Pyrimidine salvage enzymes in human tonsil lymphocytes: II. Purification and properties of deoxycytidine kinase.
Human tonsillar lymphocytes incorporate about 7-10 times more [3H]-deoxy-thymidine than [3H]-deoxycytidine at the same extracellular nucleoside concentration, and both incorporations could be inhibited by 10(-5) M arabinosyl-cytosine to 99%. On the other hand, the crude extract of these cells had about 10 times more deoxycytidine kinase than deoxythymidine kinase specific activity, as it was reported previously as well. Therefore, the differences in the labelling of these cells by [3H]-deoxycytidine and [3H]-thymidine cannot be simple due to the different levels of phosphorylating enzymes in the cells. The deoxycytidine kinase of human tonsillar lymphocytes was purified 38.6-fold by DEAE-Sephadex chromatography, and some kinetic and regulatory properties of the purified enzyme were investigated. Deoxycytidine kinase was eluted as a single peak from DEAE-Sephadex column and also from Sephadex G-100 column indicating a molecular weight of about 60 000; no isoenzymes could be detected. During the purification procedure an increase of the enzyme activity was observed suggesting the inhibition of the enzyme in the cells. An apparent Km of 13 microM for deoxycytidine and Ki of 55 microM for arabinosyl-cytosine were found for the tonsillar deoxycytidine kinase. The enzyme was strongly inhibited by dCTP, but not by the other deoxyribonucleoside triphosphates.
D002474:Cell-Free System; D002845:Chromatography / Q000379:methods; D003561:Cytarabine / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D003845:Deoxycytosine Nucleotides / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000201:enzymology*; D008970:Molecular Weight; D014066:Palatine Tonsil / Q000166:cytology; D010770:Phosphotransferases / Q000378:metabolism*
D003845:Deoxycytosine Nucleotides; D003561:Cytarabine; C024107:2'-deoxycytidine 5'-triphosphate; D010770:Phosphotransferases; D003842:Deoxycytidine Kinase