B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

More documents

Recommendations

Info

searches identified a homologous human cDNA. The bovine and human cDNAs were independently cloned, expressed in Escherichia coli, and the encoded proteins were shown to have adenosyltransferase activities similar to previously studied bacterial adenosyltransferases. Additional studies, using Western blots, showed that adenosyltransferase expression was altered in cell lines derived from patients with cblB methylmalonic aciduria when compared to cell lines from normal individuals. Two common human adenosyltransferase polymorphic variants were expressed in E. coli, purified, and biochemically characterized. Both had high specificity for ATP and comparable Km and Vmax values. An adenosyltransferase-linked cob(II)alamin reductase assay was developed and used to show that the human methionine synthase reductase functions as a cob(II)alamin reductase for adenosylcobalamin formation. A linked assay was also performed in the presence of a cob(I)alamin trapping agent, and results from these experiments suggested that human adenosyltransferase and methionine synthase reductase interact. Importantly, results from this work provide information that will allow improvements in diagnosis and treatment of methylmalonic aciduria, a devastating childhood disorder. xvi
CHAPTER 1 INTRODUCTION History of Cobalamin In 1925, Whipple and colleagues discovered that a diet of liver aided in the formation of blood cells in iron-deficient dogs. The next year, Minot and Murphy (Minot and Murphy 1926) were the first to document that patients with severe pernicious anemia (a disease that affects the formation of red blood cells) were successfully treated with a special diet consisting mainly of liver. Eight years later (in 1934) Whipple, Minot, and Murphy were awarded the Nobel Prize in Medicine and Physiology for their work on liver therapy against anemias. Following these studies, Castle (Kass 1978) concluded that the treatment of pernicious anemia by this specialized diet was dependent on the absorption of an extrinsic anti-pernicious anemia factor by an intrinsic protein in gastric juice. It is now known that Castle’s extrinsic anti-pernicious anemia factor is vitamin B12 and the intrinsic factor is a specific cobalamin transporter (synthesized by the stomach) that was shown to enhance the curing effect by vitamin B12 (Kass 1978). In 1948, two research groups led by Folkers and Smith independently isolated the red crystalline anti-pernicious anemia factor from bovine liver, and proposed the name vitamin B12 (Rickes et al. 1948, Smith and Parker 1948). The isolation of vitamin B12 inspired collaborative efforts between two laboratories (led by Alexander Todd and Dorothy Hodgkin) to elucidate its chemical structure. After 6 years, the crystal structure was resolved and finalized in the 1
Page 1 and 2: B12 METABOLISM IN HUMANS By NICOLE
Page 3 and 4: The work in this dissertation is de
Page 5 and 6: TABLE OF CONTENTS Page ACKNOWLEDGME
Page 7 and 8: General Molecular and Protein Metho
Page 9 and 10: LIST OF TABLES Table page 1-1. Aden
Page 11 and 12: 3-7. Stoichiometry of the MSR-ATR s
Page 13 and 14: DTT dithiothreitol E. coli Escheric
Page 15: Abstract of Dissertation Presented
Page 19 and 20: 3 (DMB). The DMB moiety is covalent
Page 21 and 22: 5 and deoxyadenosine. After hydroge
Page 23 and 24: 7 homocysteine recycling and methio
Page 25 and 26: methylmalonic acid. Methylmalonic a
