Relationship between thymidine transport and phosphorylation in Novikoff rat hepatoma cells as analyzed by a rapid sampling technique

Incorporation of thymidine into Novikoff rat hepatoma cells was analyzed with a rapid sampling technique which allowed collection of 12 time points in 20 sec. Transport was studied in the absence of metabolism by using either ATP-depleted cells or a thymidine kinase negative subline. Transport was a rapid, saturable, nonconcentrative process with a K_m of about 85 μM. The intracellular thymidine pool was also rapidly labeled in cells which phosphorylated thymidine, so that a group translocation process involving thymidine kinase can be ruled out. Under all conditions examined, phosphorylation, not the transport, of thymidine was the rate-determining step in its incorporation into the acid-soluble pool. Estimation of transport rates from total incorporation into cells which phosphorylate the substrate is invalid in this cell system and must be questioned in all instances.