Purification of human galactokinase and evidence for its existence as a monomer form

Abstract

A procedure for preparing a highly purified galactokinase (ATP: d-galactose 1-phosphotransferase, EC 2.7.1.6) from human erythrocytes and placenta is described, involving DEAE-Sephacel, ammonium sulfate fractionation, gel-filtration and a subsequent chromatography step on Blue Sepharose CL-6B. The final chromatography step yields a homogeneous preparation of high specific activity. The subunit molecular weight was determined to be 38 000 for both placental and erythrocyte galactokinases. Both active preparations of the native enzyme eluted from a gel filtration column gave a molecular weight of 37 000–38 000, thus suggesting the enzyme to be present in monomeric form. The isoelectric points for both crude and their respective purified enzymes was determined to be at pH 5.7. This method for purifying human galactokinase from placenta and erythrocytes represents a significant improvement over that previously reported and contradicts past evidence for the enzyme existing as a dimer.