Page 27 and 28: folate-dependent reactions includin
Page 29 and 30: tetanomorphum, P. shermanii, Euglen
Page 31 and 32: enzyme. This suggests that cob(II)a
Page 33 and 34: at the 5’-C of ATP by cob(I)alami
Page 35 and 36: iochemical studies and complementat
Page 37 and 38: 1997). Both cases of the cblD disor
Page 39 and 40: 23 The cblB complementation group i
Page 41 and 42: (Watkins et al. 2002). Some patient
Page 43 and 44: Chapter 2 of this dissertation repo
Page 45 and 46: Corrin Ring Dimethyl benzimidazole
Page 47 and 48: Classes Eliminases Dehydratases OH
Page 49 and 50: A) B) CH 3THF 33 MS-cob(I)alamin Me
Page 51 and 52: propanol dehydrogenase (PduQ) propa
Page 53 and 54: or from mutations that impair the a
Page 55 and 56: 1989). 5-bromo-4-chloro-3-indolyl-
Page 57 and 58: 41 5’-GCCGCCGGTACCGATGACGACGACAAG
Page 59 and 60: 43 Growth of Adenosyltransferase Ex
Page 61 and 62: anti-rabbit IgG (γ-chain specific)
Page 63 and 64: sequence similarity to a known ATR
Page 65 and 66: ATRs were produced as fusion protei
Page 67 and 68:
51 of the lacI repressor (not shown
Page 69 and 70:
in which proteins bind B12, the "ba
Page 71 and 72:
libraries may be particularly usefu
Page 73 and 74:
57 Figure 2-1. Multiple sequence al
Page 75 and 76:
Table 2-2. Specific activities of b
Page 77 and 78:
61 1 2 3 4 5 25 kDa Figure 2-4. Wes
Page 79 and 80:
63 (cyanocobalamin, CNCbl) and hydr
Page 81 and 82:
65 5’- triphosphate (ATP), and di
Page 83 and 84:
extracts of ATR 239K (160 mg protei
Page 85 and 86:
Milford, MA). Samples (200 µl) wer
Page 87 and 88:
Linearity of the ATR reaction 71 Th
Page 89 and 90:
73 reactions was characteristic of
Page 91 and 92:
75 MSR Produces little Cob(I)alamin
Page 93 and 94:
Johnson et al. 2001, Saridakis et a
Page 95 and 96:
AdoCbl by the MSR-ATR system. Exper
Page 97 and 98:
propionyl-CoA PCC (2S)-methylmalony
Page 99 and 100:
kDa 97 66 45 31 21 14 7 83 1 2 3 4
Page 101 and 102:
85 Table 3-3. The MSR-ATR system se
Page 103 and 104:
Absorbance 0.5 0.4 0.3 0.2 0.1 0.0
Page 105 and 106:
Wavelength, (525 nm) 0.24 0.23 0.22
Page 107 and 108:
Activity, (nmol min-1 Activity, (nm
Page 109 and 110:
al. 1988). The aldehyde is then dis
Page 111 and 112:
CoA-acylating propionaldehyde dehyd
Page 113 and 114:
97 previously published DNA sequenc
Page 115 and 116:
mM CHES (2-[N-cyclohexylamino]ethan
Page 117 and 118:
101 For the conjugation step, strai
Page 119 and 120:
103 from New England Biolabs (Bever
Page 121 and 122:
105 Propionaldehyde Dehydrogenase A
Page 123 and 124:
107 fraction. Following centrifugat
Page 125 and 126:
109 antiserum obtained was specific
Page 127 and 128:
111 biochemical evidence was presen
Page 129 and 130:
113 Table 4-1. Bacterial strains Sp
Page 131 and 132:
115 Table 4-3. Propionaldehyde dehy
Page 133 and 134:
kDa 209 80 49 35 29 117 1 2 3 4 Fig
Page 135 and 136:
CHAPTER 5 CONCLUSIONS In humans, co
Page 137 and 138:
121 substitutions that are common p
Page 139 and 140:
123 Studies also indicated that hum
Page 141 and 142:
LIST OF REFERENCES Aberg, A., S. Ha
Page 143 and 144:
127 Bradford, M. 1976. A rapid and
Page 145 and 146:
129 Fenton, W. A. and L. E. Rosenbe
Page 147 and 148:
131 Johnson, C. L. V. J., E. Pechon
Page 149 and 150:
133 Mellman, I., H. F. Willard and
Page 151 and 152:
135 Rossi, M., J. P. Glusker, L. Ra
Page 153 and 154:
137 Vlasie, M., S. Chowdhury and R.
Page 155 and 156:
BIOGRAPHICAL SKETCH Nicole Aurora L
show all

B12 METABOLISM IN HUMANS By NICOLE AURORA LEAL A ...

Create successful ePaper yourself

Delete template?

Save as template?