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

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ATR Activity (nmol min-1mg-1 ATR Activity (nmol min ) -1mg-1 ) ATR Activity (nmol min-1mg-1 ATR Activity (nmol min ) -1mg-1 ) A. 200 150 100 1 / Rate 0.012 0.009 0.006 0.003 50 0 0 100 0 0 0.05 0.1 0.15 1 / [ATP] 200 300 400 [ATP], (µM) 0.2 500 150 1 / Rate 0.0120 0.0090 86 250 200 150 100 B. C. D. 200 200 ATR Activity (nmol min-1mg-1 ATR Activity (nmol min ) -1mg-1 ) ATR Activity (nmol min-1mg-1 ATR Activity (nmol min ) -1mg-1 ) 1 / Rate 0.007 0.00525 0.0035 0.00175 0 50 0 0.1 0.2 0.3 0.4 0.5 0 0 10 20 1 / [cob(I)alamin] 30 40 50 [cob(I)alamin], (µM) 100 0.0060 0.0030 120 80 0.006 0.003 50 0 0 100 0 0 0.03 0.05 0.08 0.10 0.13 1 / [ATP] 200 300 400 500 40 0 0 10 0 0 20 0.1 0.2 0.3 0.4 1 / [cob(I)alamin] 30 40 0.5 50 [ATP], (µM) [cob(I)alamin], (µM) Figure 3-3. Determination of kinetic constants for ATR 239K and ATR 239M. Panels A and B are for ATR 239K and panels C and D are for ATR 239M. ATR assays were performed as described in the materials and methods except that the ATP and cob(I)alamin concentrations were varied as indicated. The insets are Lineweaver-Burk plots of the data shown in the larger graph. 160 1 / Rate 0.009
Absorbance 0.5 0.4 0.3 0.2 0.1 0.0 400 87 440 480 520 560 600 Wavelength, (nm) Figure 3-4. Absorbance spectra of an MSR-ATR assay. Prior to the addition of NADPH the spectrum is that of cob(I)alamin (············); 1 hour after the addition of NADPH and incubation at 37°C, the spectrum is that of AdoCbl (——); after photolysis, the spectrum is characteristic of cob(II)alamin (⎯ ⎯ ⎯).
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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
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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
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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
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methylmalonic acid. Methylmalonic a
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folate-dependent reactions includin
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tetanomorphum, P. shermanii, Euglen
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enzyme. This suggests that cob(II)a
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at the 5’-C of ATP by cob(I)alami
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iochemical studies and complementat
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1997). Both cases of the cblD disor
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23 The cblB complementation group i
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(Watkins et al. 2002). Some patient
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Chapter 2 of this dissertation repo
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Corrin Ring Dimethyl benzimidazole
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Classes Eliminases Dehydratases OH
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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: 85 Table 3-3. The MSR-ATR system se
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
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137 Vlasie, M., S. Chowdhury and R.
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BIOGRAPHICAL SKETCH Nicole Aurora L
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