Purine and pyrimidine transport and phosphoribosylation and their interaction in overall uptake by cultured mammalian cells. A re-evaluation.

The zero-trans uptake of purines and pyrimidines was measured in suspensions of Novikoff rat hepatoma, mouse L, P388 mouse leukemia, and Chinese hamster ovary cells by a rapid kinetic technique which allows the determination of uptake time points in intervals as short as 1.5 s. Kinetic parameters for purine/pyrimidine transport were determined by measuring substrate influx into cells in which substrate conversion to nucleotides was negligible either due to lack of the appropriate enzymes or to depletion of the cells of ATP (5'-phosphoribosylpyrophosphate), and by computer fitting exact, integrated rate equations derived for various carrier-mediated transport models directly to zero-trans influx data. The results indicate that different carriers function in the transport of hypoxanthine/guanine, adenine, and uracil with substrate:carrier association constants (K) at 24 degrees C of 300 to 400 muM, 2 to 3 mM, and about 14 mM, respectively, for Novikoff cells. K and Vmax for hypoxanthine transport by L and P388 cells are similar to those for Novikoff cells, but the transport capacity of Chinese hamster ovary cells is much lower and K = 1500 muM. All transport systems are completely symmetrical. Hypoxanthine transport is so rapid that an intracellular concentration of free hypoxanthine (90%) close to that in the medium is attained within 20 to 50 s of incubation at 24 degrees C, at least at extracellular concentrations below K. In cells in which conversion to nucleotides is not blocked free hypoxanthine accumulates intracellularly to steady state levels with equal rapidity and thereafter the rate of hypoxanthine uptake into total cell material is strictly a function of the rate of phosphoribosylation. The low Km systems for hypoxanthine (1 to 9 muM) and adenine (0.2 to 40 muM) uptake detected previously in many types of cells reflect the substrate saturation of the respective phosphoribosyltransferases rather than of the transport system.