Phosphorylation of selected substrates by semipurified cytoplasmic kinases of three P815 murine leukemias exhibiting collateral sensitivity (P815/TG), sensitivity (P815) or resistance (P815/ARA-C) to 1-β-d-arabinofuranosylcytosine

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