Characterization of the kinase(s) involved in the phosphorylation of - and -2'-deoxythioguanosine.

Abstract

Studies were made on the phosphorylation of α- and β-2′-deoxythioguanosine (α- and β-TGdR) in crude extracts of several mouse tumors, normal mouse tissues, calf thymus, a series of human leukemic cell samples, and some solid human tumors. In general the rate of phosphorylation was higher for β-TGdR than for α-TGdR. Deoxycytidine (CdR), 1-β-arabinofuranosylcytosine (ara-C), and deoxyguanosine (GdR) were inhibitory to the phosphorylation of α- and β-TGdR. Their effectiveness was greatest for, in decreasing order, CdR > ara-C > GdR. Human bone marrows did not appreciably phosphorylate α-TGdR, although some tumors did show such activity. Optimum conditions for these phosphorylations required an adenosine triphosphate-generating system and Mg++, and the reaction was somewhat inhibited by dithioerythritol. Purification of the kinase activity from calf thymus and from human tumor cells, by a method designed to isolate CdR kinase, yielded preparations with 220-fold higher purity as measured by ability to phosphorylate β-TGdR. The purified preparations lost all ability to phosphorylate α-TGdR. Kinetic studies showed that CdR was noncompetitive. ara-C and GdR were competitive inhibitors of β-TGdR phosphorylation. The Km value for β-TGdR was 1.8 × 10-4 m and the Ki's for GdR, CdR, and ara-C were 2.41 × 10-6, 3.60 × 10-8, and 1.69 × 10-6m, respectively. Extracts of a cell line completely unable to phosphorylate ara-C (L1210/ara-C) were able to phosphorylate both α- and β-TGdR at relatively high rates, and these substrates were not inhibited by ara-C. β-TGdR apparently can be phosphorylated by the CdR-GdR kinase of tissues. However, another kinase is apparently present in some tissues and can phosphorylate both α- and β-TGdR.