Cytidine 5' triphosphate synthetase (CTPS; E.C.6.3.4.2) catalyzes the rate limiting irreversible conversion of uridine triphosphate to cytidine triphosphate. We propose to elucidate the regulation of mammalian CTPS by purifying the enzyme from Chinese hamster liver. The chemistry, conformational changes, kinetics, cooperativity, feedback inhibition, and antigenic sites of the enzyme will be investigated. Polyclonal antibodies will be raised against the purifed CTPS and used as a tool (a) to examine directly the synthesis and accumulation of the enzyme during the cell cycle, (b) to identify specific molecular defects of mutant isolates of Chinese hamster V79 cells, and (c) to clone the CTPS structural gene. Novel selection schemes have been devised for the isolation of additional mutants, which either encode CTPS or regulate its activity. The mode of inheritance, allelism, dominance relationship, genetic complementation, gene amplification and map location of the CTPS gene(s) will be studied. The size and composition of intracellular nucleotide pools, influenced by varying the genetic and/or physiological parameters, will be determined. When the CTPS gene sequence is isolated, it will be used for an assessment of the level and translatability of CTPS-specific mRNA and the determination of the number of gene copies. The biochemical characterization of CTPS is of interest not only because of the intrinsic role of the enzyme as a synthetase, but also as a model for regulatory cooperativity patterns. The results to be obtained from the planned studies may have implications for the genetic and metabolic regulation of pyrimidine metabolism, inherited metabolic disease, tumor growth, and cancer chemotherapy in man.
| Thomas, P E; Lamb, B J; Chu, E H (1988) Purification of cytidine-triphosphate synthetase from rat liver, and demonstration of monomer, dimer and tetramer. Biochim Biophys Acta 953:334-44 |
