CMP kinase activity

Abstract: 1-β-d-Arabinofuranosylcytosine (ara-C) was phosphorylated by three successive enzymatic reactions to 1-β-d-arabinofuranosylcytosine 5′-triphosphate via the intermediate formation of 1-β-d-arabinofuranosyl-CMP and 1-β-d-arabinofuranosyl-CDP. The enzymes concerned were deoxycytidine (CdR) kinase, deoxycytidine monophosphokinase, and nucleoside diphosphokinase of calf thymus. In the initial two successive reactions, Km values for ara-C and 1-β-d-arabinofuranosyl-CMP were intermediate between those of the corresponding deoxyribosidic and ribosidic derivatives. Calf thymus extract contained a cytidine kinase which was separable from the CdR kinase in question. The former enzyme catalyzed neither ara-C nor CdR, while the latter phosphorylated ara-C as well as CdR. In the second reaction, the addition of reduced or oxidized glutathione to the enzyme preparation effects the reaction rate in varying degrees depending on the phosphate acceptor used. By adjusting the ratio between concentrations of reduced and oxidized glutathione, it was possible to abolish the dCMP and 1-β-d-arabinofuranosyl-CMP kinase activities concomitantly without causing appreciable change in CMP kinase activity. These results taken together clearly indicate that ara-C and its phosphorylated derivatives are CdR analogs rather than cytidine analogs. In the last reaction, Km values of nucleoside diphosphokinases for 1-β-d-arabinofuranosyl-CDP, 5′-dCDP, and 5′-CDP were of the same order of magnitude (10-4 m).

Abstract: Summary Pyrimidine nucleoside monophosphate kinase [deoxycytidine monophosphate:adenosine triphosphate (dCMP:ATP) phosphotransferase, EC 27414] has been purified from rat Novikoff ascites hepatoma and rat liver, each to a single major band appearing on sodium dodecyl sulfate polyacrylamide gel electrophoresis Differences exist in regard to efficiency and regulation of enzymatic activities The Km values of the tumor kinase for cytidine monophosphate (CMP) (00053 ± 00008 mm) and dCMP (0715 ± 0068 mm) are approximately one-fourth the Km values of the rat liver kinase, for CMP (0030 ± 0007 mm) and dCMP (277 ± 039 mm) The tumor dCMP kinase exhibits a lower Km for ATP (0134 ± 0008 mm) than the rat liver kinase (068 ± 009 mm) Moreover, the dCMP:CMP kinase activity ratio for the tumor enzyme is 112, while that for the rat liver enzyme is 045 The uridine monophosphate:CMP kinase activity ratio for the tumor enzyme is 193, while that for the rat liver enzyme is 268 Lower concentrations of dithiothreitol are required for 50% reactivation of the tumor dCMP kinase (100 mm) and CMP kinase (010 mm) than rat liver dCMP kinase (220 mm) and CMP kinase (057 mm) Thus, the kinase from Novikoff hepatoma exhibits properties of increased efficiency and relaxed regulation of activity which render it more suitable for a tumor, in which active DNA synthesis is ongoing