STUDIES OF THE KINETIC MECHANISM AND THE SUBUNIT STRUCTURE OF HYPOXANTHINE-GUANINE PHOSPHORIBOSYL TRANSFERASE FROM SACCHAROMYCES CEREVISIAE.
Item
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Title
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STUDIES OF THE KINETIC MECHANISM AND THE SUBUNIT STRUCTURE OF HYPOXANTHINE-GUANINE PHOSPHORIBOSYL TRANSFERASE FROM SACCHAROMYCES CEREVISIAE.
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Identifier
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AAI8212183
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identifier
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8212183
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Creator
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ALI, LINDA ZAIDOON.
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Contributor
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Donald L. Sloan
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Date
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1982
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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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Hypoxanthine-guanine phosphoribosyltransferase (HG-PRTase) has been purified to homogeneity from baker's yeast extracts using GMP-Sepharose chromatography and has been utilized to determine the molecular weight and the subunit structure of the enzyme. The native molecular weight determined by Sephadex G-100 column chromatography was 52,000. After treatment with sodium dodecylsulfate, a single band with an apparent molecular weight of 26,000 was observed using sodium dodecylsulfate polyacrylamide gel electrophoresis. Chemical cross-linking with glutaraldehyde and dimethylsuberimidate with subsequent SDS-polyacrylamide gel electrophoresis resulted in two protein species with molecular weights of 26,000 and 52,000. These results suggest that the enzyme consists of two very similar, probably identical subunits.;An assay procedure, utilizing high pressure liquid chromatography (HPLC), has been designed which allows both reactions catalyzed by hypoxanthine-guanine phosphoribosyltransferase (HG-PRTase) to be monitored simultaneously. Using this procedure and the theories described by Huang (Meth. Enzymol. 63, 486, 1979) for alternate substrate kinetic analysis, we have determined that purified HG-PRTase from yeast catalyzes the formations of both IMP and GMP through the use of an Ordered BiBi kinetic mechanism and that guanine is highly preferred over hypoxanthine as substrate in the forward reaction. This proposed kinetic mechanism has been confirmed using flow dialysis experiments in which a binary enzyme-phosphoribosyl pyrophosphate (P-Rib-PP) complex was characterized but where enzymic complexes with either guanine or hypoxanthine were not detected. Also consistent with this kinetic mechanism, was our observation that an exchange of label between {lcub}('14)C{rcub}-guanine or {lcub}('14)C{rcub}-hypoxanthine and their respective nucleotides (GMP and IMP) was not catalyzed by HG-PRTase. However, a significant exchange of label between {lcub}('32)P{rcub}-pyrophosphate and P-Rib-PP is observed upon incubation with this enzyme, suggesting that HG-PRTase may exist, in part, as a phosphoribosyl-enzyme complex in the presence of P-Rib-PP.
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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
