Kinetic studies of hypoxanthine/guanine phosphoribosyltransferase from yeast.
Item
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Title
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Kinetic studies of hypoxanthine/guanine phosphoribosyltransferase from yeast.
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Identifier
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AAI9119610
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identifier
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9119610
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Creator
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Aybar-Batista, Diogenes.
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Contributor
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Advisers: Donald L. Sloan | Steven Meshnick
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Date
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1991
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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 | Biology, Microbiology | Biology, Animal Physiology
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Abstract
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A new high performance liquid chromatographic (HPLC) procedure that improves the yield of hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) has been developed. This salt-burst elution procedure permits the rapid and efficient partial purification of virtually any globular protein by suddenly exposing it to a high ionic strength medium. For HGPRTase purification, two elution buffers were used: (a) 20mM Tris-HCl (pH 7.8) and (b) 20 mM Tris-HCl (pH 7.8) plus 1M NaCl. The enzyme was eluted with a 25 sec burst of 30% buffer b. With this technique an average of 23-fold purification was achieved in a single step. Complete recovery of activity was observed.;A survey of phosphoribosyltransferase (PRTase) activities in yeast was accomplished using reversed-phase HPLC assay procedures. The following bases were observed to be utilized during PRPP-dependent nucleotide synthesis: adenine, xanthine, hypoxanthine, guanine, uracil, orotate, nicotinamide, nicotinate and quinolinate. Gradient elution procedures have also been developed that allow the separation of PRTases assay components.;By utilizing the known equilibrium constants of Mg-PRPP complexes, a series of curves was developed which allow the determination of the concentrations of free PRPP, monomagnesium PRPP (Mg-PRPP) and dimagnesium PRPP (Mg{dollar}\sb2{dollar}-PRPP), at any pH ranging from 4.5 to 9.7 (at (MgCl{dollar}\sb2{dollar}) = 10mM). This permitted exploration of HGPRTase kinetics, taking into account the effect of pH on the substrate nature and concentration.;HGPRTase was shown to be reversibly inactivated by mercuric ion, but unaffected by the cysteine-specific modifying agent fosfomycin. Of the alternate substrates tested only xanthine proved to be a substrate for HGPRTase. Adenine, caffeine and 3-methylxanthine showed no apparent product formation over a period of 12 hr. The inhibitory effect of these bases was also investigated, and the inhibition shows mixed characteristics for all of them. This effect was investigated further for caffeine at low (200 {dollar}\mu{dollar}M) and high (1000 {dollar}\mu{dollar}M) PRPP concentrations, showing a noncompetitive character at high PRPP concentration and uncompetitive character at low PRPP concentration. (Abstract shortened with permission of author.).
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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.
