, 490as coenzyme, 87, 89f, 290tin pentose phosphate pathway, 163,164f, 165fNAD(P) + -dependent dehydrogenases, inenzyme detection, 56NADPHin cytochrome P450 reactions, 90f, 627intramitochondrial, proton-translocatingtranshydrogenase and, 99for lipogenesis, 175f, 176, 176fpentose phosphate pathway and, 163,164f, 165f, 169NADPH-cytochrome P450 reductase, 627NADPH oxidase, 621t, 622–623chronic granulomatous disease associatedwith mutations in, 623, 623fNCoA-1/NCoA-2 coactivators, 472, 472tNCoR, 472t, 473NDPs. See Ribonucleoside diphosphatesNebulin, 566tNEFA (nonesterified fatty acids). See Freefatty acidsNegative nitrogen balance, 479Negative regulators, of gene expression,374, 375t, 378, 380Negative supercoils, DNA, 306NEM-sensitive factor (NSF), 509, 510fNeonatal adrenoleukodystrophy, 503,503tNeonatal (physiologic) jaundice, 282–283Neonatal tyrosinemia, 255Neonate, hypoglycemia in, 151Nerve cells. See NeuronsNerve impulses, 428Nervous systemglucose as metabolic necessity for, 232thiamin deficiency affecting, 489NESs. See Nuclear export signalsNet charge, of amino acid, 16–17, 17fNet diffusion, 423

676 / INDEXNeuAc. See N-Acetylneuraminic acidNeural tube defects, folic acid supplementsin prevention of, 494Neuraminic acid, 110, 116Neuraminidasesdeficiency of, 532–533, 533tin glycoprotein analysis, 517influenza virus, 533Neurofilaments, 577tNeurologic diseases, protein conformationalterations and, 37Neurons, membranes ofimpulses transmitted along, 428ion channels in, 424, 425fsynaptic vesicle fusion with, 511Neuropathy, sensory, in vitamin B 6 excess,491Neutral lipids, 111Neutropenia, 610Neutrophils, 620–624activation of, 621–622biochemical features of, 620tenzymes and proteins of, 621tin infection, 620in inflammation, 620, 621tintegrins and, 620–621, 622tselectins and, 528–529, 529t, 530fproteinases of, 623–624, 624trespiratory burst and, 622–623NF-κB pathway, 468, 468f, 469fNiacin, 482t, 490, 490f. See alsoNicotinamide; Nicotinic acidin citric acid cycle, 133deficiency of, 482t, 490excess/toxicity of, 490Nick translation, 413Nickel, 496tNicks/nick-sealing, in DNA replication,332, 332fNicotinamide, 482t, 490, 490f. See alsoNiacincoenzymes derived from, 50–51dehydrogenases and, 87, 89fexcess/toxicity of, 490Nicotinamide adenine dinucleotide(NAD + ), 87, 490, 490fabsorption spectrum of, 56, 56fin citric acid cycle, 133as coenzyme, 87, 89f, 290tNicotinamide adenine dinucleotidephosphate (NADP + ), 87, 490as coenzyme, 87, 89f, 290tin pentose phosphate pathway, 163,164f, 165fNicotinic acid, 482t, 490, 490f. See alsoNiacinas hypolipidemic drug, 229NIDDM. See Non-insulin dependentdiabetes mellitusNidogen (entactin), in basal lamina, 540Niemann-Pick disease, 203tNight blindness, vitamin A deficiencycausing, 482t, 483Nitric oxide, 556, 571–573, 573f, 574t,607tclotting/thrombosis affected by, 607,607tNitric oxide synthases, 572–573, 573f, 574tNitrite, nitric oxide formation from, 572Nitrogen, amino acid (α-amino)catabolism of, 242–248end products of, 242–243urea as, 242–243, 245–247, 246fL-glutamate dehydrogenase in,244–245, 244f, 245fNitrogen balance, 479Nitroglycerin, 572NLS. See Nuclear localization signalNMR. See Nuclear magnetic resonance(NMR) spectroscopyNO. See Nitric oxideNO synthase. See Nitric oxide synthaseNoncoding regions, in recombinant DNAtechnology, 397, 398fNoncoding strand, 304Noncompetitive inhibition, competitiveinhibition differentiated from,67–69, 67f, 68f, 69fNoncovalent assemblies, in membranes, 416Noncovalent forcesin biomolecule stabilization, 6peptide conformation and, 20Nonequilibrium reactions, 128–129citric acid cycle regulation and, 135glycolysis regulation and, 140, 153–155Nonesterified fatty acids. See Free fatty acidsNonfunctional plasma enzymes, 57. See alsoEnzymesin diagnosis and prognosis, 57, 57tNonheme iron, 92, 95f, 585Nonhistone proteins, 314Non-insulin dependent diabetes mellitus(NIDDM/type 2), 161Nonoxidative phase, of pentose phosphatepathway, 163–166Nonrepetitive (unique-sequence) DNA,320, 320–321Nonsense codons, 359, 361, 363Nonsense mutations, 361Nonsteroidal anti-inflammatory drugscyclooxygenase affected by, 193prostaglandins affected by, 190Norepinephrine, 439f, 447, 447f. See alsoCatecholaminessynthesis of, 267, 267f, 445–447, 447fin thermogenesis, 217, 217fNorthern blot transfer procedure, 305–306,403, 404f, 413in gene isolation, 635tNPCs. See Nuclear pore complexesNSF. See NEM-sensitive factorNuclear export signals, 503Nuclear genes, proteins encoded by, 499Nuclear localization signal (NLS), 501,502f, 508tNuclear magnetic resonance (NMR)spectroscopyfor glycoprotein analysis, 514, 515tprotein structure demonstrated by,35–36Nuclear pore complexes, 501Nuclear proteins, O-glycosidic linkages in,518Nuclear receptor coactivators(NCoA-1/NCoA-2), 472, 472tNuclear receptor corepressor (NCoR), 472t,473Nuclear receptor superfamily, 436, 469,469–471, 471f, 472tNucleases, 8, 312active chromatin and, 316Nucleic acids. See also DNA; RNAbases of, 287–289, 288tdietarily nonessential, 293digestion of, 312structure and function of, 303–313Nucleolytic processing, of RNA, 352Nucleophile, water as, 7–8Nucleophilic attack, in DNA synthesis,328, 329fNucleoplasmin, 315Nucleoproteins, packing of, 318, 319t, 320fNucleosidases (nucleoside phosphorylases),purine, deficiency of, 300Nucleoside diphosphate kinase, 85Nucleoside triphosphatesgroup transfer potential of, 289–290,289f, 290f, 290tnonhydrolyzable analogs of, 291, 292fin phosphorylation, 85Nucleosides, 286–287, 288tNucleosomes, 314, 315–316, 315fNucleotide excision-repair of DNA, 336,337, 338fNucleotide sugars, in glycoprotein biosynthesis,516–517, 516t, 520, 521tNucleotides, 286–292, 288t. See alsoPurine; Pyrimidinesadenylyl kinase (myokinase) ininterconversion of, 84as coenzymes, 290, 290tDNA, deletion/insertion of, frameshiftmutations and, 363, 364fmetabolism of, 293–302in mRNA, 358mutations caused by changes in,361–363, 361f, 362f, 364fphysiologic functions of, 289as polyfunctional acids, 290polynucleotides, 291–292synthetic analogs of, in chemotherapy,290–291, 291fultraviolet light absorbed by, 290

INDEX / 677Nucleus (cell), importins and exportins intransport and, 501–503, 502fNutrition, 474–480. See also Dietbiochemical research affecting, 2lipogenesis regulated by, 177–178Nutritional deficiencies, 474in AIDS and cancer, 479Nutritionally essential amino acids, 124,237t, 480. See also Amino acidsNutritionally essential fatty acids, 190. Seealso Fatty acidsabnormal metabolism of, 195–196deficiency of, 191–192, 194–195Nutritionally nonessential amino acids,124, 237, 237t, 480synthesis of, 237–241O R . See Right operatorO blood group substance, 618–619, 619fO gene, 618–619O-glycosidic linkageof collagen, 537of proteoglycans, 542–543O-linked glycoproteins, 518, 518–520,519f, 520f, 520tsynthesis of, 520, 521tO-linked oligosaccharides, in mucins,519–520, 519f, 520fObesity, 80, 205, 231, 474, 478lipogenesis and, 173Octamers, histone, 315, 315fOculocerebrorenal syndrome, 512t1,25(OH) 2 -D 3 . See Calcitriol3β-OHSD. See 3β-HydroxysteroiddehydrogenaseOkazaki fragments, 327, 330, 331fOleic acid, 112, 112f, 113, 113t, 114f, 190fsynthesis of, 191, 191fOligomers, import of by peroxisomes, 503Oligomycin, respiratory chain affected by,95, 96f, 97fOligonucleotidedefinition of, 413in primary structure determination, 26Oligosaccharide:protein transferase, 523Oligosaccharide branches (antennae), 521Oligosaccharide chainsglycoprotein, 514, 515t, 581–582in N-glycosylation, 524, 525fregulation of, 526sugars in, 515, 516tglycosaminoglycans, 543Oligosaccharide processing, 521, 524, 525fGolgi apparatus in, 509regulation of, 526, 527fOligosaccharides, 102O-linked, in mucins, 519–520, 519f,520fOMP (orotidine monophosphate), 296,298fOncogenes, 1cyclins and, 334Oncoproteins, Rb protein and, 334Oncotic (osmotic) pressure, 580, 584Oncoviruses, cyclins and, 334Open complex, 345Operator locus, 377–378, 377f, 378Operon/operon hypothesis, 375, 376–378,376f, 377fOptical activity/isomer, 104ORC. See Origin replication complexORE. See Origin replication elementOri (origin of replication), 326, 327f, 413Origin replication complex, 326Origin replication element, 326Origin of replication (ori), 326, 327f,413Ornithine, 265, 266fcatabolism of, 250, 251fin urea synthesis, 245, 246–247, 246f,247Ornithine δ-aminotransferase, mutationsin, 250Ornithine-citrulline antiporter, defective,250Ornithine transcarbamoylase/L-Ornithinetranscarbamoylasedeficiency of, 247, 300in urea synthesis, 246–247, 246fOrosomucoid (α 1 -acid glycoprotein),583tOrotate phosphoribosyltransferase, 296,297, 298fOrotic aciduria, 300, 301Orotidine monophosphate (OMP), 296,298fOrotidinuria, 301Orphan receptors, 436, 471Osmotic fragility test, 617Osmotic lysis, complement in, 596Osmotic (oncotic) pressure, 580, 584Osteoarthritis, 535, 551tproteoglycans in, 548Osteoblasts, 549, 549f, 550Osteocalcin, 488, 496, 548tOsteoclasts, 549–550, 549f, 550fOsteocytes, 549, 549fOsteogenesis imperfecta (brittle bones),551–552, 551tOsteoid, 549f, 550Osteomalacia, 482t, 484, 485, 551tOsteonectin, 548tOsteopetrosis (marble bone disease), 552Osteoporosis, 485, 551t, 552Osteopontin, 548tOuabain, 106Na + -K + ATPase affected by, 428Outer mitochondrial membrane, 92, 93fprotein insertion in, 501Ovary, hormones produced by, 437,442–445, 444f, 445fOvernutrition, 478–479Oxaloacetatein amino acid carbon skeletoncatabolism, 249, 250fin aspartate synthesis, 237–238, 238fin citric acid cycle, 126, 127f, 130, 131f,133, 134f, 135Oxalosis, 170Oxidases, 86, 86–87, 87f. See also specifictypeceruloplasmin as, 587copper in, 86flavoproteins as, 86–87, 88fmixed-function, 89–90, 627. See alsoCytochrome P450 systemOxidation, 86–91definition of, 86dehydrogenases in, 87–88, 88f, 89ffatty acid, 180–189. See also Ketogenesisacetyl-CoA release and, 123–124,123f, 181–183, 181f, 182fβ, 181–183, 181f, 182fketogenesis regulation and,186–187, 187f, 188fmodified, 183, 183fclinical aspects of, 187–189hypoglycemia caused by impairmentof, 187–188in mitochondria, 180–181, 181fhydroperoxidases in, 88–89oxidases in, 86–87, 87f, 88foxygen toxicity and, 90–91, 611–613,613toxygenases in, 89–90, 90fredox potential and, 86, 87tOxidation-reduction (redox) potential, 86,87tOxidative decarboxylation, ofα-ketoglutarate, 131, 132fOxidative phase, of pentose phosphatepathway, 163, 164f, 165fOxidative phosphorylation, 83, 92–101,122. See also Phosphorylation;Respiratory chainchemiosmotic theory of, 92, 95–97, 97fclinical aspects of, 100–101muscle generation of ATP by, 573,574–576, 575f, 575tpoisons affecting, 92, 95, 96fOxidative stress, 612Oxidoreductases, 49, 86. See also specific typedeficiency of, 100Oxidosqualene:lanosterol cyclase, 220, 222fOxygenbinding, 42, 42f. See also OxygenationBohr effect and, 44, 45fhistidines F8 and E7 in, 40, 41fhemoglobin affinities (P 50 ) for, 42–43,43fmyoglobin in storage of, 40, 41–42, 42f,573

678 / INDEXOxygen (cont.)reductive activation of, 627transport of, ferrous iron in, 40–41Oxygen dissociation curve, for myoglobinand hemoglobin, 41–42, 42fOxygen radicals. See Free radicalsOxygen toxicity, superoxide free radicaland, 90–91, 611–613, 613t. Seealso Free radicalsOxygenases, 86, 89–90Oxygenation of hemoglobinconformational changes and, 42, 43f, 44fapoprotein, 422,3-bisphosphoglycerate stabilizing,45, 45fhigh altitude adaptation and, 46mutant hemoglobins and, 46Oxysterols, 119P i , in muscle contraction, 561, 561fP 50 , hemoglobin affinity for oxygen and,42–43, 43fp53 protein/p53 gene, 339p160 coactivators, 472, 472tp300 coactivator/CPB/p300, 461, 468,469, 469f, 472, 472tP450 cytochrome. See Cytochrome P450systemP450scc (cytochrome P450 side chaincleavage enzyme), 438, 440f, 442p/CIP coactivator, 472, 472tP component, in amyloidosis, 590P-selectin, 529tPAC (P1-based) vector, 401–402, 402t, 413PAF. See Platelet-activating factorPAGE. See Polyacrylamide gelelectrophoresisPain, prostaglandins in, 190Palindrome, 413Palmitate, 173, 173–174Palmitic acid, 112tPalmitoleic acid, 113t, 190fPalmitoylation, in covalent modification,mass increases and, 27tPancreatic insufficiency, in vitamin B 12deficiency, 492Pancreatic islets, insulin produced by, 160Pancreatic lipase, 475, 476fPanproteinase inhibitor, α 2 -macroglobulinas, 590Pantothenic acid, 173, 482t, 495, 495iin citric acid cycle, 133coenzymes derived from, 51deficiency of, 482tPapain, immunoglobulin digestion by, 591PAPS. See Adenosine 3′-phosphate-5′-phosphosulfateParallel β sheet, 32, 33fParathyroid hormone (PTH), 438, 450,451fstorage/secretion of, 453, 454tsynthesis of, 450, 451fParoxysmal nocturnal hemoglobinuria,432t, 528, 530t, 531, 531fPartition chromatography, for protein/peptide purification, 21Passive diffusion/transport, 423, 423t, 424fPasteur effect, 157pBR322, 402, 402t, 403fPCR. See Polymerase chain reactionPDH. See Pyruvate dehydrogenasePDI. See Protein disulfide isomerasePECAM-1, 529, 529tPedigree analysis, 409, 410fPellagra, 482t, 490Penicillamine, for Wilson disease, 589Pentasaccharide, in N-linked glycoproteins,521, 522fPentose phosphate pathway, 123, 163–166,164f, 165f, 167fcytosol as location for reactions of, 163enzymes of, 156terythrocyte hemolysis and, 169–170,613impairment of, 169–170NADPH produced by, 163, 164f,165ffor lipogenesis, 175f, 176, 176fnonoxidative phase of, 163–166oxidative phase of, 163, 164f, 165fribose produced by, 163, 164fPentoses, 102, 102tin glycoproteins, 109tphysiologic importance of, 104–105,105tPentosuria, essential, 163, 170PEPCK. See PhosphoenolpyruvatecarboxykinasePepsin, 477in acid-base catalysis, 52Pepsinogen, 477Peptidases, in protein degradation, 242,243fPeptide bonds. See also Peptidesformation of, 7, 368partial double-bond character of, 19–20,20fPeptides, 14–20, 439f. See also Aminoacids; Proteinsabsorption of, 477amino acids in, 14, 19, 19fformation of, L-α-amino acids in, 14as hormone precursors, 449–453intracellular messengers used by,457–468, 461t, 463tas polyelectrolytes, 19purification of, 21–24Peptidyl prolyl isomerase, 508Peptidylglycine hydroxylase, vitamin C ascoenzyme for, 496Peptidyltransferase, 368, 370tPeriodic acid-Schiff reagent, in glycoproteinanalysis, 515tPeriodic hyperlysinemia, 258Periodic paralysishyperkalemic, 569thypokalemic, 569tPeripheral proteins, 420–421, 421fPeripherin, 577tPermeability coefficients, of substances inlipid bilayer, 418, 419fPernicious anemia, 482t, 492Peroxidases, 88, 192Peroxidation, lipid, free radicals producedby, 118–119, 120fPeroxins, 503Peroxisomal-matrix targeting sequences(PTS), 503, 508tPeroxisomes, 89, 503absence/abnormalities of, 503, 503tin Zellweger’s syndrome, 188, 503biogenesis of, 503in fatty acid oxidation, 182–183Pfeiffer syndrome, 551tPFK-1. See PhosphofructokinasePGHS. See Prostaglandin H synthasePGIs. See ProstacyclinsPGs. See ProstaglandinspH, 9–13. See also Acid-base balanceamino acid net charge and, 16, 17fbuffering and, 11–12, 12f. See alsoBufferscalculation of, 9–10definition of, 9enzyme-catalyzed reaction rate affectedby, 64, 64fisoelectric, amino acid net charge and, 17Phage lambda, 378–383, 379f, 380f, 381f,382fPhagesfor cloning in gene isolation, 635tin recombinant DNA technology, 401Phagocytic cells, respiratory burst of,622–623Phagocytosis, 429Pharmacogenetics, 630, 631–632Pharmacogenomics, 632, 638Phasing, nucleosome, 315–316Phenobarbital, warfarin interaction and,cytochrome P450 inductionaffecting, 628Phenylalanine, 16tcatabolism of, 255–258, 255fin phenylketonuria, 255, 255frequirements for, 480in tyrosine synthesis, 239, 240fPhenylalanine hydroxylasedefect in, 255localization of gene for, 407tin tyrosine synthesis, 239, 240fPhenylethanolamine-N-methyltransferase(PNMT), 447, 447f

INDEX / 679Phenylisothiocyanate (Edman reagent), inprotein sequencing, 25, 26fPhenylketonuria, 255–258Phi (ϕ) angle, 31, 31fPhosphagens, 83, 84fPhosphatasesacid, diagnostic significance of, 57talkalinein bone mineralization, 550isozymes of, diagnostic significance of,57tin recombinant DNA technology,400tPhosphate transporter, 99, 99fPhosphates/phosphorus, 496texchange transporters and, 99, 99f, 100,101fin extracellular and intracellular fluid,416tfree energy of hydrolysis of, 82–83, 82thigh-energy, 83. See also ATPin energy capture and transfer, 82–83,82f, 82t, 83fas “energy currency” of cell, 83–85,84f, 85fsymbol designating, 83transport of, creatine phosphateshuttle in, 100, 101flow-energy, 83Phosphatidate, 198f, 199in triacylglycerol synthesis, 197, 197f,198, 198f, 199Phosphatidate phosphohydrolase, 198f, 199Phosphatidic acid, 114, 115f, 416–417,417fPhosphatidic acid pathway, 476f, 477Phosphatidylcholines (lecithins), 114–115,115fin cytochrome P450 system, 617membrane asymmetry and, 420synthesis of, 197, 197f, 198fPhosphatidylethanolamine (cephalin), 115,115fmembrane asymmetry and, 420synthesis of, 197, 197fPhosphatidylglycerol, 115, 115fPhosphatidylinositol/phosphatidylinositide,115, 115fin blood coagulation, 601GPI-linked glycoproteins and, 527.See also Glycosylphosphatidylinositol-anchored(GPIanchored/GPI-linked)glycoproteinsmetabolism of, 464–465, 464f, 465fas second messenger/second messengerprecursor, 115, 115f, 437, 437t,457, 463–465, 463t, 464f, 465fsynthesis of, 197, 197f, 198fPhosphatidylinositol 4,5-bisphosphate, 115,464–465, 465fin neutrophil activation, 621–622in platelet activation, 606–607, 606fPhosphatidylinositol 3-kinase (PI-3 kinase)in insulin signal transmission, 465, 466fin Jak/STAT pathway, 467Phosphatidylserine, 115, 115fin blood coagulation, 601membrane asymmetry and, 420Phosphocreatine, in muscle, 556Phosphodiester, 291Phosphodiesterases, 291in calcium-dependent signaltransduction, 463in cAMP-dependent signal transduction,461, 462fcAMP hydrolyzed by, 147Phosphoenolpyruvate, 156tfree energy of hydrolysis of, 82tin gluconeogenesis, 133, 134f, 156tPhosphoenolpyruvate carboxykinase(PEPCK), 133, 134fin gluconeogenesis regulation, 133, 134f,153, 154fPhosphofructokinase/phosphofructokinase-1, 156tin gluconeogenesis regulation, 157in glycolysis, 137, 138f, 156tregulation and, 140muscle, deficiency of, 143, 152tPhosphofructokinase-2, 157, 158fPhosphoglucomutase, in glycogenbiosynthesis, 145, 146f6-Phosphogluconate dehydrogenase, 156t,163, 164f, 165f3-Phosphoglyceratein glycolysis, 137, 138fin serine synthesis, 238, 238fPhosphoglycerate kinase, in glycolysis, 137,138fin erythrocytes, 140, 140fPhosphoglycerate mutase, in glycolysis, 137,138fPhosphoglycerides, in membranes,416–417, 417fPhosphoglycerolslysophospholipids in metabolism of, 116,116fsynthesis of, 197f, 198f, 199Phosphohexoseisomerase, in glycolysis, 137,138fPhosphoinositide-dependent kinase-1(PDK1), in insulin signaltransmission, 465Phospholipase A 1 , 200, 201fPhospholipase A 2 , 200, 201fin platelet activation, 606f, 607Phospholipase C, 200, 201fin calcium-dependent signal transduction,464–465, 464f, 465fin Jak/STAT pathway, 467in respiratory burst, 623Phospholipase Cβ, in platelet activation,606, 606fPhospholipase D, 200, 201fPhospholipasesin glycoprotein analysis, 515tin phosphoglycerol degradation andremodeling, 200–201, 201fPhospholipids, 111, 205digestion and absorption of, 475–477,476fglycerol ether, synthesis of, 199, 200fin lipoprotein lipase activity, 207–208in membranes, 114–116, 115f,416–417, 417f, 419, 511membrane asymmetry and, 420, 511in multiple sclerosis, 202as second messenger precursors, 197synthesis of, 198fPhosphoprotein phosphatases, in cAMPdependentsignal transduction,462, 462fPhosphoproteins, in cAMP-dependentsignal transduction, 461, 462fPhosphoric acid, pK/pK a value of, 12tPhosphorus. See PhosphatesPhosphorylasein glycogen metabolism, 145–146,146fregulation of, 148–150, 150–151,150f, 151fliver, 147deficiency of, 152tmuscle, 147absence of, 152tactivation ofcalcium/muscle contraction and,148cAMP and, 147–148, 149fPhosphorylase a, 147, 149fPhosphorylase b, 147, 149fPhosphorylase kinasecalcium/calmodulin-sensitive, inglycogenolysis, 148deficiency of, 152tprotein phosphatase-1 affecting, 147Phosphorylase kinase a, 148, 149fPhosphorylase kinase b, 148, 149fPhosphorylationin covalent modification, 76, 77–79, 78f,78tmass increases and, 27tmultisite, in glycogen metabolism, 151oxidative. See Oxidative phosphorylationin respiratory burst, 623Photolysis reaction, in vitamin D synthesis,445Photosensitivity, in porphyria, 274Phototherapy, cancer, porphyrins in, 273Phylloquinone, 482t, 486, 488f. See alsoVitamin KPhysical map, 633, 634f

