[Isolation and properties or uridine kinase from Zajdela hepatoma cells].

Uridine kinase (ATP: uridine-5-phosphotransferase, EC 2.7.1.48) was isolated from cytosol of rat Zajdela ascite hepatoma cells by fractionation with ammonium sulfate and gel-filtration on Sephadex G-200. The enzyme has a pH optimum of 7.2 - 7.8; Km for uridine is 4.8 . 10(-5) M, that for ATP - 1.9 . 10(-4) M. The optimal ratio of ATP of Mg2+ is 2.6. The enzyme activity is inhibited by end products of pyrimidine biosynthesis with Ki for CTP of 6.0 . 10(-4) M and for UTP of 1.2 . 10(-3) M. The Ki values for uridine competitive analogs, i. e. 6-azauridine, 5-bromuridine and 5-azacytidine are equal to 4.0 . 10(-4) M, 1.5 . 10(-3) M and 2.5 . 10(-3) M, respectively. Further purification of the enzyme on Sepharose 4B allowed to obtain the most active, although heterogeneous fractions purified 86-fold, with specific activity of 11.2 mkmole/hour per mg of protein. Using electrofocusing, uridine kinase was found to consist of two major and one minor active fractions with pH of 6.2, 6.7 and 6.35, respectively. Chromatography on DEAE-cellulose DE-32 resulted in two major active fractions of the enzyme, differing in thermal stability and inhibition by CTP. It may be concluded that Zajdela ascite hepatoma cells contain at least two isoforms of uridine kinase.