Cell cycle related studies on thymidine kinase and its isoenzymes in Ehrlich ascites tumours

Thymidine kinase (TK) activity was measured in relation to the cell cycle of in vivo growing ascites tumour cells. The cells were synchronized by means of centrifugal elutriation and the cell cycle composition of the cell fractions was determined by flow cytometry. TK activity was low in G1, increased during S phase and declined in G2. A half-life of TK activity of about 45 min was found throughout the cell cycle. Four isoenzymes at pI values of 4.1, 5.3, 6.9 and 8.3, denoted as isoenzymes 1-4, were identified using isoelectric focusing. Isoenzymes 3 and 4 were responsible for the profound cell cycle related changes in the TK activity. Corresponding isoenzymes were also found in the fetal mouse liver. In the adult mouse liver isoenzyme 2 was the dominating isoenzyme. The half-life of the isoenzymes was in the same range as for the total TK activity. We conclude that the low TK activity in G1 is due to degradation of the enzyme in G2 at a normal rate combined with an arrest in the synthesis of TK. We also conclude that isoenzyme 4 and the intermediate isoenzyme 3, which had earlier been suggested to be a mitochondrial form of TK, in fact represent cytoplasmatic forms of TK. According to cell cycle and pI studies, isoenzyme 2 belongs to the mitochondrial form. Studies with various phosphor donors and specific substrates, however, indicate that it also contains a cytoplasmic component.