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

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Purification of the Human ATR Variants 70 Results The human ATR catalyzes the transfer of a 5'-deoxyadenosyl group from ATP to the central cobalt atom of cob(I)alamin to form AdoCbl (Chapter 2, Leal et al. 2003). Two common polymorphic variants of the human ATR having either lysine or methionine as amino acid 239 (ATR 239M and 239K) were recently shown to be present in 54% and 46% of 72 control cell lines, respectively (Dobson et al. 2002). We previously produced ATR 239K as a GST-fusion protein in E. coli (Leal et al. 2003). Here, subcloning and site-directed mutagenesis were used to construct E. coli expression strains that produce high levels of ATR 239K and 239M without fusion tags and lacking their predicted mitochondrial targeting sequences. Both ATR variants were produced at high levels and purified to apparent homogeneity from soluble cell extracts of recombinant E. coli. The purification consisted of three steps including ammonium sulfate precipitation and ceramic-hydroxyapatite and Mono Q chromatography (Table 3-1). Both ATR variants precipitated between 40 and 50% ammonium sulfate saturation and eluted from the hydroxyapatite and Mono Q columns at 110-135 mM potassium phosphate and 114-130 mM KCl. ATR 239K and 239M were purified 4.5 and 5.4-fold with specific activities of 220 and 190 nmol min -1 mg -1 proteins, respectively. The yields were 14 and 9%. During the purification, inclusion of KCl in chromatography and storage buffers increased enzyme stability. SDS-PAGE was used to follow the purification of both ATR variants. Since purification of both enzymes proceeded similarly, only the gel used to assess the purity of ATR 239K is depicted (Figure 3-2). Following the final Mono Q chromatography step, both variants were homogenous following staining with Coomassie Brilliant Blue.
Linearity of the ATR reaction 71 The effect of ATR concentration on activity was determined. Results showed that the rate of adenosylation was proportional to ATR concentration ranging from 5 µg to 115 µg for both variants and linear regression yielded R 2 values of 0.9995 and 0.9993 for ATR 239K and 239M, respectively. ATR Reaction Requirements To determine the ATR reaction requirements, key assay components were individually omitted. For both variants, there was no detectable activity in the absence of ATR, ATP, HOCbl, or titanium (III) citrate (the reducing agent used to produce cob(I)alamin). In the complete reaction mixture, there was 215 and 194 nmol min -1 mg -1 activity for ATR 239K and 239M, respectively. In the absence of MgCl2, activity decreased by 20% for both variants. There was no significant difference in activity in the absence of KCl. Alternative Nucleotide Donors The specificity of the ATR variants for ATP, CTP, GTP, UTP, ADP, and AMP was examined. ATP was found to be the best substrate giving a specific activity of 207 and 197 nmol min -1 mg -1 for ATR 239K and 239M, respectively. These values were set as 100% activity. When CTP, GTP, and UTP were tested as substrates for ATR 239K there was 9, 16, or 8% activity, respectively (Table 3-2). Similar results were found for ATR 239M (6, 14, or 6% activity with CTP, GTP, and UTP, respectively). For both ATR variants there was no detectable activity when AMP or ADP was substituted for ATP. Km and Vmax Values for ATR 239K and 239M Lineweaver-Burk double-reciprocal plots were used to calculate Km and Vmax values (Segel 1976). For ATR 239K and ATR 239M, the Km values for ATP were 6.3 µM and
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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
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: Milford, MA). Samples (200 µl) wer
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
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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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