Glycoprotein biosynthesis: Purification and characterization of a glycoprotein: Galactosyl transferase from Ehrlich ascites tumor cell membranes

Abstract

An enzyme was found in the smooth membranes (Golgi apparatus) of Ehrlich ascites tumor cells which transfers galactose from UDP-galactose to specific glycoprotein receptors. It was purified 46-fold by gel filtration and DEAE-cellulose chromatography following solubilization with Triton X-100. The enzyme shows an absolute requirement for Mn 2+, for which the optimal concentration is 0.02 m. The pH optimum lies between 6.4 and 7.2; a temperature optimum of 24 ° was found. K m values with respect to UDP-galactose and receptor were 5 × 10 −6 and 3.3 × 10 −4 m, respectively. The transfer of galactose to receptor is highly specific, recognizing N-acetylglucosamine either as the monosaccharide or in glycosidic linkage as part of the glycoprotein to give the 4- O-β-galactopyranosyl- N-acetylglucosaminne linkage. The receptor was prepared from α 1-glycoprotein in which sialic acid and 80% of its galactose residues had been removed by successive treatment with neuraminidase and β-galactosidase. It was found that 80% of the theoretical receptor sites (galactose removed) of the α 1-glycoprotein receptor would react after prolonged incubation. The glycoprotein:galactosyl transferase was localized in the smooth internal membranes following fractionation of the Ehrlich ascites tumor membranes on a sucrose gradient. This enzyme is intimately bound to these membranes but is dissociated and rendered soluble with nonionic detergents or phospholipase A, agents which disrupt the nonpolar bonding in membranes. Addition of Triton X-100 to the alcohol-washed enzyme increased the enzyme activity over 2-fold.