Transport and countertransport of thymidine in ATP depleted and thymidine kinase-deficient Novikoff rat hepatoma and mouse L cells: evidence of a high Km facilitated diffusion system with wide nucleoside specificity.

Incubation of cultured Novikoff rat hepatoma and mouse L cells in a glucose-free basal medium containing 5 mM KCN and 5 mM iodoacetate for about 10 minutes resulted in a complete depletion of the cells of ATP. ATP-depleted wild type cells or thymidine kinase-deficient sublines of Novikoff or L cells took up thymidine rapidly from the medium without concentrating it intracellularly, and exhibited countertransport of thymidine. Thus uptake was by facilitated diffusion. This transport system differs from the substrate-specific, low-Km (0.5 muM] thymidine transport system previously described for various types of cultured cells in that it exhibits an at least 100-fold higher Km and transports equally well various ribo- and deoxyribonucleosides. The results suggest that the rate-limiting step in thymidine incorporation into the nucleotide pool by wild type cells is phosphorylation rather than transport, or that the cells possess two transport systems, a facilitated diffusion system with low substrate specificity and a second system which involves substrate phosphorylation by thymidine kinase.