Decline of Thymidine Kinase Activity in Stationary Phase Mouse Fibroblast Cells.

It has become increasingly apparent that t,hymidine kinase (ATP: thymidine 5’-phosphotransferase, EC 2.7.1.21) is a site of specific regulatory control of the levels of uracil and thymine deoxyribonucleotides within cells (1, 2). The synthesis of thymidine kinase is subject to control by repression (3, 4) and the activity of the enzyme can be strikingly inhibited by thymidine triphosphate (end product inhibition) (1, 5-9). Both pox and herpes simplex viruses induce the synthesis of thymidine kinase in animal cell cultures, suggesting that the enzyme has survival value for these viruses (3,4, 10-15). During the mitotic cycle of Lilium longijlorum, thymidine kinase activity appears at a precisely defined time preceding deoxyribonucleic acid synthesis and persists for more than 24 hours but subsequently disappears (16, 17). Tissues with mitotic activity such as wheat and corn seedlings after 2 days of germination show high enzyme activity although no activity is observed in wheat embryos or in corn germinated for 25 hours (18). A marked rise in kinase activit’y in liver tissue occurs at about the time DNA synthesis begins (19, 20) and x-irradiation in viva, before or shortly after partial hepatectomy, not only blocks DNA synthesis, but thymidine kinase does not appear at the expected time (21). The present study and experiments by Weissman, Smellie, and Paul (19) show that thymidine kinase activity increases rapidly during the accelerated growth phase of mouse fibroblast cells (strain L), but declines before the completion of cell growth. Thymidine kinase activity also diminishes rapidly when protein synthesis is inhibited by treating cells with puromycin or p-fluorophenylalanine. In exploring the mechanisms underlying this enzyme decline, three hypotheses ha,ve been considered: (a) thymidine kinase is unstable. Hence, when new enzyme synthesis is arrested and when substrate concentrations are low, the pre-existing enzyme undergoes conformational changes to an inactive form (22); (b) inhibitors or enzymes (or both) which interfere with the thymidine kinase assay increase in cells whose growth or protein synthesis has been inhibited; and (c) thymidine kinase “leaks” from stationary phase tissue culture cells. The validity of the first hypothesis is supported by the observations that preparations of partially purified thymidine kinase are very labile when incubated in vitro at 38” in Tris buffer at pH 7 to 8 in the absence of substrates, and that thymidine, adenosine triphosphate, or thymidine triphosphate protect the enzyme against thermal inactivation (23, 24). This study