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

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mitochondrial targeting sequence (Leclerc et al. 1999). There have been no studies 18 documenting the role of MSR in the mitochondria. Banerjee and Olteanu (2001) showed that MSR is a monomeric protein of 77 kDa, that when purified contains stoichiometric amounts of FAD and FMN. In an NADPH-dependent reaction, purified MSR reduced cob(II)alamin to cob(I)alamin for activation of MS in vitro (Olteanu and Banerjee 2001). In these same studies, it was found that the reduction of cobalamin bound to MS by MSR is dependent on electrostatic interactions. Inherited Disorders of Cobalamin Metabolism In humans, inherited disorders in the metabolic pathways of both AdoCbl and CH3Cbl synthesis result in methylmalonic aciduria and homocystinuria. Methylmalonic aciduria is caused by a block in the pathway of propionyl-CoA metabolism which causes an accumulation methylmalonyl-CoA that is hydrolyzed to methylmalonic acid, and excreted into the blood and urine (Rosenblatt and Cooper 1990). Clinically, methylmalonic aciduria presents itself by lethargy, recurrent vomiting, dehydration, respiratory distress, feeding difficulties, failure to thrive, hypotonia, and death (Ciani et al. 2000). Homocystinuria is the result of a block in the methylation cycle for methionine synthesis, and results in an accumulation of homocysteine, which is excreted into the blood and urine (Rosenblatt and Cooper 1990). Homocystinuria presents itself by megaloblastic anemia, delayed development, neurological disorders, cardiovascular disease, and death (Kapadia 1995). All of the disorders involved in cobalamin metabolism are inherited as autosomal recessive traits (Rosenblatt and Fenton 1999). There have been no reported heterozygotes with the presentation of these diseases. Inborn errors of cobalamin metabolism have been classified into eight distinct complementation groups, cblA to cblH (Figure 1-5). This classification is based on
iochemical studies and complementation analysis of human cultured fibroblast cells (Fenton and Rosenberg 1978a). Complementation analysis examines the uptake and 19 conversion of labeled precursors (propionate, CH3THF) by fused and unfused fibroblast cell lines derived from two patients with cobalamin disorders. In these studies, fused cells that stimulated the incorporation of labeled precursors complemented each other and were assigned to different complementation groups. If the fused cells were unable to stimulate incorporation of the precursors they were assigned to the same complementation group (Gravel et al. 1975). An assay that has helped to determine if defects are due to decreased synthesis of one or both cobalamin coenzymes involves incubating the cultured fibroblasts with labeled HOCbl and measuring the cofactors formed against control cell lines. Combined Methylmalonic Aciduria and Homocystinuria Defects in cellular cobalamin metabolism resulting in both methylmalonic aciduria and homocystinuria are defined by the complementation groups cblF, cblC, and cblD. Cultured fibroblasts from patients in these groups were analyzed and shown to have a deficiency in the conversion of CNCbl or HOCbl to AdoCbl or CH3Cbl cofactors (Qureshi et al. 1994). The absence of these cofactors renders both MCM and MS inactive. There are over 100 patients with combined methylmalonic aciduria and homocystinuria. The cblC complementation group has more than 100 patients, the cblD group has only two patients who are siblings, and the cblF group has six patients (Watkins and Rosenblatt 2001). Individuals with the cblF disorder have impaired MCM and MS activities. The total intracellular pools of AdoCbl and CH3Cbl are reduced when compared to control cells. Cultured cells incubated with [ 57 Co]-labeled CNCbl showed an accumulation of
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 and 16: Abstract of Dissertation Presented
Page 17 and 18: CHAPTER 1 INTRODUCTION History of C
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: at the 5’-C of ATP by cob(I)alami
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
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