Isolation, purification and partial characterization of galactokinase from Chinese hamster liver

1. Galactokinase was purified from Chinese hamster liver. The purification process consisted of an initial biphasic partition separation followed by column chromatography on DEAE-cellulose. DEAE-Sephadex, Sephacryl S-200 and hydroxyapatite. 2. The enzyme was stabilized during the purification procedure by the inclusion of 10% glycerol, 1 mM phosphate and 20 mM beta-mercaptoethanol in all the buffer solutions. 3. Chromatography on hydroxyapatite separated two forms of galactokinase, one of which was purified to homogeneity using gel electrophoresis. 4. The purified galactokinase has a mol. wt of approximately 60,000 as determined by SDS gel electrophoresis and molecular sieving column chromatography. 5. The pH optimum is 7.4 and the Km for galactose is 1.16 x 10(-4) M.