508 PHOSPHORIBOSYLPYROPHOSPHATE (PRPP) SYNTHESIS IN CULTURED HUMAN FIBROBLASTS

Abstract

An investigation of the regulation of purine metabolism is important in understanding the accelerated purine synthesis and abnormalities in PRPP metabolism that occur in the Lesch-Nyhan (LN) syndrome. PRPP levels in cultured human fibroblasts increased from 0.48±.11 nmoles/mg protein(±1SD)to 0.75±.15(p<.01)when hypoxanthine was removed from the medium. This is less than levels observed in LN cells(1-1.8). Without hypoxanthine, PRPP amidotransferase, and not hypoxanthine-guanine phosphoribosyltransferase is utilized to supply cellular purine needs. Two to three fold increase in PRPP synthetase occured in cells starved for purines by growth in medium lacking purines and containing aminopterin, an inhibitor of purine synthesis. Enzyme activity did not increase when the aggregation of PRPP synthetase was prevented by homogenization in a buffer containing 0.05mM ATP and lmM MgS04. These results suggest that the aggregation of PRPP synthetase has physiological significance in providing higher PRPP levels for de novo purine synthesis. ADP inhibition of PRPP synthetase was the same in control and purine starved cells. Purine levels and PRPP synthetase increased in a biphasic pattern following removal of thymidine inhibition of mitosis and at S phase of the cell cycle. Adjustment of PRPP levels by change in PRPP synthetase activity and aggregation is a sensitive cellular mechanism in the regulation of purine metabolism.