Purification and Properties of Galactokinase from Human Red Blood Cells

Abstract 1. Galactokinase from normal human red blood cells was purified 3300- to 3900-fold by ion exchange chromatography, ammonium sulfate precipitation, and dextran gel filtration with a yield of 14 to 19%. Analytical disc electrophoresis of the purified enzyme revealed a single protein band. 2. The Km of purified galactokinase for galactose is 100 to 150 µm; for ATP, 200 to 500 µm. The pH optimum for enzymic phosphorylation of galactose is in the range of pH 7.7 to 7.9. In electrofocusing experiments the isoelectric point of galactokinase was found to be 5.7. The molecular weight of galactokinase was determined to be 53,000 to 57,000 by means of dextran gel chromatography and sodium dodecyl sulfatepolyacrylamide electrophoresis. The enzyme seems to have a dimeric structure consisting of monomers with a molecular weight of 25,000 to 27,000. 3. The biochemical properties of galactokinase from human red blood cells are similar to the characteristics of galactokinase from other mammalian tissues, but differ considerably from galactokinase of yeast and Escherichia coli.