680 / INDEXPhysiologic (neonatal) jaundice, 282–283Phytanic acid, Refsum’s disease caused byaccumulation of, 188Phytase, 477Phytic acid (inositol hexaphosphate), calciumabsorption affected by, 477Pi, 589. See also α 1 -AntiproteinasepI (isoelectric pH), amino acid net chargeand, 17PI-3 kinasein insulin signal transmission, 465, 466fin Jak/STAT pathway, 467PIC. See Preinitiation complexPIG-A gene, mutations of in paroxysmalnocturnal hemoglobinuria, 531,531f“Ping-Pong” mechanism, in facilitateddiffusion, 427, 427fPing-pong reactions, 69–70, 69fPinocytosis, 429–430PIP 2 , in absorptive pinocytosis, 430Pituitary hormones, 437. See also specifictypeblood glucose affected by, 161pK/pK aof amino acids, 15–16t, 17, 17f, 18environment affecting, 18, 18tmedium affecting, 13of weak acids, 10–11, 11–12, 12t, 13, 17PKA. See Protein kinase APKB. See Protein kinase BPKC. See Protein kinase CPKU. See PhenylketonuriaPlacenta, estriol synthesis by, 442Plaque hybridization, 403. See alsoHybridizationPlasma, 580Plasma cells, immunoglobulins synthesizedin, 591Plasma enzymes. See also Enzymesdiagnostic significance of, 57, 57tPlasma lipoproteins. See LipoproteinsPlasma membrane, 415, 426–431, 426f.See also Membranescarbohydrates in, 110mutations in, diseases caused by, 431,432tPlasma proteins, 514, 580–591, 581f, 583t.See also specific type andGlycoproteinsin bone, 548tconcentration of, 580electrophoresis for analysis of, 580, 582ffunctions of, 583, 583thalf life of, 582in inflammation, 621tpolymorphism of, 582synthesis ofin liver, 125, 581on polyribosomes, 581transport, 454–455, 454t, 455t, 583tPlasma thromboplastin antecedent(PTA/factor XI), 599f, 600, 600tdeficiency of, 601Plasma thromboplastin component(PTC/factor IX), 599f, 600, 600tcoumarin drugs affecting, 604deficiency of, 604Plasmalogens, 116, 117f, 199, 200fPlasmids, 400–401, 401f, 402, 402t, 403f,413for cloning in gene isolation, 635tPlasmin, 604–605, 604fPlasminogen, 604activators of, 604–605, 604f, 605, 605f,607tPlatelet-activating factor, 197, 621tsynthesis of, 198f, 199, 200fPlatelets, activation/aggregation of, 598,605–607, 606faspirin affecting, 607–608Pleckstrin, in platelet activation, 607PLP. See Pyridoxal phosphatePNMT. See Phenylethanolamine-N-methyltransferasepOH, in pH calculation, 9Point mutations, 361recombinant DNA technology indetection of, 408–409, 408f, 409tPoisons, oxidative phosphorylation/respiratory chain affectedby, 92, 95, 96fPol IIphosphorylation of, 350–351in preinitiation complex formation,351–352in transcription, 350–351Polarityof DNA replication/synthesis, 330–331of protein synthesis, 364of xenobiotics, metabolism and, 626Poly(A) tail, of mRNA, 309, 355–356in initiation of protein synthesis, 365Polyacrylamide gel electrophoresis, forprotein/peptide purification,24, 24f, 25fPolyadenylation sites, alternative, 394Polyamines, synthesis of, 265–266, 266fPolycistronic mRNA, 376Polycythemia, 46Polydystrophy, pseudo-Hurler, 532, 546tPolyelectrolytes, peptides as, 19Polyfunctional acids, nucleotides as, 290Polyisoprenoids, in cholesterol synthesis,220, 221fPolyisoprenol, in N-glycosylation, 521–522Polymerase chain reaction (PCR), 57,405–406, 406f, 413, 414in gene isolation, 635tin microsatellite repeat sequencedetection, 322in primary structure determination, 26PolymerasesDNA, 326, 327–328, 327f, 328, 328tin recombinant DNA technology, 400tRNA, DNA-dependent, in RNAsynthesis, 342–343, 342f, 343tPolymorphisms, 407acetyltransferase, 630cytochrome P450, 628, 630tmicrosatellite, 322, 411, 413plasma protein, 582restriction fragment length. SeeRestriction fragment lengthpolymorphismssingle nucleotide, 414Polynucleotide kinase, in recombinantDNA technology, 400tPolynucleotides, 291–292posttranslational modification of, 289Polyol (sorbitol) pathway, 172Polypeptidesreceptors for, 436sequencing ofcleavage in, 25, 26tSanger’s determination of, 24–25Polyphosphoinositide pathway, plateletactivation and, 605–607Polyprenoids, 118, 119fPolyribosomes (polysomes), 310, 370protein synthesis on, 498, 499f, 500f,506plasma proteins, 581signal hypothesis of binding of,503–505, 504t, 505fPolysaccharides, 102, 107–110, 108f, 109f.See also specific typePolysomes. See PolyribosomesPolytene chromosomes, 318, 318fPolyunsaturated fatty acids, 112, 113t. Seealso Fatty acids; Unsaturated fattyacidsdietary, cholesterol levels affected by, 227eicosanoids formed from, 190, 192,193f, 194fessential, 190, 190fsynthesis of, 191, 191f, 192fPOMC. See Pro-opiomelanocortin(POMC) peptide familyPompe’s disease, 152tPorcine stress syndrome, 565Porphobilinogen, 270, 273f, 275fPorphyrias, 274–278, 277f, 277tPorphyrinogens, 272accumulation of in porphyria, 274–278Porphyrins, 270–278, 271f, 272fabsorption spectra of, 273–274, 277fheme synthesis and, 270–273, 273f,274f, 275f, 276freduced, 272spectrophotometry in detection of,273–274Positive nitrogen balance, 479

INDEX / 681Positive regulators, of gene expression, 374,375t, 378, 380Posttranslational processing, 30, 37–39,38f, 371of collagen, 537–538, 537tin membrane assembly, 511–512Posttranslational translocation, 499Potassium, 496tin extracellular and intracellular fluid,416, 416tpermeability coefficient of, 419fPower stroke, 561PPI. See Peptidyl prolyl isomerasePP i . See Pyrophosphate, inorganicPR. See Progesterone, receptors forPravastatin, 229PRE. See Progestin response elementPre-β-lipoproteins, 205, 206t, 210Precursor proteins, amyloid, 590Pregnancyestriol synthesis in, 442fatty liver of, 188hypoglycemia during, 161iron needs during, 586Pregnancy toxemia of ewes (twin lambdisease)fatty liver and, 212ketosis in, 188Pregnenolone, 440fin adrenal steroidogenesis, 438–440,440f, 441fin testicular steroidogenesis, 442, 443fPreinitiation complex, 343, 351–352assembly of, 351–352in protein synthesis, 365, 366fPrekallikrein, 599f, 600Premenstrual syndrome, vitamin B 6 inmanagement of, sensoryneuropathy and, 491Prenatal diagnosis, recombinant DNAtechnology in, 409Preprocollagen, 537Preprohormone, insulin synthesized as, 449,450fPreproparathyroid hormone (preproPTH),450, 451fPreproprotein, albumin synthesized as, 583Preproteins, 498, 581Presequence. See Signal peptidePreventive medicine, biochemical researchaffecting, 2Primaquine-sensitive hemolytic anemia, 613Primary structure, 21–29, 31. See alsoProtein sequencingamino acid sequence determining, 18–19Edman reaction in determination of, 25,26fgenomics in analysis of, 28molecular biology in determination of,25–26of polynucleotides, 291–292proteomics and, 28–29Sanger’s technique in determination of,24–25Primary transcript, 342Primases, DNA, 327, 327f, 328tPrimosome, 328, 414Prion diseases (transmissible spongiformencephalopathies), 37Prion-related protein (PrP), 37Prions, 37Proaccelerin (factor V), 600t, 601, 602fProaminopeptidase, 477Probes, 402, 414. See also DNA probesfor gene isolation, 635tProbucol, 229Procarcinogens, 626Processivity, DNA polymerase, 328Prochymotrypsin, activation of, 77, 77fProcollagen, 371, 496, 537Procollagen aminoproteinase, 537Procollagen carboxyproteinase, 537Procollagen N-proteinase, disease caused bydeficiency of, 538tProconvertin (factor VII), 599f, 600t, 601coumarin drugs affecting, 604Prodrugs, 626Proelastase, 477Proenzymes, 76rapid response to physiologic demandand, 76Profiling, protein-transcript, 412Progesterone, 439f, 440fbinding of, 455, 455treceptors for, 471synthesis of, 438, 442, 445fProgesterone (∆ 4 ) pathway, 442, 443fProgestin response element, 459tProgestins, binding of, 455Prohormones, 371Proinsulin, 449, 450fProkaryotic gene expression. See also Geneexpressioneukaryotic gene expression comparedwith, 391–395, 392tas model for study, 375unique features of, 375–376Prolactin, 437localization of gene for, 407treceptor for, 436Proline, 16taccumulation of (hyperprolinemia),249–250catabolism of, 249–250, 251fsynthesis of, 238, 239fProline dehydrogenase, block of prolinecatabolism at, 249–250Proline hydroxylase, vitamin C as coenzymefor, 496Proline-cis,trans-isomerase, protein foldingand, 37, 37fProlyl hydroxylase reaction, 240, 240f, 535Promoter recognition specificity, 343Promoters, in transcription, 342, 342falternative use of in regulation, 354–355,355f, 393–394bacterial, 345–346, 345feukaryotic, 346–349, 347f, 348f, 349f,384Promotor site, in operon model, 377f, 378Proofreading, DNA polymerase, 328Pro-opiomelanocortin (POMC) peptidefamily, 452–453, 453f. See alsospecific typePro-oxidants, 612. See also Free radicalsProparathyroid hormone (proPTH), 450,450fPropionateblood glucose and, 159in gluconeogenesis, 154f, 155metabolism of, 155, 155fPropionic acid, 112tPropionyl-CoAfatty acid oxidation yielding, 182methionine in formation of, 259, 259fPropionyl-CoA carboxylase, 155, 155fProproteins, 37–38, 76, 371Propyl gallate, as antioxidant/foodpreservative, 119Prostacyclins, 112clinical significance of, 196clotting/thrombosis affected by, 607, 607tProstaglandin E 2 , 112, 113fProstaglandin H synthase, 192Prostaglandins, 112, 113f, 190, 192cyclooxygenase pathway in synthesis of,192, 192–194, 193f, 194fProstanoids, 112, 119clinical significance of, 196cyclooxygenase pathway in synthesis of,192, 192–194, 193f, 194fProsthetic groups, 50in catalysis, 50–51, 51fProtamine, 603Proteases/proteinases, 8, 477, 624t. See alsospecific typeα 2 -macroglobulin binding of, 590in cartilage, 553as catalytically inactive proenzymes,76–77mucin resistance to, 520of neutrophils, 623–624, 624tin protein degradation, 242, 243f, 477Staphylococcus aureus V8, for polypeptidecleavage, 25, 26tProtein 4.1, in red cell membranes, 615f,616f, 616t, 617Protein C, in blood coagulation, 600t, 603Protein disulfide isomerase, protein foldingand, 37, 508Protein-DNA interactions, bacteriophagelambda as paradigm for,378–383, 379f, 380f, 381f, 382f

