Activation of hepatic glycogen phosphorylase in anoxic liver [proceedings].

was centrifuged at 20000g for 20min and the supernatant filtered through a column (3.5cmx0.85cm) of Sephadex G-25 (fine grade) to remove all molecules of mol.wt. less than 5000. Cell extracts were incubated at 37°C with [3H]thymidine, ATP and MgClz and the phosphorylated products collected on DE-81 (DEAE-cellulose) filters to be assayed for radioactivity. The assay was shown t o be linear with added extract protein and with time of incubation. Hypoxanthine/guanine phosphoribosyltransferase and adenine phosphoribosyltransferase activities were determined in the same cell extracts by the methods previously published (Green & Martin, 1973). Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were determined by the method of Glock & McLean (1953). We found that thymidine kinase activity is increased 2-3-fold by physiological concentrations of oestrogen or insulin and it is decreased by hormones and anti-hormones that inhibit cell division. The dose-response curve for the increase in enzyme activity caused by oestradiol-17/? is the same as that for the increase in [3H]thymidine incorporation. The effect on thymidine kinase appears t o be relatively specific, since the activities of the purine-salvage-pathway enzymes hypoxanthine/guanine phosphoribosyltransferase and adenine phosphori bosyltransferase and of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase are unaffected by hormone treatment. The effect of oestrogen on [3H]thymidine incorporation cannot wholly be explained by its stimulation of thymidine kinase activity, however. Oestrogen does stimulate division of MCF-7 cells as measured by increase in cell number per dish, although the effect does not become apparent until after 6 days in the presence of the hormone. Stimulation of [3H]thymidine incorporation does not occur before24h after the addition of oestradiol, but the increase in thymidine kinase activity is significant by 12 h. This latter result would seem to indicate that thymidine incorporation is regulated at some step beyond thymidine kinase so that this regulatory point is not affected by oestradiol until later times in the response. Thymidine kinase is a pyrimidine ‘salvage’ enzyme and as such is not on the direct pathway for synthesis of pyrimidine nucleotides de novo. In view of this it is difficult t o see why it should be subject to hormonal regulation; nevertheless, in these cells a t least, it seems to be induced by oestradiol and the increase in its activity is one of the earliest responses to the hormone.