Abstract
Partial separation and purification of the kinases phosphorylating 2'-deoxycytidine (EC 2.7.1.74) (D-K), pyrimidine nucleoside-5'-monophosphate (EC 2.7.4.14) (PM-K) and nucleoside-5'-diphosphate (EC 2.7.4.6) (NDP-K) were achieved by high performance liquid chromatography on Micro Pak TSK-gel 3000 SW columns. Using the standard conditions for all three kinases investigated, the following observations were made: a comparison of the D-K activities using deoxycytidine (dCyd) or l-β- d-arabinofuranosylcytosine (ara-C) as substrate in peak fractions derived from homogenates of murine neoplasms P815, either sensitive (P815) or resistant to ara-C (P815/ara-C) or resistant to 6-thioguanine (P815/TG), revealed comparable specific activities for dCyd and somewhat lower specific activities for ara-C in fractions derived from P815 and P815/TG cells, while substantially reduced specific activities were observed for both substrates in fractions derived from P815/ara-C cells. The 5'-monophosphate of ara-C (ara-CMP) exhibited a higher specific activity than 2'-deoxycytidine-5'-monophosphate (dCMP) in peak fractions with PM-K activity derived from all three cell lines. The 5'-diphosphate of dCyd (dCDP) was phosphorylated to comparable extents by peak fractions with NDP-K activity derived from all three cell lines. The 5'-diphosphate of ara-C (ara-CDP) is a substrate with specific activities comparable to dCDP in peak fractions with NDP-K activity derived from P815/ara-C and P815/TG cell lines, but with somewhat lower specific activities than the dCDP substrate in P815-derived fractions. The ratios of total enzyme activities recovered after injection of a crude P815 cell homogenate were: 1:260:20,000 for D-K (dCyd as substrate), PM-K (dCMP as substrate) and NDP-K (CDP as substrate); their yield was 100% for D-K and NDP-K, and 40% for PM-K activities. The purification achieved ranged from 5 to 33 times, with substantial reductions in the number of bands observed in disc electrophoresis when compared with those in the crude extracts. Experiments evaluating the inhibitory activity of ara-C and its 5′-mono-, di- and triphosphates upon the phosphorylation by these semipurified kinases of dCR and its 5′-phosphates revealed the possibility that ara-CDP and, less so, ara-CTP effectively inhibit the phosphorylation of dCDP to dCTP in the de novo biosynthesis of the latter and, thus, provide insufficient amounts of dCTP for DNA synthesis.
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