Transport of hypoxanthine in fibroblasts with normal and mutant hypoxanthine-guanine phosphoribosyltransferase

Abstract

Hypoxanthine transport has been studied in cultured human fibroblasts with normal and mutant H-G PRT. Transport is dependent on cell density, the activity of H-G PRT, and de novo purine synthesis. Transport is decreased in control cultures at high density and in cell strains with a mutant H-G PRT. Cell strains from patients with the Lesch-Nyhan syndrome with almost no H-G PRT activity demonstrate almost no hypoxanthine transport. Preincubation of cells with aminopterin, an inhibitor of de novo purine synthesis, increased hypoxanthine transport in actively growing control cell strains and in two mutant H-G PRT cell strains from patients with hyperuricemia, but had little effect on cells from patients with the Lesch-Nyhan syndrome. Aminopterin affected the kinetics of transport in the two mutant cell strains from patients with hyperuricemia, but did not affect the kinetics of transport in control cells. These combined results suggest that intracellular H-G PRT is associated with hypoxanthine transport in human fibroblasts. Isolated, washed red cell membrane ghosts do not demonstrate H-G PRT activity. Red cell membranes do not preferentially bind enough hypoxanthine in equilibrium dialysis experiments to suggest the presence of a membrane carrier protein. Oxypurine levels are elevated in cell strains from patients within H-G PRT mutations. Studies with aminopterin suggest that the H-G PRT mutation and not the accelerated purine synthesis in these cells is primarily responsible for this elevation.