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

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A. 58 kDa 1 2 3 4 5 6 7 kDa 1 2 3 4 5 6 7 116.25 97.4 97.4 66.2 66.2 45 31 21.5 14.4 45 31 21.5 Figure 2-2. SDS-PAGE analysis of cell extracts from bovine (A) and human (B) ATR expression strains. Panels A and B lane 1, molecular mass markers. Panel A, lanes 2 thru 4 are soluble extracts of BE237 (T7 expression vector without insert), BE255 (bovine ATR + MTS), and BE256 (bovine ATR without MTS). Panel A, lanes 5 thru 7 are inclusion body fractions from strains BE237, BE255, and BE256. Panel B, lanes 2 thru 4 are soluble extracts from BE237 (T7 expression vector without insert), BE257 (human ATR + MTS), and BE258 (human ATR without MTS), respectively. Lanes 5 thru 7 are inclusion body fractions from strains BE237, BE257, and BE258. B.
Table 2-2. Specific activities of bovine and human ATP:cob(I)alamin ATRs Strain S/I * 59 Total activity nmol min -1 Total activity % Specific activity nmol min -1 mg -1 protein BE255 S 2021 95 47 (bovine ATR+MTS) I 107 5 85.7 BE256 S 3364 84 44.1 (bovine ATR without MTS) I 632 16 30 BE257 S 2445 52 37.5 (human ATR+MTS) I 2296 48 60.8 BE258 S 5481 57 63 (human ATR without MTS) I 4144 43 98 * S=soluble fraction, I=inclusion body fraction
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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
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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
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: 57 Figure 2-1. Multiple sequence al
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
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109 antiserum obtained was specific
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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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