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

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106 control strain BE237 (which is isogenic to BE273 except that it contains the expression plasmid without insert) was analyzed by SDS-PAGE (Figure 4-1). Both the soluble and inclusion-body fractions of cell extracts were examined. High amounts of a protein with a molecular mass near 50 kDa were found in the inclusion-body fraction of cell extracts from expression strain BE273, and moderate amounts of a 50-kDa protein were also found in the soluble fraction of cell extracts from this strain (Figure 4-1, lanes 3 and 5). This is near the predicted molecular mass of His8-PduP (50 kDa). In contrast, the soluble and inclusion-body fractions from control strain BE237 (expression vector without insert) contained relatively little protein of 50 kDa molecular mass (Figure 4-1, lanes 2 and 4) indicating that the observed 50-kDa protein produced by BE273 was His8-PduP. The cell extracts analyzed by SDS-PAGE (Figure 4-1) were tested for propionaldehyde dehydrogenase activity. Substantial activity (2.4 µmol min -1 mg -1 protein) was found in inclusion bodies isolated from strain BE273, and in the soluble fraction of this strain (0.19 µmol min -1 mg -1 protein). However, little activity was found in either the soluble or inclusion-body fractions of control strain BE237 (0.02 µmol min -1 mg -1 protein). For the inclusion-body fraction from the expression strain, propionaldehyde dehydrogenase activity was linear with protein concentration (data not shown). Furthermore, when the substrates, propionaldehyde, NAD + or HS-CoA were omitted from the assay mixture, no activity was detected. Thus, these data provided additional evidence that PduP is a CoA-acylating propionaldehyde dehydrogenase. Purification of Recombinant His8-PduP Protein It was observed that following storage of purified inclusion bodies at 4 °C for 24 hours a significant amount of active His8-PduP protein was eluted into the soluble
107 fraction. Following centrifugation of this preparation at 31,000 x g for 30 min., approximately 0.5 mg of soluble protein remained in the supernatant. Enzyme assays showed that this supernatant contained 9.22 µmol min -1 mg -1 propionaldehyde dehydrogenase activity. This sample was further purified by nickel-affinity chromatography. The purity of the resulting preparation was analyzed by SDS-PAGE followed by staining with Coomassie. Results indicated that the His8-PduP obtained was highly purified (Figure 4-1, lane 6) and enzyme assays showed that the preparation was enriched in propionaldehyde dehydrogenase activity, with a specific activity of 15.2 µmol min -1 mg -1 protien. The reaction requirements for purified PduP were the same as those described above for the partially purified enzyme. These results demonstrated that the His8-PduP protein had propionaldehyde dehydrogenase activity. Propionyl-CoA is a Product of the PduP Reaction The fact that CoA-SH was required for the propionaldehyde dehydrogenase activity of PduP suggested that propionyl-CoA was a reaction product. Several additional experiments were conducted to test this possibility. Propionaldehyde dehydrogenase reactions containing purified PduP protein were allowed to proceed to completion. Then, reverse-phase HPLC was used to identify CoA-SH and CoA-derivatives present in reaction mixtures. A single CoA compound with a retention time of 10.2 min was detected. Further analyses showed that this compound co-eluted with authentic propionyl-CoA following co-injection and resolution via C18 reverse-phase HPLC. This indicated that propionyl-CoA was a product of the PduP aldehyde dehydrogenase reaction. Next, the reaction product identified as propionyl-CoA by reverse-phase HPLC was analyzed by MALDI-TOF MS. Three major peaks at m/z 824.8, 848.1, and 878.1
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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
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propanol dehydrogenase (PduQ) propa
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or from mutations that impair the a
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1989). 5-bromo-4-chloro-3-indolyl-
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41 5’-GCCGCCGGTACCGATGACGACGACAAG
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43 Growth of Adenosyltransferase Ex
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anti-rabbit IgG (γ-chain specific)
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sequence similarity to a known ATR
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ATRs were produced as fusion protei
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51 of the lacI repressor (not shown
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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: 105 Propionaldehyde Dehydrogenase A
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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