Selective inactivation of the deoxyadenosine phosphorylating activity of pure human deoxycytidine kinase: stabilization of different forms of the enzyme by substrates and biological detergents.
Deoxycytidine kinase, purified from human leukemic spleen to apparent homogeneity, is a multisubstrate enzyme that also phosphorylates purine deoxyribonucleosides [Bohman & Eriksson (1988) Biochemistry 27, 4258-4265]. In the present investigation we show that the stability and temperature dependence of dCyd kinase activity differed appreciably from the dAdo kinase activity of the same pure enzyme. Selective inactivation of dAdo activity was observed upon an incubation of the enzyme at both 4 and 37 degrees C. The half-life of dAdo activity at 4 degrees C increased from 36 to 84 h, when the protein concentration was increased by addition of bovine serum albumin. However, the half-life of dCyd activity increased from 72 h to more than 7 days under the same conditions. dCyd activity was stable for at least 6 h at 37 degrees C while the half-life of dAdo activity was 2 h. The presence of substrates like ATP, dTTP, or dAdo stabilized dAdo activity at both temperatures, and full maintenance of both activities at 37 degrees C was obtained by the addition of the zwitterionic detergent CHAPS. Furthermore, thermal inactivation of the dAdo activity occurred at a lower temperature (48 degrees C) as compared to the dCyd activity (54 degrees C). The presence of protease inhibitors had no effect on enzyme inactivation, nor was there a difference in the subunit structure of the selectively inactivated enzyme as compared to the fully active form, as revealed by size-exclusion chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)
Kierdaszuk
B
B
Department of Biochemistry I, Karolinska Institute, Stockholm, Sweden.
Eriksson
S
S
D002793:Cholic Acids / Q000494:pharmacology; D003839:Deoxyadenosines / Q000378:metabolism*; D003842:Deoxycytidine Kinase / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism*; D013696:Temperature
D002793:Cholic Acids; D003839:Deoxyadenosines; D003902:Detergents; D010770:Phosphotransferases; D003842:Deoxycytidine Kinase; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate