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Title
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NICOTINATE PHOSPHORIBOSYL TRANSFERASE FROM YEAST: A NEW HIGH PRESSURE LIQUID CHROMATOGRAPHY ASSAY PROCEDURE, A NEW PURIFICATION PROCEDURE AND A KINETIC ANALYSIS.
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Identifier
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AAI8203286
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identifier
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8203286
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Creator
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HANNA, LEWIS S.
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Contributor
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Dr. Donald L. Sloan
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Date
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1981
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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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Nicotinate phosphoribosyltransferase (N(,a)PRTase) catalyzes the formation of nicotinate mononucleotide (N(,a)MN) from phosphoribosyl-(alpha)-pyrophosphate (PRPP) and nicotinic acid (N(,A)). The enzyme from several sources hydrolyzes 1 mole of ATP to ADP and inorganic phosphate for every 1 mole of N(,a)MN formed. This thesis describes the development of a highly sensitive, reliable and fast assay procedure, utilizing high pressure liquid chromatography (HPLC), for N(,a)PRTase from baker's yeast. The concomitant utilization of the substrates ATP and N(,A) is followed as is the formation of the products ADP and N(,a)MN. The assay procedure enabled me to demonstrate the 1:1 stoichiometry between the hyrolysis of ATP and the formation of N(,a)MN. Moreover, it enabled me to investigate the kinetics of the N(,a)PRTase catalyzed reaction faster and with greater accuracy than the previously published procedure which made use of radioactive nicotinic acid.;A purification procedure which provided a very good yield of purified enzyme has been developed employing Phosphocellulose, Hydroxylapatite, Blue-Sepharose CL-6B and DEAE-cellulose chromatographic steps. The procedure produced a 5% yield (10 mg per 6 pounds of yeast) of enzyme with a specific activity of 4.4 units per mg (1 unit = umole of N(,a)MN formed per min). The molecular weight was determined to be 45,000 by SDS-gel electrophoresis.;Kinetic analysis of the N(,a)PRTase-catalyzed reaction yielded the following results. Utilizing the HPLC assay procedure, double reciprocal initial velocity plots were constructed using data obtained by varying one substrate at different concentrations of the second substrate while maintaining the third substrate constant (6 families of plots) and by varying one substrate at different concentrations of the other two substrates which were maintained at a constant ratio (3 families of plots). In addition, product inhibition studies, with the product pyrophosphate, was performed: (1) at various concentrations of ATP and unsaturated and saturated conditions of N(,A), (2) at various concentrations of N(,A) and unsaturated and saturated conditions of ATP and (3) at various concentrations of PRPP at saturated conditions of N(,A). During these studies I observed a significant substrate inhibition at high concentrations of PRPP. In addition I demonstrated that purified N(,a)PRTase possesses an ATPase activity only in the presence of either product (pyrophosphate or N(,a)MN) and in the absence of PRPP. Exchange studies between {lcub}('14)C{rcub}-N(,A)/N(,a)MN in the presence and absence of ATP and PRPP and exchange studies between {lcub}('32)P{rcub}-PP(,i)/PRPP in the presence and absence of ATP and N(,A) were accomplished. Binding studies of {lcub}('14)C{rcub}-N(,A) to N(,a)PRTase were also performed in the presence and absence of ATP and PRPP, and effects of Cr('III)ATP and Cr('III)PP(,i) on the N(,a)PRTase-catalyzed reaction were initiated. Cr('III)ATP was observed to be a competitive inhibitor of Mg-ATP function whereas Cr('III)PP(,i) and the product MgPP(,i) inhibited the N(,a)PRTase activity differently.;All of the above kinetic studies implicate a Uni Uni Bi Ter Ter Quad Ping-Pong system, in which the second and third products dissociate at random, as the kinetic mechanism for the N(,a)PRTase reaction. The K(,m) values for ATP, PRPP and N(,A) were calculated to be 70 (+OR-) 10 uM, 24 (+OR-) 3 uM and 23 (+OR-) 4 uM respectively, whereas a V(,max) value of 2.5 (+OR-) 0.4 x 10('-2) umol N(,a)MN/min and a value for K(,i(PRPP)) of 5 (+OR-) 1 uM were determined.
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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
