Pyrimidine salvage enzymes in human tonsil lymphocytes. I. Separation and properties of thymidine kinase isoenzymes.
Two forms of deoxythymidine kinase were separated from human tonsillar lymphocytes on DEAE-Sephadex column (peak 1 and peak 2 isoenzymes) and identified by gel electrophoresis. Both isoenzymes were found in freshly prepared lymphocytes as well as in lymphocytes cultured in the presence or absence of phytohaemagglutinin, but freshly prepared and phytohaemagglutinin-stimulated cells contained higher activity of peak 1 isoenzyme than the cultured, non-stimulated ones. Kinetic properties and feed-back inhibition of the two separated isoenzymes were compared. The apparent Km values of isoenzyme 1 and 2 were 4 microM and 5 microM for thymidine and were 0.15 mM and 0.12 mM for ATP, respectively. Isoenzyme 1 was found to be more sensitive to heat denaturation at 55 degrees C, and to feedback inhibition by dTTP than isoenzyme 2. On the contrary, isoenzyme 1 was more resistant to dCTP inhibition than the other one. It is concluded that the isoenzyme 1 activity correlates with the synthesis of DNA, while the activity of isoenzyme 2 seems to be constant. The high activity of isoenzyme 1 in freshly isolated tonsil lymphocytes supports their active proliferation in the organ.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002845:Chromatography / Q000379:methods; D004586:Electrophoresis / Q000379:methods; D005246:Feedback; D006358:Hot Temperature; D006801:Humans; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology*; D014066:Palatine Tonsil / Q000166:cytology; D013937:Thymidine Kinase / Q000302:isolation & purification / Q000378:metabolism*
D007527:Isoenzymes; D000255:Adenosine Triphosphate; D013937:Thymidine Kinase