Properties of the thymidine transport system of chinese hamster ovary cells as probed by nitrobenzylthioinosine

Summary The transport of thymidine into Chinese hamster ovary cells grown in suspension culture was measured under conditions in which thymidine was not metabolized, namely, when cells had been depleted of ATP. The system transporting thymidine was saturable (K _m ^zt =70M), rapid (50% of transmembrane equilibrium level attained within 8 sec), and was apparently shared by other nucleosides, but not thymine or hypoxanthine. 6(4-nitrobenzyl]thio)-9--d-ribofuranosylpurine, nitrobenzylthioinosine, inhibited thymidine transport in a simple, noncompetitive fashion with an apparentK _i =1.0 nM (based on total concentration of inhibitor, which significantly overestimates that of free inhibitor). The rate of expression of inhibition was slow (t _1/2=17 sec) relative to the rate of association of thymidine with its transporter, and thymidine partially protected the transport system against inhibition by nitrobenzylthioinosine. The dissociation constant for the inhibitortransporter complex was estimated at about 0.1 nM, and the number of binding sites per cell at about 6×10⁴. HeLa, P388 murine leukemia, and mouse L cells were as sensitive to nitrobenzylthioinosine inhibition of thymidine transport as Chinese hamster ovary cells; Novikoff rat hepatoma cells were much less sensitive.