Regulation of purine nucleotide synthesis: Effects of inosine on normal and hypoxantine-guanine phosphoribosyltransferase-deficient fibroblasts

Abstract

Incubation of normal and hypoxanthine-guanine phosphoribosyltransferase-deficient (mutant) human fibroblasts with inosine results in increased intracellular concentration of 5-phosphoribosyl 1-pyrophosphate ( PP-ribose- P). The magnitude of this increase is dependent on the concentration of the nucleoside and results from donation of the ribose moiety of inosine to the ribosyl phosphate moiety of PP-ribose- P through ribose phosphate intermediates. During incubation, rates of purine nucleotide synthesis de novo, estimated by incorporation of [ 14C] formate into formylglycinamide ribotide, are diminished in both normal and mutant cells: 5 mM inosine inhibits purine syntheiss by 60–80% in normal cells and 2–20% in hypoxanthine-guanine phosphoribosyltransferase-deficient cells. The rates of purine synthesis in both normal and mutant cells are increased, however, during incubation with methylene blue at concentrations (50–100 μM) which result in more modest increases in ribose 5-phosphate and PP-ribose- P concentrations than are observed with inosine. Saturation of the PP-ribose- P amidotransferase reaction by PP-ribose- P does not appear, therefore, to explain the failure of increased PP-ribose- P concentration to stimulate the rate of purine synthesis in either type of fibroblast during incubation with inosine. Although the dissociation between PP-ribose- P concentration and the rate of purine nucleotide synthesis in normal fibroblasts incubated with inosine may be explained at least in part by an accompanying increase in intracellular concentrations of purine nucleotide feedback inhibitors, purine nucleotide concentrations are unchanged in mutant cells during incubation with inosine; these cells, in addition, show minimal (<3% of normal) incorporation of labeled hypoxanthine or the hypoxanthine moiety of inosine into purine nucleotides. The effect of inosine on purine synthesis de novo in hypoxanthine-guanine phosphoribosyltransferasedeficient fibroblasts is not explained in full by consideration of the concentrations of purine nucleotides and of PP-ribose- P, the factors frequently invoked as antagonistic regulators controlling the rate of this process.