title: Regulation of purine synthesis de novo in human fibroblasts by purine nucleotides and phosphoribosylpyrophosphate.
abstract: Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.
mesh_terms: D001377:Azaserine / Q000494:pharmacology; D002478:Cells, Cultured; D005347:Fibroblasts / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D007041:Hypoxanthine Phosphoribosyltransferase / Q000502:physiology; D010428:Pentosephosphates / Q000502:physiology*; D010754:Phosphoribosyl Pyrophosphate / Q000096:biosynthesis / Q000502:physiology*; D011685:Purine Nucleotides / Q000378:metabolism*; D011687:Purines / Q000096:biosynthesis*; D012268:Ribose-Phosphate Pyrophosphokinase / Q000502:physiology
chemical_list: D010428:Pentosephosphates; D011685:Purine Nucleotides; D011687:Purines; D005973:Glutamine; D010754:Phosphoribosyl Pyrophosphate; D001377:Azaserine; D007041:Hypoxanthine Phosphoribosyltransferase; D012268:Ribose-Phosphate Pyrophosphokinase