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

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OD 600 1.0 0.5 0.1 60 0 10 20 30 40 Time (hours) Figure 2-3. Complementation of an ATR-deficient bacterial mutant for AdoCbl-dependent growth on 1,2-propanediol by a plasmid that expresses the human ATR. Cells were grown in minimal 1,2-propanediol medium supplemented with HOCbl and 0.1 mM IPTG. Growth was determined by measuring the absorbance of cultures at 600 nm (OD600), and data are shown as a semi-log plot. , Wild-type (S. enterica serovar Typhimurium LT2); , BE265, ATR-deficient/pNL135(human ATR under plac control); , BE263, ATR-deficient /pLAC22 (vector without insert).
61 1 2 3 4 5 25 kDa Figure 2-4. Western blot analysis of ATR expression by normal and cblB mutant cell lines. Extracts were prepared from cultured fibroblast lines obtained from a normal individual and three cblB patients. Lane 1: cell extract from the MCH45 control cell line. Lanes 2 thru 4: cell extracts from cblB mutant cell lines WG1680, WG1879 and WG2127, respectively. 220 µg of protein was loaded into each of lanes 1 thru 4 (It was necessary to load relatively high amounts of protein because of the low abundance of the ATR enzyme in skin fibroblasts). Lane 5 contained 100 pg recombinant human ATR lacking the mitochondrial targeting sequence and affinity tags. The arrow shows the location of the recombinant ATR enzyme (approximately 25 kDa). The experiment was performed twice with similar results.
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B12 METABOLISM IN HUMANS By NICOLE
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The work in this dissertation is de
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TABLE OF CONTENTS Page ACKNOWLEDGME
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General Molecular and Protein Metho
Page 9 and 10:
LIST OF TABLES Table page 1-1. Aden
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3-7. Stoichiometry of the MSR-ATR s
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DTT dithiothreitol E. coli Escheric
Page 15 and 16:
Abstract of Dissertation Presented
Page 17 and 18:
CHAPTER 1 INTRODUCTION History of C
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3 (DMB). The DMB moiety is covalent
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5 and deoxyadenosine. After hydroge
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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: Table 2-2. Specific activities of b
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
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113 Table 4-1. Bacterial strains Sp
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115 Table 4-3. Propionaldehyde dehy
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kDa 209 80 49 35 29 117 1 2 3 4 Fig
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CHAPTER 5 CONCLUSIONS In humans, co
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121 substitutions that are common p
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123 Studies also indicated that hum
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LIST OF REFERENCES Aberg, A., S. Ha
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127 Bradford, M. 1976. A rapid and
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129 Fenton, W. A. and L. E. Rosenbe
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131 Johnson, C. L. V. J., E. Pechon
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133 Mellman, I., H. F. Willard and
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135 Rossi, M., J. P. Glusker, L. Ra
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137 Vlasie, M., S. Chowdhury and R.
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BIOGRAPHICAL SKETCH Nicole Aurora L
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