STUDIES OF NICOTINAMIDE ADENINE DINUCLEOTIDE METABOLISM IN YEAST.
Item
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Title
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STUDIES OF NICOTINAMIDE ADENINE DINUCLEOTIDE METABOLISM IN YEAST.
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Identifier
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AAI8801778
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identifier
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8801778
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Creator
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YAN, CONG.
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Contributor
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Donald L. Sloan
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Date
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1987
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Language
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English
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Publisher
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City University of New York.
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Subject
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Chemistry, Biochemistry
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Abstract
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Nicotinamide phosphoribosyltransferase (N{dollar}\sb{lcub}\rm m{rcub}{dollar}PRTase; 2.4.2.12) and quinolinate phosphoribosyltransferase (QPRTase; 2.4.2.19) activities were detected for the first time in yeast and were partially purified and characterized. In addition, the yeast nicotinamide deamidase (YNDase; 3.5.1.19) was purified to homogeneity, as determined by the criteria of gel electrophoresis. The enzyme was then characterized in detail. Polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) gel filtration molecular weight studies showed that YNDase is a monomeric protein with a molecular weight of 34,000. Our kinetic study of this enzyme showed that the K{dollar}\sb{lcub}\rm m{rcub}{dollar} of nicotinamide is 34 {dollar}\mu{dollar}M and that there exists an activity-dependent pK equal to 7.8. A pH study indicated that this enzyme has an unusually broad range of pH stability. Chemical modification analysis with N-ethylmaleimide (NEM) revealed that a cysteine residue is located at active center. YNDase was also inactivated by diethylpyrocarbonate (DEP) and the enzyme activity could be protected by substrate. A substrate analogue study showed that nicotinaldehyde is a noncompetitive inhibitor of YNDase. When we incubated YNDase with nicotinaldehyde in the presence of sodium borohydride, a Schiff base was formed, which indicates that a lysine residue is located near to the active center to facilitate the enzymatic reaction. Amino acid composition analysis was also accomplished and some substrate specificity studies have been performed, revealing a high degree of specificity of YNDase for nicotinamide. In the yeast system, we carried out some studies concerning ADP-ribosylation of proteins. NAD glycohydrolase and ADPR phosphodiesterase activities were discovered. Finally a sensitive HPLC assay method was designed and implemented in our research.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biochemistry