682 / INDEXProtein folding, 36–37, 37fchaperones and, 499, 507–508, 508tafter denaturation, 36Protein kinase A (PKA), 460, 462fProtein kinase B (PKB), in insulin signaltransmission, 465, 466fProtein kinase C (PKC)in calcium-dependent signaltransduction, 464, 464fin platelet activation, 606f, 607Protein kinase D1, in insulin signaltransmission, 466f, 467Protein kinase-phosphatase cascade, assecond messenger, 437, 437tProtein kinases, 77in cAMP-dependent signal transduction,460–461, 462fin cGMP-dependent signal transduction,463deficiency of, 151–152DNA-dependent, in double-strand breakrepair, 338in glycogen metabolism, 147–148, 149f,151, 151fin hormonal regulation, 436, 465–468of lipolysis, 215, 216fin initiation of protein synthesis, 365in insulin signal transmission, 465–467,466fin Jak/STAT pathway, 467, 467fin NF-κB pathway, 468, 468fin protein phosphorylation, 77, 78fProtein-lipid respiratory chain complexes,93Protein-losing gastroenteropathy, 582Protein phosphatase-1, 147, 148, 149f, 151,151fProtein phosphatases, 77. See alsoPhosphatasesProtein profiling, 412Protein-RNA complexes, in initiation,365–367, 366fProtein S, in blood coagulation, 600t, 603Protein sequencingEdman reaction in, 25, 26fgenomics and, 28mass spectrometry in, 27, 27f, 27tmolecular biology in, 25–26peptide purification for, 21–24polypeptide cleavage and, 25, 26tproteomics and, 28–29purification for, 21–24, 22f, 23f, 24f,25fSanger’s method of, 24–25Protein sorting, 498–513chaperones and, 507–508, 508tcotranslational insertion and, 505–506,506fdisorders due to mutations in genesencoding, 512t, 513Golgi apparatus in, 498, 500f, 507, 509importins and exportins in, 501–503,502fKDEL amino acid sequence and,506–507, 508tmembrane assembly and, 511–513,512f, 512tmitochondria in, 499–501, 501fperoxisomes/peroxisome disorders and,503, 503tprotein destination and, 507, 507f, 508tretrograde transport and, 507signal hypothesis of polyribosome bindingand, 503–505, 504t, 505fsignal sequences and, 492f, 498–499,499ftransport vesicles and, 508–511, 509t,510fProtein turnover, 74, 242membranes affecting, 511rate of enzyme degradation and, 74Proteinases. See Proteases/proteinasesProteins. See also specific type and Peptidesβ-turns in, 32, 34facute phase, 583, 583tnegative, vitamin A as, 483–484L-α-amino acids in, 14asymmetry of, membrane assembly and,511, 512fbinding, 454–455, 454t, 455tcatabolism of, 242–248classification of, 30configuration of, 30conformation of, 30peptide bonds affecting, 20core, 542, 543fin glycosaminoglycan synthesis,542–543degradation of, to amino acids, 242, 243fdenaturation ofprotein refolding and, 36temperature and, 63dietarydigestion and absorption of, 477metabolism of, in fed state, 232requirements for, 479–480dimeric, 34domains of, 33–34encoding of by human genome, 636,637tin extracellular and intracellular fluid,416, 416tfibrous, 30collagen as, 38function of, bioinformatics inidentification of, 28–29fusion, in enzyme study, 58, 59fglobular, 30Golgi apparatus in glycosylation andsorting of, 509import of, by mitochondria, 499–501,501tloss of in trauma/infection, 480in membranes, 419, 420t, 514. See alsoGlycoproteins; Membraneproteinsratio of to lipids, 416, 416fmodular principals in construction of, 30monomeric, 34phosphorylation of, 76, 77–79, 78f, 78t.See also Phosphorylationposttranslational modification of, 30,37–39, 38f, 371purification of, 21–24receptors as, 431, 436soluble, 30structure of, 31–36diseases associated with disorders of,37folding and, 36–37, 37fhigher orders of, 30–39molecular modeling and, 36nuclear magnetic resonance spectroscopyin analysis of, 35–36primary, 21–29, 31. See also Primarystructureprion diseases associated withalteration of, 37quaternary, 33–35, 35fsecondary, 31, 31–33, 31f, 32f, 33f,34fsupersecondary motifs and, 33tertiary, 33–35, 35fx-ray crystallography in analysis of, 35synthesis of, 358–373. See also Proteinsortingamino acids in, 124, 124felongation in, 367–370, 368fenvironmental threats affecting, 370in fed state, 232genetic code/RNA and, 307–308,309t, 358–363. See also Geneticcodeinhibition of by antibiotics, 371–372,372finitiation of, 365–367, 366f, 367fby mitochondria, 499–501, 501tmodular principles in, 30polysomes in, 370, 498, 499fposttranslational processing and, 371in ribosomes, 126, 127frecognition and attachment (charging)in, 360, 360frecombinant DNA techniques for,407reticulocytes in, 611termination of, 369f, 370translocation and, 368viruses affecting, 370–371, 371ftransmembraneion channels as, 423–424, 425f, 426tin red cells, 615–616, 615f, 616f,616t

INDEX / 683transport, 454–455, 454t, 455txenobiotic cell injury and, 631Proteoglycans, 109, 535, 538, 542–549,542f. See alsoGlycosaminoglycansin bone, 548tcarbohydrates in, 542, 542f, 543fin cartilage, 551, 553disease associations and, 548–549functions of, 547–549, 548tgalactose in synthesis of, 167–169, 170flink trisaccharide in, 518Proteolysisin covalent modification, 76, 76–77, 77fin prochymotrypsin activation, 77, 77fProteome/proteomics, 28–29, 414,636–637, 637–638Prothrombin (factor II), 600t, 601, 602factivation of, 601coumarin drugs affecting, 487, 604in vitamin K deficiency, 487Prothrombinase complex, 601Proton acceptors, bases as, 9Proton donors, acids as, 9Proton pump, respiratory chain complexesas, 96, 96f, 97fProton-translocating transhydrogenase, assource of intramitochondrialNADPH, 99Protons, transport of, by hemoglobin, 44,45fProtoporphyrin, 270, 272fincorporation of iron into, 271–272, 272fProtoporphyrin III, 271, 276fProtoporphyrinogen III, 271, 276fProtoporphyrinogen oxidase, 271, 275f, 276fProvitamin A carotenoids, 482–483Proximal histidine (histidine F8)in oxygen binding, 40, 41freplacement of in hemoglobin M, 46Proximity, catalysis by, 51PrP (prion-related protein), 37PRPPin purine synthesis, 294, 295fin pyrimidine synthesis, 296, 298f, 299PRPP glutamyl amidotransferase, 294, 295fPRPP synthetase, defect in, gout caused by,299Pseudo-Hurler polydystrophy, 532, 546t,547Pseudogenes, 325, 414Psi (ψ) angle, 31, 31fPstI, 399tPstI site, insertion of DNA at, 402, 403fPTA. See Plasma thromboplastin antecedentPTC. See Plasma thromboplastincomponentPteroylglutamic acid. See Folic acidPTH. See Parathyroid hormonePTSs. See Peroxisomal-matrix targetingsequences“Puffs,” polytene chromosome, 318, 318fPulsed-field gel electrophoresis, for geneisolation, 635tPumps, 415in active transport, 427–428, 428fPurification, protein/peptide, 21–24Purine nucleoside phosphorylase deficiency,300Purines/purine nucleotides, 286–290, 286f,289fdietarily nonessential, 293metabolism of, 293–302disorders of, 300gout as, 299uric acid formation and, 299, 299fsynthesis of, 293–294, 294f, 295f, 296f,297fcatalysts in, 293, 294fpyrimidine synthesis coordinated with,299“salvage” reactions in, 294, 295f, 297fultraviolet light absorbed by, 290Puromycin, 372, 372fPutrescine, in polyamine synthesis, 266fPyranose ring structures, 103f, 104Pyridoxal phosphate, 50, 491, 491fin heme synthesis, 270in urea biosynthesis, 243Pyridoxine/pyridoxal/pyridoxamine(vitamin B 6 ), 482t, 491, 491fdeficiency of, 482t, 491xanthurenate excretion in, 258, 258fexcess/toxicity of, 491Pyrimethamine, 494Pyrimidine analogs, in pyrimidinenucleotide biosynthesis, 297Pyrimidines/pyrimidine nucleotides,286–290, 286f, 289fdietarily nonessential, 293metabolism of, 293–302, 301fdiseases caused by cataboliteoverproduction and, 300–301water-soluble metabolites and, 300,301fprecursors of, deficiency of, 300–301synthesis of, 296–299, 298fcatalysts in, 296purine synthesis coordinated with, 299regulation of, 297–299, 298fultraviolet light absorbed by, 290Pyrophosphatase, inorganicin fatty acid activation, 85, 180in glycogen biosynthesis, 145, 146fPyrophosphatefree energy of hydrolysis of, 82tinorganic, 85, 85fPyrrole, 40, 41fPyruvate, 123formation of, in amino acid carbonskeleton catabolism, 250–255,252f, 253foxidation of, 134, 135f, 140–142, 141f,142f, 143t. See also Acetyl-CoA;Glycolysisclinical aspects of, 142–143enzymes in, 156tgluconeogenesis and, 153, 154fPyruvate carboxylase, 133, 134f, 156tin gluconeogenesis regulation, 133, 134f,153, 156tPyruvate dehydrogenase, 134, 135f, 140,141f, 156tdeficiency of, 143regulation of, 141–142, 142facyl-CoA in, 141–142, 142f, 178thiamin diphosphate as coenzyme for,488Pyruvate dehydrogenase complex, 140Pyruvate kinase, 156tdeficiency of, 143, 619gluconeogenesis regulation and, 157in glycolysis, 137–139, 138f, 156tregulation and, 140Q (coenzyme Q/ubiquinone), 92, 95fQ 10 (temperature coefficient), enzymecatalyzedreactions and, 63QT interval, congenitally long, 432tQuaternary structure, 33–35, 35fof hemoglobins, allosteric properties and,42–46stabilizing factors and, 35R groups, amino acid properties affected by,18, 18tpK/pK a , 18R (relaxed) state, of hemoglobin,oxygenation and, 43, 43f, 44fRab protein family, 511RAC3 coactivator, 472, 472tRadiation, nucleotide excision-repair ofDNA damage caused by, 337Radiation hybrid mapping, 635tRan protein, 501, 502f, 503Rancidity, peroxidation causing, 118Rapamycin, mammalian target of (mTOR),in insulin signal transmission,466f, 467RAR. See Retinoic acid receptorRARE. See Retinoic acid response elementRate constant, 62K eq as ratio of, 62–63Rate of degradation (k deg ), control of, 74Rate-limiting reaction, metabolism egulatedby, 73Rate of synthesis (k s ), control of, 74Rb protein. See Retinoblastoma proteinReactant concentration, chemical reactionrate affected by, 62Reactive oxygen species. See Free radicals

