STUDIES OF THE EFFECTS OF MAGNESIUM ION ON THE STRUCTURE AND MECHANISM OF OROTATE PHOSPHORIBOSYL TRANSFERASE AND NICOTINATE PHOSPHORIBOSYL TRANSFERASE FROM YEAST.
Item
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Title
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STUDIES OF THE EFFECTS OF MAGNESIUM ION ON THE STRUCTURE AND MECHANISM OF OROTATE PHOSPHORIBOSYL TRANSFERASE AND NICOTINATE PHOSPHORIBOSYL TRANSFERASE FROM YEAST.
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Identifier
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AAI8312359
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identifier
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8312359
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Creator
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MAYER, LEONARD.
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Contributor
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Donald Sloan
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Date
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1983
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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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Biophysics, General
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Abstract
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The purpose of this investigation was to explore the role that Mg('2+) plays in the structure and mechanism of orotate phosphoribosyltransferase (OPRTase) and nicotinate phosphoribosyltransferase (NPRTase) using nuclear magnetic relaxation (NMR) and SDS acrylamide gel electrophoresis. Crosslinking of OPRTase by glutaraldehyde (GTA) in the presence of Mg('2+) did reveal by SDS electrophoresis the existence of a dimeric form of yeast OPRTase. At low concentrations of GTA (0.05%) in the absence of Mg('2+), detectable dimer was not observed while at high concentrations of GTA (0.2%) in the absence of Mg('2+), the dimer was detectable as a band on SDS electrophoresis gels. The appearance of the crosslinked dimer depended on the concentration of GTA employed and on whether or not Mg('2+) was present. Mg('2+) enhances the appearance of the dimer. Mg('2+)-phosphoribosylpyrophosphate (PRPP) did not protect the enzyme against crosslinking but may have protected against inactivation by GTA. Whereas GTA inactivates OPRTase, dimethyl suberimidate (DMS) does not. Crosslinking of NPRTase at all concentrations of GTA or DMS tested did not occur. Neither GTA nor DMS succeeded in rendering NPRTase inactive. Neither Mg('2+), Mg('2+)-PRPP, nor Mg('2+)-adenosine triphosphate (ATP) affected the electrophoretic pattern or the activity of NPRTase treated with either GTA or DMS. From the SDS electrophoresis results, it has been proposed that the OPRTase monomer is in equilibrium with a dimeric form and that Mg('2+) promotes the extent of dimer formation. In contrast, NPRTase exists as a monomer in the native form and does not interconvert to higher molecular weight forms.;Proton-NMR was employed to characterize the formation of OMP-metal ion, PRPP-metal ion, and OPRTase-substrate-metal ion complexes. Moreover, when Mg('2+) was added to solutions of PRPP, OMP, and OPRTase, the changes in the proton resonance amplitudes of PRPP (decreases) and OMP (increases) suggest that a new equilibrium between this substrate/product pair has been established. Such an alteration in the equilibrium in the absence of orotate and PP(,i), the other substrate/product pair, can only occur if the kinetic mechanism is Bi Bi Ping Pong. Thus the NMR experiments provide independent evidence of a mechanism that has been suggested from kinetic analysis.
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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
