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

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(bovine ATR), BE257 (human ATR + MTS), and BE258 (human ATR). Of the four 50 bovine ATR extracts tested, the highest specific activity measured was 85.7 nmol min -1 mg -1 found in the inclusion body fraction from strain BE255 (ATR + MTS). The highest specific activity found in the human ATR extracts tested was 98 nmol min -1 mg -1 in the inclusion body fraction containing human ATR without its presumptive MTS. For each ATR fusion protein, the majority of the activity was found in the soluble fraction (Table 2-2, column 4). For both the human and bovine cell extracts, ATR activity was linear with protein concentration (data not show). Furthermore, when the substrates, ATP and/or cob(I)alamin were excluded from the assay mixture, no activity was detected. Also, as expected, cell extracts from strain BE237 (T7 expression vector without insert) expressed no measurable ATR activity. Complementation of an S. enterica Mutant Deficient in ATR Activity by a Human Adenosyltransferase cDNA Clone We previously showed that an S. enterica strain deficient for both the CobA and PduO ATRs (cobA pduO) was unable to grow on 1,2-propanediol due to an ATR deficiency (Johnson et al. 2001). To test whether the human ATR cDNA could complement this defect, we compared the growth of the wild-type strain, BE265 (pduO cobA /pLAC22-human ATR) and BE263 (pduO cobA /pLAC22-no insert) on 1,2-propanediol minimal medium (Figure 2-3). The doubling times of the wild-type and strain BE265 (pduO cobA /pLAC22-human ATR cDNA) were found to be comparable; they were 8.4 and 8.9 h, respectively. Control experiments showed that strain BE263, which carried the pLAC22 vector without insert, grew minimally on 1,2-propanediol, and that growth of strain BE265 (pduO cobA/pNL135-human ATR) required the addition of IPTG as was expected since expression of the human ATR in this strain is under control
51 of the lacI repressor (not shown). Thus, clearly, the human ATR cDNA complemented the ATR-deficient bacterial mutant for AdoCbl-dependent growth on 1,2-propanediol. These results provided further evidence that human cDNA BC005054 encodes an ATR enzyme, and also showed that this enzyme can function as an ATR under physiological conditions albeit in a heterologous host. Level of Human Adenosyltransferase Enzyme in Normal and cblB Mutant Human Skin Fibroblasts Above, we presented biochemical and genetic evidence that human cDNA BC005054 encodes an ATR enzyme. If defects in this enzyme underlie cblB methylmalonic aciduria, its expression or stability might be altered in cblB mutant cell lines. To examine the expression of this enzyme in normal and cblB mutant human skin fibroblasts, Western blots were performed on lysates from these cells using antibody against recombinant human ATR described above. Figure 2-4 shows a representative experiment. Lane 5 contained recombinant human ATR lacking both the mitochondrial targeting sequence and the expression tags. In this lane, a single protein band with an approximate molecular mass of 25 kDa was recognized by the anti-ATR antibody as expected. In lane 1, which contained cell extracts from normal skin fibroblasts, the anti-human ATR antibodies recognized two bands. One of these bands was similar in molecular mass to the recombinant human ATR (approximately 25 kDa) and the second band was of lower mass (approximately 23 kDa). Neither the 25 kDa nor the 23 kDa band was observed in blots performed with pre-immune serum (data not shown) indicating that these bands most likely represent two processed forms of the human ATR. Interestingly, ATR-reactive polypeptides of these molecular masses were differentially expressed in the three cblB cell lines tested (lanes 2-3). The cblB mutants WG1680 and
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B12 METABOLISM IN HUMANS By NICOLE
Page 3 and 4:
The work in this dissertation is de
Page 5 and 6:
TABLE OF CONTENTS Page ACKNOWLEDGME
Page 7 and 8:
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
Page 13 and 14:
DTT dithiothreitol E. coli Escheric
Page 15 and 16: Abstract of Dissertation Presented
Page 17 and 18: CHAPTER 1 INTRODUCTION History of C
Page 19 and 20: 3 (DMB). The DMB moiety is covalent
Page 21 and 22: 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: ATRs were produced as fusion protei
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 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
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103 from New England Biolabs (Bever
Page 121 and 122:
105 Propionaldehyde Dehydrogenase A
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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.
Page 155 and 156:
BIOGRAPHICAL SKETCH Nicole Aurora L
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