684 / INDEXRearrangements, DNAin antibody diversity, 325–326, 393,593–594recombinant DNA technology indetection of, 409, 409trecA, 381, 382fReceptor-associated coactivator 3 (RAC3coactivator), 472, 472tReceptor-effector coupling, 435–436Receptor-mediated endocytosis, 429f,430Receptors, 431, 436. See also specific typeactivation of in signal generation,456–457, 458fnuclear, 436, 469, 469–471, 471f, 472tRecognition domains, on hormonereceptors, 435Recombinant DNA/recombinant DNAtechnology, 396–414, 635tbase pairing and, 396–397blotting techniques in, 403, 404fchimeric molecules in, 397–406cloning in, 400–402, 401f, 402t, 403fdefinition of, 414DNA ligase in, 399–400DNA sequencing in, 404, 405fdouble helix structure and, 396, 397in enzyme study, 58, 59fgene mapping and, 406–407, 407tin genetic disease diagnosis, 407–412,408f, 409t, 410f, 411fhybridization techniques in, 403–404libraries and, 402oligonucleotide synthesis in, 404–405organization of DNA into genes and,397, 398f, 399tpolymerase chain reaction in, 405–406,406fpractical applications of, 406–412restriction enzymes and, 397–400, 399t,400f, 400t, 401fterminology used in, 413–414transcription and, 397, 398fRecombinant erythropoietin (epoetinalfa/EPO), 526, 610Recombinant fusion proteins, in enzymestudy, 58, 59fRecombination, chromosomal, 323–324,323f, 324fRecruitment hypothesis, of preinitiationcomplex formation, 352Red blood cells, 609–610, 610–619. Seealso Erythrocytesrecombinant DNA technology in studyof, 624Red thrombus, 598Red (slow) twitch fibers, 574–576, 575tRedox (oxidation-reduction) potential, 86,87tof respiratory chain components, 92–93,94f, 95fRedox state, 184Reduced porphyrins, 272Reducing equivalentsin citric acid cycle, 130–133, 132fin pentose phosphate pathway, 166respiratory chain in collection and oxidationof, 92–93, 93f, 94f, 95f5α-Reductase, 442, 444fReduction, definition of, 86Reductive activation, of molecular oxygen,627Refsum’s disease, 188, 503, 503tRegional asymmetries, membrane, 420Regulated secretion, 498Regulatory proteins, binding of to DNA,motifs for, 387–390, 388t, 389f,390f, 391fRegurgitation hyperbilirubinemia, 282Relaxation phaseof skeletal muscle contraction, 561, 564of smooth muscle contractioncalcium in, 571nitric oxide in, 571–573, 573fRelaxed (R) state, of hemoglobin,oxygenation and, 43, 43f, 44fReleasing factors (RF1/RF3), in proteinsynthesis termination, 369f, 370Remnant removal disease, 228tRenal glomerulus, laminin in basal laminaof, 540–542Renal threshold for glucose, 161Renaturation, DNA, base pair matchingand, 305–306Renin, 451, 452fRenin-angiotensin system, 451–452, 452fRepeat sequences, 637amino acid, 519, 520fshort interspersed (SINEs), 321–322,414Repetitive-sequence DNA, 320, 321–322Replication/synthesis. See DNA,replication/synthesis of;RNA, synthesis ofReplication bubbles, 331–333, 331f, 332f,333fReplication fork, 327–328, 327fReporter genes, 385–386, 387f, 388fRepression, enzymeenzyme synthesis control and, 74in gluconeogenesis regulation, 155–157Repressor protein/gene, lambda (cI),379–383, 380f, 381f, 382fRepressors, 348in gene expression, 374, 377, 378, 385tissue-specific expression and, 385Reproduction, prostaglandins in, 190Respiration, 86Respiratory burst, 479, 622–623Respiratory chain, 92–101. See alsoOxidative phosphorylationclinical aspects of, 100–101collection and oxidation of reducingequivalents and, 92–93, 93f, 94f,95fdehydrogenases in, 87energy for metabolism provided by,93–95, 98foxidative phosphorylation at level of, 94poisons affecting, 92, 95, 96fas proton pump, 96, 96f, 97fredox potential of components of,92–93, 94f, 95fsubstrates for, citric acid cycle providing,131,131fRespiratory control, 81, 94–95, 97, 97t,98f, 134–135Respiratory distress syndrome, surfactantdeficiency causing, 115, 202Restriction endonucleases/enzymes, 312,397–399, 399t, 400f, 414in recombinant DNA technology,399–400, 399t, 400f, 400t, 401fRestriction enzymes. See RestrictionendonucleasesRestriction fragment length polymorphisms(RFLPs), 57, 409–411, 411fin forensic medicine, 411Restriction map, 399Retention hyperbilirubinemia, 282Reticulocytes, in protein synthesis, 611Retinagyrate atrophy of, 250retinaldehyde in, 483, 484fRetinal. See also RetinolRetinaldehyde, 482, 483fRetinitis pigmentosa, essential fatty aciddeficiency and, 192Retinoblastoma protein, 333Retinoic acid, 482, 483f. See also Retinolfunctions of, 483receptors for, 471, 483Retinoic acid receptor (RAR), 471, 483Retinoic acid response element, 459tRetinoid X receptor (RXR), 470, 470f, 471,483Retinoids, 482–484, 483f, 484f. See alsoRetinolRetinol, 482, 482t, 483f, 484f. See alsoVitamin Adeficiency of, 482tfunctions of, 482t, 483, 484fRetinol-binding protein, 583tRetrograde transport, 505, 510from Golgi apparatus, 507Retroposons/retrotransposons, 321, 637Retroviruses, reverse transcriptases in, 308,332–333Reverse cholesterol transport, 210, 211f,219, 224Reverse transcriptase/reverse transcription,308, 333, 414in recombinant DNA technology, 400t

INDEX / 685Reversed-phase high-pressurechromatography, for protein/peptide purification, 23–24Reversible covalent modifications, 77–79,78f, 78t. See also PhosphorylationReye’s syndrome, orotic aciduria in, 300RFLPs. See Restriction fragment lengthpolymorphismsRFs. See Releasing factorsRheumatoid arthritis, glycosylationalterations in, 533Rho-dependent termination signals, 344,346, 346fRhodopsin, 483, 484fRiboflavin (vitamin B 2 ), 86, 482t, 489–490in citric acid cycle, 133coenzymes derived from, 50–51, 489, 490deficiency of, 482t, 490dehydrogenases dependent on, 87Ribonucleases, 312Ribonucleic acid. See RNARibonucleoside diphosphates (NDPs),reduction of, 294, 297fRibonucleosides, 286, 287fRibonucleotide reductase complex, 294,297fRibose, 102in nucleosides, 286, 287fpentose phosphate pathway inproduction of, 123, 163, 166D-Ribose, 104f, 105t, 286Ribose phosphate, pentose phosphate pathwayin production of, 163,164fRibose 5-phosphate, in purine synthesis,293–294, 295fRibose 5-phosphate ketoisomerase, 163,165fRibosomal dissociation, in proteinsynthesis, 365, 366fRibosomal RNA (rRNA), 307–308,310–311, 341, 342t. See alsoRNAas peptidyltransferase, 368, 370tprocessing of, 355Ribosomes, 310, 312tbacterial, 371–372protein synthesis in, 126, 127fdissociation and, 370Ribozymes, 308, 311, 356D-Ribulose, 105t, 106fRibulose 5-phosphate 3-epimerase, 163,165fRichner-Hanart syndrome, 255Ricin, 372, 518tRickets, 482t, 484, 551tRight operator, 379–383, 380f, 382fRigor mortis, 562, 564RNA, 303, 306–312, 341–357as catalyst, 356in chromatin, 314classes/species of, 307–308, 309t, 341,342tcomplementarity of, 306, 309fheterogeneous nuclear (hnRNA), 310gene regulation and, 354messenger (mRNA), 307, 309–310,310f, 311f, 341, 342t, 359alternative splicing and, 354, 354f,393–394, 636codon assignments in, 358, 359tediting of, 356expression of, detection of in geneisolation, 635tmodification of, 355–356nucleotide sequence of, 358mutations caused by changes in,361–363, 361f, 362f, 364fpolycistronic, 376recombinant DNA technology and,397relationship of to chromosomal DNA,321fstability of, regulation of geneexpression and, 394–395, 394ftranscription starting point and,342variations in size/complexity of, 397,399tmodification of, 355–356processing of, 352–355alternative, in regulation of geneexpression, 354, 355f, 393–394in protein synthesis, 307–308, 309tribosomal (rRNA), 307–308, 310–311,341, 342tas peptidyltransferase, 368, 370tprocessing of, 355small nuclear (snRNA), 308, 309t, 311,341, 342t, 414small stable, 311splicing, 352–354, 414alternative, in regulation of geneexpression, 354, 354f,393–394, 636recombinant DNA technology and,397, 398fstructure of, 306–312, 308f, 309f, 311f,312fsynthesis of, 341–352initiation/elongation/termination in,342, 342f, 343–344, 344ftransfer (tRNA), 308, 310, 312f, 341,342t, 360–361, 361faminoacyl, in protein synthesis, 368anticodon region of, 359processing and modification of, 355,356suppressor, 363xenobiotic cell injury and, 631RNA editing, 356RNA polymerase III, 343tRNA polymerases, DNA-dependent, inRNA synthesis, 342–343, 342f,343tRNA primer, in DNA synthesis, 328, 329f,330fRNA probes, 402, 414RNAP. See RNA polymerasesRNase. See RibonucleasesROS (reactive oxygen species). See FreeradicalsRotor syndrome, 283Rough endoplasmic reticulumglycosylation in, 524–525, 525fin protein sorting, 498, 499f, 500fprotein synthesis and, 370routes of protein insertion into,505–507, 506fsignal hypothesis of polyribosomebinding to, 503–505, 504t, 505frRNA. See Ribosomal RNART-PCR, 414RXR. See Retinoid X receptorRyanodine, 563Ryanodine receptor, 563, 564fmutations in gene for, diseases caused by,564–565, 565f, 630tRYR. See Ryanodine receptorS 50 , 67S phase of cell cycle, DNA synthesis during,333–335, 334f, 335tSaccharopine, in lysine catabolism, 256f,258Salt (electrostatic) bonds (salt bridges/linkages), 7oxygen binding rupturing, Bohr effectprotons and, 44–45, 45f“Salvage” reactionsin purine synthesis, 294, 295f, 297fin pyrimidine synthesis, 296Sanfilippo syndrome, 546tSanger’s methodfor DNA sequencing, 404, 405ffor polypeptide sequencing, 24–25Sanger’s reagent (1-fluoro-2,4-dinitrobenzene),for polypeptidesequencing, 25Sarcolemma, 556Sarcomere, 556–557, 557fSarcoplasm, 556of cardiac muscle, 566Sarcoplasmic reticulum, calcium level inskeletal muscle and, 563–564,563f, 564fSaturated fatty acids, 111, 112, 112tin membranes, 417, 418fSaturation kinetics, 64f, 66sigmoid substrate, Hill equation inevaluation of, 66–67, 67fScavenger receptor B1, 210, 211f

686 / INDEXScheie syndrome, 546tSchindler disease, 532–533, 533tScrapie, 37Scurvy, 482t, 496collagen affected in, 38–39, 496,538–539SDS-PAGE. See Sodium dodecylsulfate-polyacrylamide gelelectrophoresisSe gene, 618Sec1 proteins, 511Sec61p complex, 504Second messengers, 76, 436–437, 437t,457–468, 461t, 463t. See alsospecific typecalcium as, 436–437, 437t, 457cAMP as, 147, 436, 437t, 457, 458–462,460t, 462fcGMP as, 290, 436, 437t, 457, 462–463diacylglycerol as, 464, 465finositol trisphosphate as, 464–465, 464f,465fprecursors ofphosphatidylinositol as, 115, 115fphospholipids as, 197Secondary structure, 31, 31–33, 32f, 33f,34fpeptide bonds affecting, 31, 31fsupersecondary motifs and, 33Secretor (Se) gene, 618Secretory component, of IgA, 595fSecretory granules, protein entry into, 507,507fSecretory (exocytotic) pathway, 498Secretory vesicles, 498, 500fD-Sedoheptulose, 106fSelectins, 528–530, 529f, 529t, 530fSelectivity/selective permeability,membrane, 415, 423–426,423t, 424f, 425f, 426tSelenium, 496tin glutathione peroxidase, 88, 166Selenocysteine, synthesis of, 240, 240fSelenophosphate synthetase/synthase, 240,240fSelf-assemblyin collagen synthesis, 537of lipid bilayer, 418Self-association, hydrophobic interactionsand, 6–7Sensory neuropathy, in vitamin B 6 excess,491Sepharose-lectin column chromatography,in glycoprotein analysis, 515tSequential displacement reactions, 69, 69fSerine, 15tcatabolism of, pyruvate formation and,250, 252fconserved residues and, 54, 55tin cysteine and homoserine synthesis,239, 239fin glycine synthesis, 238, 239fphosphorylated, 264synthesis of, 238, 238ftetrahydrofolate and, 492–494, 493fSerine 195, in covalent catalysis, 53–54, 54fSerine hydroxymethyltransferase, 250, 252f,493–494Serine protease inhibitor, 589. See alsoα 1 -AntiproteinaseSerine proteases. See also specific typeconserved residues and, 54, 55tin covalent catalysis, 53–54, 54fzymogens of, in blood coagulation, 600,600t, 601Serotonin, 266–267, 621tSerpin, 589. See also α 1 -AntiproteinaseSerum prothrombin conversion accelerator(SPCA/factor VII), 599f, 600t,601coumarin drugs affecting, 604Sex (gender), xenobiotic-metabolizingenzymes affected by, 630Sex hormone-binding globulin(testosterone-estrogen-bindingglobulin), 455, 455t, 583tSGLT 1 transporter protein, 475, 475fSGOT. See Aspartate aminotransferaseSGPT. See Alanine aminotransferaseSH2 domains. See Src homology 2 (SH2)domainsSHBG. See Sex hormone-binding globulinShort interspersed repeat sequences(SINEs), 321–322, 414Shoshin beriberi, 489Shotgun sequencing, 634SI nuclease, in recombinant DNAtechnology, 400tSialic acids, 110, 110f, 116, 169, 171fin gangliosides, 171f, 201, 203fin glycoproteins, 109t, 516tSialidosis, 532–533, 533t, 546, 546tSialoprotein, bone, 548t, 550Sialyl-Lewis X , selectins binding, 530, 530fSialylated oligosaccharides, selectinsbinding, 530Sickle cell disease, 363, 619pedigree analysis of, 409, 410frecombinant DNA technology indetection of, 408–409Side chain cleavage enzyme P450(P450scc), 438, 440f, 442Side chains, in porphyrins, 270, 271fSigmoid substrate saturation kinetics, Hillequation in evaluation of, 66–67,67fSignal. See also Signal peptidegeneration of, 456–457, 458f, 459f, 459tin recombinant DNA technology, 414transmission of. See also Signaltransductionacross membrane, 415, 431Signal hypothesis, of polyribosome binding,503–505, 504t, 505fSignal peptidase, 504, 505fSignal peptide, 498, 503–504, 508talbumin, 583in protein sorting, 498–499, 499f, 500f,503–504, 505, 505fSignal recognition particle, 504Signal sequence. See Signal peptideSignal transducers and activators oftranscription (STATs), 467, 467fSignal transduction, 456–473GPI-anchors in, 528hormone response to stimulus and, 456,457fintracellular messengers in, 457–468,461t, 463t. See also specific typein platelet activation, 606, 606fsignal generation and, 456–457, 458f,459f, 459ttranscription modulation and, 468–473,470f, 471f, 472tSilencers, 348recombinant DNA technology and, 397Silencing mediator for RXR and TR(SMRT), 472t, 473Silent mutations, 361Silicon, 496tSimple diffusion, 423, 423t, 424fSimvastatin, 229SINEs. See Short interspersed repeatsequencesSingle displacement reactions, 69, 69fSingle nucleotide polymorphism (SNP), 414Single-pass membrane proteins,glycophorins as, 615–616,615f, 616f, 616tSingle-stranded DNA, replication from,326. See also DNA,replication/synthesis ofSingle-stranded DNA-binding proteins(SSBs), 326, 327, 327f, 328tSister chromatid exchanges, 325, 325fSister chromatids, 318, 319fSite-directed mutagenesis, in enzyme study,58Site-specific DNA methylases, 398Site specific integration, 324Sitosterol, for hypercholesterolemia, 229Size exclusion chromatography, forprotein/peptide purification,21–22, 23fSK. See StreptokinaseSkeletal muscle, 556, 568t. See also Muscle;Muscle contractionglycogen stores in, 573metabolism in, 125, 125flactate production and, 139as protein reserve, 576slow (red) and fast (white) twitch fibersin, 574–576, 575t

