Comparisons of the orotate phosphoribosyl transferase and hypoxanthine/guanine phosphoribosyl transferase activities in yeast.
Item
-
Title
-
Comparisons of the orotate phosphoribosyl transferase and hypoxanthine/guanine phosphoribosyl transferase activities in yeast.
-
Identifier
-
AAI8801693
-
identifier
-
8801693
-
Creator
-
Chung, Sung H.
-
Contributor
-
Adviser: Donald L. Sloan
-
Date
-
1987
-
Language
-
English
-
Publisher
-
City University of New York.
-
Subject
-
Chemistry, Biochemistry
-
Abstract
-
Both orotate phosphoribosyltransferase (OPRTase) and hypoxanthine/guanine phosphoribosyltransferase (HGPRTase) have been purified from Baker's yeast and analyzed kinetically using a modification of published HPLC procedures. Because these two enzymes exist in the cytosol of yeast and might compete for the limiting (1-13 {dollar}\mu{dollar}M) concentration of phosphoribosyl 1-pyrophosphate (PRibPP), we elected to examine both equilibrium and steady-state effects of one enzymatic reaction on the other with HPLC. First, under the condition of equivalent mass concentrations of OPRTase and HGPRTase, the initial rate of orotidine monophosphate synthesis and the equilibrium state were greatly affected by the presence of HGPRTase activity. In contrast, the presence of the OPRTase activity had no effect on the HGPRTase-catalyzed reaction under the same conditions. Second, to examine a competition by these enzymes for PRibPP in vivo, we have established that the total activities (units/ml) of OPRTase and HGPRTase in yeast cell extracts were 740 units/ml and 450 units/ml, respectively. These relative activities were then employed in an in vitro reaction competition analysis. The results were similar to those obtained from experiments where equivalent OPRTase and HGPRTase activities were employed and revealed profound initial velocity and equilibrium effects of one reaction on the other. Thus a real competition between these enzymes for PRibPP may occur in the yeast cell cytosol, as determined by this unique HPLC competition assay procedure.;Several pyrimidine base analogs and xanthine derivatives were examined, to determine whether they are alternative substrates or inhibitors of yeast OPRTase and HGPRTase, respectively. These studies showed that both enzymes have a fairly selective specificities for their base substrates. Most of these compounds studied were relatively poor inhibitors or had no inhibitory or substrate capabilities.;Non-reacting molecular sieve HPLC studies suggested the occurrence of a concentration-dependent equilibrium between monomeric and dimeric HGPRTase forms. However, the monomeric form of HGPRTase was observed to dominate in solution, in the presence of Mg-PRibPP in the eluting buffer. According to previously described cross linking studies and this HPLC study, the catalytically active form of yeast HGPRTase may be the monomeric form of the enzyme. (Abstract shortened with permission of author.).
-
Type
-
dissertation
-
Source
-
PQT Legacy CUNY.xlsx
-
degree
-
Ph.D.
