Properties of deoxythymidine kinase partially purified from noninfected and virus-infected mouse fibroblast cells

Abstract

Deoxythymidine kinase, partially purified from noninfected LM mouse fibroblast cells, was rapidly inactivated when incubated in the absence of substrates at 38°C. The half-life of the enzyme was 29–43 minutes whether prepared from LM cells in the logarithmic phase of growth, the phase of negative growth acceleration, the stationary phase, or from LM cells which had been incubated for 24 hours with 50 μg deoxythymidine per milliliter prior to harvest. The deoxythymidine kinase induced in LM (TK −) cells by herpes simplex virus was also very unstable. In contrast, deoxythymidine kinase prepared from vaccinia-infected LM or LM (TK −) cells was considerably more stable with a half-life greater than 300 minutes. The addition of ATP (15 m M), dTTP (0.025 m M), or deoxythymidine (0.05 to 0.1 m M) significantly protected the LM cell enzyme against thermal inactivation. However, charcoal treatment of the vaccinia-induced enzyme did not decrease the stability of that enzyme. Immunological differences and differences in apparent Michaelis constants for deoxyuridine were also observed between the vaccinia-induced deoxythymidine kinase and the enzyme from noninfected LM cells. However, the temperature characteristics of the reactions, calculated from the V m values at 38° and 30°, were similar, and these enzymes were nearly equal in susceptibility to inhibition by dTTP. With either enzyme, the phosphorylation of deoxyuridine was competitively inhibited by fluorodeoxyuridine, bromodeoxyuridine, iododeoxyuridine, or deoxythymidine. The phosphorylation of deoxythymidine was inhibited by low concentrations of bromodeoxyuridine or iododeoxyuridine, and by high concentrations of deoxyuridine or fluorodeoxyuridine.