INDEX / 687Skinessential fatty acid deficiency affecting,194–195mutant keratins and, 578vitamin D 3 synthesis in, 445, 446f, 484,485fSleep, prostaglandins in, 190Sliding filament cross-bridge model, ofmuscle contraction, 557–559,558fSlow acetylators, 630Slow-reacting substance of anaphylaxis, 196Slow (red) twitch fibers, 574–576, 575tSly syndrome, 546tSmall intestinecytochrome P450 isoforms in, 627monosaccharide digestion in, 475, 475fSmall nuclear RNA (snRNA), 308, 309t,311, 341, 342t, 414Small nucleoprotein complex (snurp), 353Small stable RNA, 311SmokingCYP2A6 metabolism of nicotine and,628cytochrome P450 induction and, 628nucleotide excision-repair of DNAdamage caused by, 337Smooth endoplasmic reticulum, cytochromeP450 isoforms in,627Smooth muscle, 556, 568tactin-myosin interactions in, 572tcontraction ofcalcium in, 570–571, 571fmyosin-based regulation of, 570myosin light chain phosphorylation in,570relaxation ofcalcium in, 571nitric oxide in, 571–573, 573fSMRT, 472t, 473SNAP (soluble NSF attachment factor)proteins, 509, 510fSNAP 25, 511SNARE proteins, 509, 510f, 511SNAREpins, 511SNP. See Single nucleotide polymorphismsnRNA. See Small nuclear RNASnurp (small nucleoprotein [snRNP]complex), 353Sodium, 496tin extracellular and intracellular fluid,416, 416tpermeability coefficient of, 419fSodium-calcium exchanger, 463Sodium dodecyl sulfate-polyacrylamide gelelectrophoresisfor protein/peptide purification, 24, 24f,25fred cell membrane proteins determinedby, 614–615, 615fSodium-potassium pump (Na + -K + ATPase),427–428, 428fin glucose transport, 428, 429fSolubility point, of amino acids, 18Soluble NSF attachment factor (SNAP)proteins, 509, 510f, 511Solutions, aqueous, K w of, 9Solvent, water as, 5, 6fSorbitol, in diabetic cataract, 172Sorbitol dehydrogenase, 167, 169fSorbitol intolerance, 172Sorbitol (polyol) pathway, 172Soret band, 273Southern blot transfer procedure, 305–306,403, 404f, 414Southwestern blot transfer procedure, 403,414SPARC (bone) protein, 548tSparteine, CYP2D6 in metabolism of,628SPCA. See Serum prothrombin conversionacceleratorSpecific acid/base catalysis, 51–52Specificity, enzyme, 49, 50fSpectrin, 615, 615f, 616f, 616t, 617abnormalities of, 617Spectrometrycovalent modifications detected by, 27,27f, 27tfor glycoprotein analysis, 514, 515tSpectrophotometryfor NAD(P) + -dependent dehydrogenases,56, 56ffor porphyrins, 273–274Spectroscopy, nuclear magnetic resonance(NMR)for glycoprotein analysis, 514, 515fprotein structure demonstrated by,35–36Spermidine, synthesis of, 265–266, 266fSpermine, synthesis of, 265–266, 266fSpherocytosis, hereditary, 432t, 617, 617fSphingolipidoses, 202–203, 203tSphingolipids, 197metabolism of, 201–202, 202f, 203fclinical aspects of, 202–203, 203tin multiple sclerosis, 202Sphingomyelins, 116, 116f, 201, 202fin membranes, 417membrane asymmetry and, 420Sphingophospholipids, 111Sphingosine, 116, 116fSpina bifida, folic acid supplements inprevention of, 494Spliceosome, 353, 414Spongiform encephalopathies, transmissible(prion diseases), 37Squalene, synthesis of, in cholesterolsynthesis, 219, 221f, 222fSqualene epoxidase, in cholesterol synthesis,220, 222fSR-B1. See Scavenger receptor B1SRC-1 coactivator, 472, 472tSrc homology 2 (SH2) domainsin insulin signal transmission, 465, 466f,467in Jak/STAT pathway, 467, 467fSRP. See Signal recognition particleSRS-A. See Slow-reacting substance ofanaphylaxisssDNA. See Single-stranded DNAStaphylococcus aureus V8 protease, forpolypeptide cleavage, 25, 26tSTAR. See Steroidogenic acute regulatoryproteinStarch, 107, 108fglycemic index of, 474hydrolysis of, 474Starling forces, 580Starvation, 80clinical aspects of, 236fatty liver and, 212ketosis in, 188metabolic fuel mobilization in, 232–234,234f, 234ttriacylglycerol redirection and, 208Statin drugs, 229STATs (signal transducers and activators oftranscription), 467, 467fStearic acid, 112tSteely hair disease (Menkes disease), 588Stem cells, differentiation of to red bloodcells, erythropoietin in regulationof, 610, 611fStereochemical (-sn-) numbering system,114, 115fStereoisomers. See also Isomerismof steroids, 117, 118fSteroid nucleus, 117, 117f, 118fSteroid receptor coactivator 1 (SRC-1coactivator), 472, 472tSteroid sulfates, 201Steroidogenesis. See Steroids, synthesis ofSteroidogenic acute regulatory protein(STAR), 442Steroids, 117–118, 117f, 118f, 119f. Seealso specific typeadrenal. See also Glucocorticoids;Mineralocorticoidssynthesis of, 438–442, 440f, 441fcalcitriol as, 484receptors for, 436stereoisomers of, 117, 118fstorage/secretion of, 453, 454tsynthesis of, 123f, 124, 438, 438–445,439t, 440f, 441ftransport of, 454–455, 455tvitamin D as, 484Sterol 27-hydroxylase, 226Sterols, 117in membranes, 417Stickler syndrome, 553

688 / INDEXSticky end ligation/sticky-ended DNA, 299,398, 400f, 401f, 414“Sticky foot,” 527“Sticky patch,” in hemoglobin S, 46, 46fStoichiometry, 60Stokes radius, in size exclusionchromatography, 21Stop codon, 369f, 370Stop-transfer signal, 506Strain, catalysis by, 52Streptokinase, 605, 605f, 606tStreptomycin, 106Striated muscle, 556, 557, 557f. See alsoCardiac muscle; Skeletal muscleactin-myosin interactions in, 572tStroke, with mitochondrial encephalopathyand lactic acidosis (MELAS),100–101Strong acids, 9Strong bases, 9Structural proteins, 535Stuart-Prower factor (factor X), 599f, 600,600tactivation of, 599–600, 599fcoumarin drugs affecting, 604Substrate analogs, competitive inhibitionby, 67–68, 67fSubstrate level, phosphorylation at, 94Substrate shuttlescoenzymes as, 50in extramitochondrial NADH oxidation,99, 100fSubstrate specificity, of cytochrome P450isoforms, 627Substrates, 49competitive inhibitors resembling,67–68, 67fconcentration of, enzyme-catalyzedreaction rate affected by, 64,64f, 65fHill model of, 66–67, 67fMichaelis-Menten model of, 65–66,66fconformational changes in enzymescaused by, 52, 53fmultiple, 69–70Succinate, 131–133, 132fSuccinate dehydrogenase, 87, 132f,133inhibition of, 67–68, 67fSuccinate semialdehyde, 267, 268fSuccinate thiokinase (succinyl-CoAsynthetase), 131, 132fSuccinic acid, pK/pK a value of, 12tSuccinyl-CoA, in heme synthesis, 270–273,273f, 274f, 275f, 276fSuccinyl-CoA-acetoacetate-CoA transferase(thiophorase), 133, 186, 186fSuccinyl-CoA synthetase (succinatethiokinase), 131, 132fSucrase-isomaltase complex, 475Sucrose, 106–107, 107f, 107tglycemic index of, 474Sugars. See also Carbohydratesamino (hexosamines), 106, 106fglucose as precursor of, 169, 171fin glycosaminoglycans, 109, 169, 171fin glycosphingolipids, 169, 171finterrelationships in metabolism of,171fclassification of, 102, 102tdeoxy, 106, 106f“invert,” 107isomerism of, 102–104, 103fnucleotide, in glycoprotein biosynthesis,516–517, 516t“Suicide enzyme,” cyclooxygenase as, 194Sulfateactive (adenosine 3′-phosphate-5′-phosphosulfate), 289, 289f, 629in glycoproteins, 515in mucins, 520Sulfatide, 116Sulfation, of xenobiotics, 629Sulfo(galacto)-glycerolipids, 201Sulfogalactosylceramide, 201accumulation of, 203Sulfonamides, hemolytic anemiaprecipitated by, 613Sulfonylurea drugs, 188Sulfotransferases, in glycosaminoglycansynthesis, 543Sunlight. See Ultraviolet lightSupercoils, DNA, 306, 332, 333fSuperoxide anion free radical, 90–91,611–613, 613t. See also Freeradicalsproduction of in respiratory burst, 622Superoxide dismutase, 90–91, 119,611–613, 613t, 622Supersecondary structures, 33Suppressor mutations, 363Suppressor tRNA, 363Surfactant, 115, 197deficiency of, 115, 202SV40 viruses, cancer caused bySwainsonine, 527, 527tSymport systems, 426, 426fSyn conformers, 287, 287fSynaptobrevin, 511Syntaxin, 511Synthesis, rate of (k s ), control of, 74t 1/2 . See Half lifeT 3 . See TriiodothyronineT 4 . See ThyroxineT m . See Melting temperature/transitiontemperatureT lymphocytes, 591t-PA. See Tissue plasminogen activatorTΨC arm, of tRNA, 310, 312f, 360, 361ft-SNARE proteins, 509, 511T (taut) state, of hemoglobin2,3-bisphosphoglycerate stabilizing, 45,45foxygenation and, 43, 43f, 44fT tubular system, in cardiac muscle, 566T-type calcium channel, 567TAFs. See TBP-associated factorsTalin, 540, 541fTandem, 414Tandem mass spectrometry, 27Tangier disease, 228tTaqI, 399tTarget cells, 434–435, 435treceptors for, 435, 436fTargeted gene disruption/knockout, 412Tarui’s disease, 152tTATA binding protein, 346, 349f, 350,351TATA box, in transcription control, 345,345f, 346, 347f, 348, 348f, 351tTaurochenodeoxycholic acid, synthesis of,226fTaut (T) state, of hemoglobin2,3-bisphosphoglycerate stabilizing, 45,45foxygenation and, 43, 43f, 44fTay-Sachs disease, 203tTBG. See Thyroxine-binding globulinTBP. See TATA binding proteinTBP-associated factors, 346, 350, 351TΨC arm, of tRNA, 310, 312f, 360, 361fTEBG. See Testosterone-estrogen-bindingglobulinTelomerase, 318Telomeres, 318, 319fTemperaturechemical reaction rate affected by, 62,62fenzyme-catalyzed reaction rate affectedby, 63in fluid mosaic model of membranestructure, 422Temperature coefficient (Q 10 ), enzymecatalyzedreactions and, 63Template binding, in transcription, 342,342fTemplate strand DNA, 304, 306, 307ftranscription of in RNA synthesis,341–343, 342fTenase complex, 600–601Terminal transferase, 400t, 414Terminationchainin glycosaminoglycan synthesis, 543in transcription cycle, 342, 342fof protein synthesis, 369f, 370of RNA synthesis, 342, 342f, 344, 344fTermination signals, 359for bacterial transcription, 346, 346ffor eukaryotic transcription, 349–350

