Purification and Properties of Thymidine Monophosphate Kinase from Mouse Hepatoma

Thymidylate kinase (deoxythymidine 5'-monophosphate kinase) has been purified some 7,500-fold from extracts of mouse ascites hepatoma. The stability of the enzyme was maintained during purification by the constant presence of substrate (dTMP) and 2-mercaptoethanol and by the elimination of substrate-destroying phosphatase activity. Thiol was essential for the protective effect of dTMP. Also effective as enzyme stabilizers were dTDP and dTTP. The purified enzyme catalyzed the reversible reaction: dTMP + ATP (Mg++)/⇌ dTDP + ADP Interfering enzyme activities were not detectable, although the purified enzyme was not entirely physically homogeneous. An approximation of the molecular size of the principal component was of the order of 35,000. It has been shown that the dTMP kinase of mouse hepatoma is subject to an extraordinary product inhibition by ADP (Km:Ki ratio of 5). The inhibition appeared to be a mixed type in which binding of ADP to the enzyme tends to prevent dissociation of the enzyme complex to its products and, at the same time, interferes with the binding of ATP. The potency of the ADP inhibition and its wide ranging effect on dTMP kinase activity have been discussed as a basis for enzyme regulation.