dTMP synthetase levels in parenchymal cells

The effect of methotrexate (MTX) on thymidylate syn thetase activity during liver regeneration was examined with parenchymal cells isolated 22 and 44 hr after partial hepatectomy and cultured as a monolayer. The synthetase activity in these cells decreased with a half-life of 18 to 24 hr. but if MTX (1.5 x l0' to 1.5 x l0@ M) was present in the culture media, this decline could be delayed for at least 48 hr. In contrast, thymidine kinase activity decreased at a rate which was unaffected by MTX. Dihydrofolate reduc tase was inhibited at all concentrations of MTX used to block the decrease in synthetase activity. Folic acid at l0@ M, although less effective than MTX, also delayed the decrease in synthetase activity. The addition of cyclohexi mide, puromycin, or actinomycin D to the culture media did not alter the response of the synthetase to MTX. The latter studies, coupled with those indicating that the rapid loss of synthetase activity in crude extracts could be prevented by MTX or, more effectively, by MTX plus deoxyuridine 5'-monophosphate, suggest that the primary effect of MTX on thymidylate synthetase in vivo is that of enzyme stabilization. Similar stablizing effects were obtained in liver cell extracts with l0@ M deoxyuridine 5'-monophos phate in combination with 10 @ M folate or l0 M dihydrofolate.