INDEX / 689Tertiary structure, 33–35, 35fstabilizing factors and, 35Testes, hormones produced by, 437, 442,443f. See also specific typeTestosterone, 439f, 440fbinding of, 455, 455tmetabolism of, 442, 444fsynthesis of, 442, 443fTestosterone-estrogen-binding globulin (sexhormone-binding globulin), 455,455t, 583tTetracycline (tet) resistance genes, 402,403fTetrahedal transition state intermediate, inacid-base catalysis, 52, 53fTetrahydrofolate, 492, 493–494, 493fTetraiodothyronine (thyroxine/T 4 ), 438,447storage/secretion of, 453, 454tsynthesis of, 447–449, 448ftransport of, 454, 454tTetramershemoglobin as, 42histone, 314–315, 315Tetroses, 102, 102tTf. See TransferrinTFIIA, 350TFIIB, 350TFIID, 346, 350, 351in preinitiation complex formation, 352TFIIE, 350TFIIF, 350TFIIH, 350TFPI. See Tissue factor pathway inhibitorTfR. See Transferrin receptorThalassemias, α and β, 47, 610trecombinant DNA technology indetection of, 408f, 409, 409tThanatophoric dysplasia, 551tTheca cells, hormones produced by, 442Theobromine, 289Theophylline, 289hormonal regulation of lipolysis and, 215Thermodynamicsbiochemical (bioenergetics), 80–85. Seealso ATPglycolysis reversal and, 153–155laws of, 80–81hydrophobic interactions and, 7Thermogenesis, 217, 217fdiet-induced, 217, 478Thermogenin, 217, 217fThiamin (vitamin B 1 ), 482t, 488–489, 489fin citric acid cycle, 133coenzymes derived from, 51deficiency of, 482t, 489pyruvate metabolism affected by, 140,143, 489Thiamin diphosphate, 140, 166, 488–489,489fThiamin pyrophosphate, 50Thiamin triphosphate, 489Thick (myosin) filaments, 557, 558fThin (actin) filaments, 557, 558f, 559fThioesterase, 1736-Thioguanine, 290, 291fThiokinase (acyl-CoA synthetase)in fatty acid activation, 180, 181fin triacylglycerol synthesis, 199, 214f,215Thiol-dependent transglutaminase. SeeTransglutaminaseThiol ester plasma protein family, 590Thiolase, 181, 182f, 184in mevalonate synthesis, 219, 220fThiophorase (succinyl-CoAacetoacetate-CoAtransferase),133, 186, 186fThioredoxin, 294Thioredoxin reductase, 294, 297fThreonine, 15tcatabolism of, 253f, 255phosphorylated, 264requirements for, 480Thrombin, 601, 602, 603fantithrombin III affecting, 603–604circulating levels of, 602–603conserved residues and, 55tformation of fibrin and, 601–602, 603fin platelet activation, 606, 606fThrombolysislaboratory tests in evaluation of, 608t-PA and streptokinase in, 605, 605f,606tThrombomodulin, in blood coagulation,600t, 603, 607, 607tThrombosis, 598–608. See also Coagulationantithrombin III in prevention of,603–604circulating thrombin levels and, 602–603endothelial cell products in, 607, 607thyperhomocysteinemia and, folic acidsupplements in prevention of, 494phases of, 598in protein C or protein S deficiency, 603t-PA and streptokinase in managementof, 605, 605f, 606ttypes of thrombi and, 598Thromboxane A 2 , 113fin platelet activation, 606f, 607Thromboxanes, 112, 113f, 190, 192clinical significance of, 196cyclooxygenase pathway in formation of,192, 193fThymidine, 288tbase pairing of in DNA, 303, 304, 305fThymidine monophosphate (TMP), 288tThymidine-pseudouridine-cytidine (TΨC)arm, of tRNA, 310, 312f, 360,361fThymidylate, 303Thymine, 288tThyroglobulin, 447, 449Thyroid-binding globulin, 454, 583tThyroid hormone receptor-associatedproteins (TRAPs), 472t, 473Thyroid hormone response element, 459tstorage/secretion of, 453, 454tThyroid hormones, 437, 438in lipolysis, 215, 216freceptors for, 436, 471synthesis of, 447–449, 448ftransport of, 454, 454tThyroid-stimulating hormone (TSH), 437,438, 439f, 449Thyroperoxidase, 449Thyrotropin-releasing hormone (TRH),438, 439fThyroxine (T 4 ), 438, 447storage/secretion of, 453, 454tsynthesis of, 447–449, 448ftransport of, 454, 454tThyroxine-binding globulin, 454, 454tTIF2 coactivator, 472, 472tTiglyl-CoA, catabolism of, 261fTIM. See Translocase-of-the-innermembraneTimnodonic acid, 113tTin, 496tTissue differentiation, retinoic acid in, 483Tissue factor complex, 601Tissue factor (factor III), 599f, 600t, 601Tissue factor pathway inhibitor, 601Tissue plasminogen activator (alteplase/t-PA), 604–605, 605, 605f,606t, 607tTissue-specific gene expression, 385Titin, 566tTMP (thymidine monophosphate), 288f,288tTocopherol, 482t, 486, 486f. See alsoVitamin Eas antioxidant, 91, 119, 486, 487fdeficiency of, 482tTocotrienol, 486, 486f. See also Vitamin ETolbutamide, 188TOM. See Translocase-of-the-outermembraneTopogenic sequences, 506Topoisomerases, DNA, 306, 328t, 332,332fTotal iron-binding capacity, 586Toxemia of pregnancy of ewes, ketosis and,188Toxic hyperbilirubinemia, 283Toxopheroxyl free radical, 486TpC. See Troponin CTpI. See Troponin ITpT. See Troponin TTR activator molecule 1 (TRAM-1coactivator), 472, 472tTRAM (translocating chain-associatedmembrane) protein, 504

690 / INDEXTRAM-1 coactivator, 472, 472tTrans fatty acids, 113–114, 192Transaldolase, 166Transaminases. See AminotransferasesTransamination, 124, 124fin amino acid carbon skeleton catabolism,249–250, 249f, 250f, 251fcitric acid cycle in, 133–134, 134fin urea biosynthesis, 243–244, 243fTranscortin (corticosteroid-bindingglobulin), 454–455, 455tTranscript profiling, 412Transcription, 306, 350–352, 351t, 414activators and coactivators in control of,351, 351tbacterial promoters in, 345–346, 345fcontrol of fidelity and frequency of,344–350eukaryotic promoters in, 346–349, 347f,348f, 349fin gene expression regulation, 383–387,391, 392t. See also Geneexpressionhormonal regulation of, 457, 458f,468–473, 470f, 471f, 472tinitiation of, 342–343, 342fNF-κB in regulation of, 468, 469fnuclear receptor coregulators in,471–473, 472trecombinant DNA technology and, 397,398fretinoic acid in regulation of, 483reverse, 414in retroviruses, 308, 332–333in RNA synthesis, 306, 307f, 341–343,342fTranscription complex, eukaryotic, 306,350–352, 351tTranscription control elements, 351, 351tTranscription domains, definition of,387Transcription factors, 351, 351tnuclear receptor superfamily, 469–471,471f, 472tTranscription start sites, alternative,393–394Transcription unit, 342, 345fTranscriptional intermediary factor 2 (TIF2coactivator), 472, 472tTranscriptome information, 412, 414Transfection, identification ofenhancers/regulatory elementsand, 386Transfer RNA (tRNA), 308, 310, 312f,341, 342t, 360–361, 361f. Seealso RNAaminoacyl, in protein synthesis, 368anticodon region of, 359processing and modification of, 355, 356suppressor, 363Transferases, 50Transferrin, 478, 583t, 584–586, 585f,585tTransferrin receptor, 586Transfusion, ABO blood group and, 618Transgenic animals, 385, 411–412, 414enhancers/regulatory elements identifiedin, 386Transglutaminase, in blood coagulation,600, 600t, 602, 603fTranshydrogenase, proton-translocating, assource of intramitochondrialNADPH, 99Transient insertion signal. See SignalpeptideTransition mutations, 361, 361fTransition state intermediate, tetrahedal, inacid-base catalysis, 52, 53fTransition states, 61Transition temperature/meltingtemperature (T m ), 305, 422Transketolase, 163–166, 165f, 170erythrocyte, in thiamin nutritional statusassessment, 489thiamin diphosphate in reactionsinvolving, 166, 170, 488–489Translation, 358, 414Translocase-of-the-inner membrane, 499Translocase-of-the-outer membrane, 499Translocating chain-associated membrane(TRAM) protein, 504Translocation, protein, 499Translocation complexes, 499Translocon, 504Transmembrane proteins, 419ion channels as, 423–424, 425f, 426tin red cells, 615–616, 615f, 616f, 616tTransmembrane signaling, 415, 431in platelet activation, 606, 606fTransmissible spongiform encephalopathies(prion diseases), 37Transport proteins, 454–456, 454t, 455t,583tTransport systems/transporters. See alsospecific typeactive, 423, 423t, 424f, 426–427,427–428, 428fADP/ATP, 95, 98fATP-binding cassette, 210, 211fin cotranslational insertion, 506, 506fdisorders associated with mutations ingenes encoding, 512t, 513exchange, 98–100, 98f, 99ffacilitated diffusion, 423, 423t, 424f,426–427, 427, 427fglucose. See Glucose transportersin inner mitochondrial membrane,98–100, 98f, 99fmembrane, 426–431, 426ffor nucleotide sugars, 517Transport vesicles, 498, 508–511, 509t,510fTransposition, 324–325retroposons/retrotransposons and, 321,637Transthyretin, 583t, 590Transverse asymmetry, 511Transversion mutations, 361, 361fTRAPs, 472t, 473Trauma, protein loss and, 480TRE. See Thyroid hormone responseelementTrehalase, 475Trehalose, 107tTRH. See Thyrotropin-releasing hormoneTriacylglycerols (triglycerides), 114, 115f,205digestion and absorption of, 475–477,476fexcess of. See Hypertriacylglycerolemiainterconvertability of, 231in lipoprotein core, 205, 207fmetabolism of, 123, 123f, 125–126,126fin adipose tissue, 214–215, 214ffatty liver and, 212, 213fhepatic, 211–212, 213fhigh-density lipoproteins in,209–211, 211fhydrolysis in, 197reduction of serum levels of, drugs for,229synthesis of, 198f, 199transport of, 207, 208f, 209f, 210fTricarboxylate anions, transporter systemsfor, 98–99lipogenesis regulation and, 178Tricarboxylic acid cycle. See Citric acidcycleTriglycerides. See TriacylglycerolsTriiodothyronine (T 3 ), 438, 447storage/secretion of, 453, 454tsynthesis of, 447–449, 448ftransport of, 454, 454tTrimethoprim, 494Trinucleotide repeat expansions, 322Triokinase, 167, 169fTriose phosphates, acylation of, 123Trioses, 102, 102tTriphosphates, nucleoside, 287, 287fTriple helix structure, of collagen, 38, 38f,535–539, 536fTriplet code, genetic code as, 358, 359ttRNA. See Transfer RNATropocollagen, 38, 38fTropoelastin, 539Tropomyosin, 557, 559f, 562in red cell membranes, 616tas striated muscle inhibitor, 563Troponin/troponin complex, 557, 559f,562as striated muscle inhibitor, 563Troponin C, 562

