Enzyme kinetics of thymidine kinase isoenzymes of Ehrlich ascites tumour.

Three isoenzymes of thymidine kinase (TK) identified on Ehrlich ascites tumour corresponding to pl values of 5.3, and 6.9 and 8.3 were studied with respect to their kinetic properties. The isoenzymes were separated by thin-layer isoelectric focusing and measured in the presence of thymidine (TdR) as substrate, adenosinetriphosphate (ATP) as phosphor donor and deoxythymidinetriphosphate (dTTP) as inhibitor. Exponentially growing cells and growth inhibited cells were used with high proportions of the isoenzymes at pl values of 6.9 and 8.3, and 5.3 respectively. The concentrations of the former were further enhanced by the use of S-phase cells isolated by elutriator centrifugation. The isoenzymes were identified as TK1-onc, earlier found in human leukemia cells. In order to confirm the existence of TK1, particular at pl value 5.3, the ability of the isoenzymes to use different phosphor donors (ATP and uridinetriphosphate (UTP)) and substrates (TdR and deoxycytidine (dCdR) was also studied. These results showed that the isoenzymes at pl 6.9 and 8.3 correspond to TK1, while the isoenzyme at pl 5.3 is heterogeneous. Further, high resolution isoelectric focusing confirmed the existence of two isoenzymes at pl values of 5.1 and 5.3, representing mitochondrial (TK2) and cytoplasmic (TK1) isoenzymes.