INDEX / 691Troponin I, 562Troponin T, 562Trypsin, 477conserved residues and, 55tin digestion, 477for polypeptide cleavage, 25, 26tTrypsinogen, 477Tryptophan, 16t, 266–267, 490catabolism of, 257f, 258, 258fdeficiency of, 490niacin synthesized from, 490permeability coefficient of, 419frequirements for, 480Tryptophan oxygenase/L-tryptophanoxygenase (tryptophanpyrrolase), 89, 257f, 258TSEs. See Transmissible spongiformencephalopathiesTSH. See Thyroid-stimulating hormoneα-Tubulin, 577β-Tubulin, 577γ-Tubulin, 577Tumor cells, migration of, hyaluronic acidand, 548Tumor suppressor genes, p53, 339Tunicamycin, 527, 527tβ-Turn, 32, 34fTwin lamb disease. See Pregnancy toxemiaof ewesTwitch fibers, slow (red) and fast (white),574–576, 575tTwo-dimensional electrophoresis, proteinexpression and, 28TXs. See ThromboxanesTyk-2, in Jak-STAT pathway, 467Type A response, in gene expression, 374,375fType B response, in gene expression,374–375, 375fType C response, in gene expression, 375,375fTyrosine, 15t, 16t, 267, 267fcatabolism of, 254f, 255epinephrine and norepinephrine formedfrom, 267, 267fin hemoglobin M, 46in hormone synthesis, 438, 439–449,439tphosphorylated, 264requirements for, 480synthesis of, 239, 240fTyrosine aminotransferase, defect in, intyrosinemia, 255Tyrosine hydroxylase, catecholaminebiosynthesis and, 446, 447fTyrosine kinasein insulin signal transmission, 465–467,466fin Jak/STAT pathway, 467, 467fTyrosinemia, 255Tyrosinosis, 255Ubiquinone (Q/coenzyme Q), 92, 95f, 118in cholesterol synthesis, 220, 221fUDP-glucose. See Uridine diphosphateglucoseUDPGal. See Uridine diphosphate galactoseUDPGlc. See Uridine diphosphate glucoseUFA (unesterified fatty acids). See Free fattyacidsUlcers, 474Ultraviolet lightnucleotide absorption of, 290nucleotide excision-repair of DNAdamage caused by, 337vitamin D synthesis and, 484, 485fUMP (uridine monophosphate), 288f, 288tUncouplers/uncoupling proteinsin respiratory chain, 95, 96fchemiosmotic theory of action of, 97undernutrition and, 479Undernutrition, 474, 478–479Unequal crossover, 324, 324fUnesterified fatty acids. See Free fatty acidsUniport systems, 426, 426fUnique-sequence (nonrepetitive) DNA,320, 320–321Universal donor/universal recipient, 618Unsaturated fatty acids, 111, 112, 113t. Seealso Fatty acidscis double bonds in, 112–114, 114fdietary, cholesterol levels affected by,227eicosanoids formed from, 190, 192,193f, 194fessential, 190, 190f, 193abnormal metabolism of, 195–196deficiency of, 191–192, 194–195prostaglandin production and, 190in membranes, 417, 418fmetabolism of, 190–192oxidation of, 183structures of, 190fsynthesis of, 191, 191fUnwinding, DNA, 326, 326–327RNA synthesis and, 344Uracil, 288tdeoxyribonucleosides of, in pyrimidinesynthesis, 296–297, 298fUrate, as antioxidant, 119Ureaamino acid metabolism and, 124, 124fnitrogen catabolism producing,242–243, 245–247, 246fpermeability coefficient of, 419fsynthesis of, 243–244, 243f, 244fmetabolic disorders associated with,247–248gene therapy for, 248Uric acid, 289purine catabolism in formation of, 299,299fUridine, 287f, 288tUridine diphosphate N-acetylgalactosamine(UDP-GalNAc), 516tUridine diphosphate N-acetylglucosamine(UDP-GlcNAc), 516tUridine diphosphate galactose (UDPGal),167, 516–517, 516tUridine diphosphate galactose (UDPGal)4-epimerase, 167, 170finherited defects in, 172Uridine diphosphate glucose(UDP/UDPGlc), 145, 147f,516, 516tin glycogen biosynthesis, 145, 146fUridine diphosphate glucose dehydrogenase,166, 168fUridine diphosphate glucosepyrophosphorylase, 166, 168fin glycogen biosynthesis, 145, 146fUridine diphosphate-glucuronate/glucuronicacid, 166–167, 168f, 290Uridine diphosphate xylose (UDP-Xyl),516tUridine monophosphate (UMP), 288f,288tUridine triphosphate (UTP), in glycogenbiosynthesis, 145, 146fUridyl transferase deficiency, 172Urobilinogensconjugated bilirubin reduced to, 281,282fin jaundice, 284, 284tnormal values for, 284tUrocanic aciduria, 250Urokinase, 605, 605fUronic acid pathway, 163, 166–167, 168fdisruption of, 170Uronic acids, 109in heparin, 545, 545fUroporphyrinogen I, 271, 274f, 275fUroporphyrinogen I synthase, in porphyria,277tUroporphyrinogen III, 271, 274f, 275fUroporphyrinogen decarboxylase, 271,275fin porphyria, 277tUroporphyrins, 270, 271f, 272fspectrophotometry in detection of,273–274UTP, in phosphorylation, 85V8 protease, for polypeptide cleavage, forpolypeptide cleavage, 25, 26tv i . See Initial velocityV max . See Maximal velocityV region/segment. See Variable regions/segmentsv-SNARE proteins, 509, 511Valeric acid, 112tValine, 15tcatabolism of, 259, 260f, 262f

692 / INDEXValine (cont.)interconversion of, 240requirements for, 480Valinomycin, 99Van der Waals forces, 7Vanadium, 496tVariable numbers of tandemly repeatedunits (VNTRs), in forensicmedicine, 411Variable regions/segments, 591–592, 594fgene for, 593DNA rearrangement and, 325–326,393, 593–594immunoglobulin heavy chain, 591, 592f,594fimmunoglobulin light chain, 325–326,393, 591, 592f, 594fVascular system, nitric oxide affecting,571–573, 573f, 574tVasodilators, 556nitric oxide as, 571–573, 573f, 574tVDRE. See Vitamin D response elementVector, 414cloning, 400–402, 401f, 402t, 403f, 414expression, 402Vegetarian diet, vitamin B 12 deficiency and,491Velocityinitial, 64inhibitors affecting, 68, 68f, 69fmaximal (V max )allosteric effects on, 75–76inhibitors affecting, 68, 68f, 69fMichaelis-Menten equation indetermination of, 65–66, 66fsubstrate concentration and, 64, 64fVery low density lipoprotein receptor, 208Very low density lipoproteins, 125, 205,206t, 207hepatic secretion of, dietary and hormonalstatus and, 211–212, 213fmetabolism of, 125, 126f, 207–209, 210fin triacylglycerol transport, 207, 208f,210fVesiclescoating, 509, 510fbrefeldin A affecting, 510–511secretory, 498, 500ftargeting, 509, 510ftransport, 498, 508–511, 509t, 510fVimentins, 577t, 578Vinculin, 540, 541fViral oncogenes. See OncogenesViruses, host cell protein synthesis affectedby, 370–371, 371fVision, vitamin A in, 482t, 483, 484fVitamin A, 482–484, 482t, 483f, 484fdeficiency of, 482t, 483–484excess/toxicity of, 484functions of, 482t, 483in vision, 482t, 483Vitamin B complex. See also specific vitaminin citric acid cycle, 133coenzymes derived from, 50–51, 51fVitamin B 1 (thiamin), 482t, 488–489, 489fin citric acid cycle, 133coenzymes derived from, 51deficiency of, 482t, 489pyruvate metabolism affected by, 140,143, 489Vitamin B 2 (riboflavin), 86, 482t, 489–490in citric acid cycle, 133coenzymes derived from, 50–51, 489,490deficiency of, 482t, 490dehydrogenases dependent on, 87Vitamin B 6 (pyridoxine/pyridoxal/pyridoxamine), 482t, 491, 491fdeficiency of, 482t, 491xanthurenate excretion in, 258, 258fexcess/toxicity of, 491Vitamin B 12 (cobalamin), 482t, 491–492,492fabsorption of, 491–492intrinsic factor in, 477, 491–492deficiency of, 482t, 492functional folate deficiency and, 492,494in methylmalonic aciduria, 155Vitamin B 12 -dependent enzymes, 292f, 492Vitamin C (ascorbic acid), 163, 482t,495–496, 496fas antioxidant, 119in collagen synthesis, 38, 496, 535deficiency of, 482t, 496collagen affected in, 38–39, 496,538–539iron absorption and, 478, 496supplemental, 496Vitamin D, 482t, 484–486in calcium absorption, 477, 484,484–485deficiency of, 482t, 484, 485ergosterol as precursor for, 118, 119fexcess/toxicity of, 485–486metabolism of, 484–485, 485freceptor for, 471Vitamin D 2 (ergocalciferol), 484Vitamin D 3 (cholecalciferol)synthesis of in skin, 445, 446f, 484, 485fin vitamin D metabolism, 484, 485fVitamin D-binding protein, 445Vitamin D receptor-interacting proteins(DRIPs), 472t, 473Vitamin D response element, 459tVitamin E, 482t, 486, 486fas antioxidant, 91, 119, 486, 487fdeficiency of, 482t, 486Vitamin H. See BiotinVitamin K, 482t, 486–488, 488f, 604calcium-binding proteins and, 487–488,488fin coagulation, 486–488, 488fcoumarin anticoagulants affecting,604deficiency of, 482tVitamin K hydroquinone, 487, 488fVitamins, 2, 481–496, 482t. See also specificvitaminin citric acid cycle, 133digestion and absorption of, 477–478lipid- (fat) soluble, 482–488absorption of, 475water-soluble, 488–496VLA-1/VLA-5/VLA-6, 622tVLDL. See Very low density lipoproteinsVNTRs. See Variable numbers of tandemlyrepeated unitsVoltage-gated channels, 424, 568tvon Gierke’s disease, 152t, 300Von Willebrand factor, in plateletactivation, 605Warfarin, 486, 604phenobarbital interaction and,cytochrome P450 inductionaffecting, 628vitamin K affected by, 487Water, 2, 5–9as biologic solvent, 5, 6fbiomolecular structure and, 6–7, 6tdissociation of, 8–9in hydrogen bonds, 5, 6fas nucleophile, 7–9permeability coefficient of, 419fstructure of, 5, 6fWater solubility, of xenobiotics, metabolismand, 626Watson-Crick base pairing, 7, 303Waxes, 111Weak acids, 9buffering capacity of, 11–12, 12fdissociation constants for, 10–11, 12Henderson-Hasselbalch equationdescribing behavior of, 11, 12fphysiologic significance of, 10–11pK/pK a values of, 10–13, 12t,Weak bases, 9Wernicke-Korsakoff syndrome, 482tWernicke’s encephalopathy, 489Western blot transfer procedure, 403, 404f,414White blood cells, 620–624. See also specifictypegrowth factors regulating production of,610recombinant DNA technology in studyof, 624White thrombus, 598White (fast) twitch fibers, 574–576, 575tWhole genome shotgun approach, 634Williams syndrome, 539

INDEX / 693Wilson disease, 432t, 587–589ceruloplasmin levels in, 587gene mutations in, 432t, 588–589Wobble, 361X-linked disorders, RFLPs in diagnosis of,411X-ray diffraction and crystallography,protein structuredemonstrated by, 35Xanthine, 289Xanthine oxidase, 87deficiency of, hypouricemia and, 300Xanthurenate, excretion of in vitamin B 6deficiency, 258, 258fXenobiotics, metabolism of, 626–632conjugation in, 626, 628–630cytochrome P450 system/hydroxylationin, 626–628, 629tfactors affecting, 630pharmacogenetics in drug research and,631–632responses to, 630–631, 630t,631ttoxic, 631, 631fXeroderma pigmentosum, 337Xerophthalmia, vitamin A deficiency in,482t, 483XP. See Xeroderma pigmentosumXylose, in glycoproteins, 516tD-Xylose, 104f, 105tD-Xylulose, 106fL-Xylulose, 105taccumulation of in essential pentosuria,170YAC vector. See Yeast artificial chromosome(YAC) vectorYeast artificial chromosome (YAC) vector,401–402, 402tfor cloning in gene isolation, 635tYeast cells, mitochondrial protein importstudied in, 499Z line, 556, 557f, 558fZellweger’s (cerebrohepatorenal) syndrome,188, 503, 503tZinc, 496tZinc finger motif, 387, 388t, 390, 390fin DNA-binding domain, 470Zona fasciculata, steroid synthesis in, 440Zona glomerulosa, mineralocorticoidsynthesis in, 438Zona pellucida, glycoproteins in, 528Zona reticularis, steroid synthesis in,440ZP. See Zona pellucidaZP1–3 proteins, 528Zwitterions, 16Zymogens, 76, 477in blood coagulation, 600, 600t, 601rapid response to physiologic demandand, 76ZZ genotype, α 1 -antiproteinase deficiencyandin emphysema, 589in liver disease, 590