Characterization and specific assay for a galactoside β‐3‐galactosyltransferase of human kidney

Two galactosyltransferases identified as UDP-galactose:lactose (lactosylceramide) alpha-4- and beta-3-galactosyltransferases [Bailly P. et al. (1986) Biochem. Biophys. Res. Commun. 141, 84-91] have been characterized in human kidney microsomes. Using methyl beta-D-galactoside as acceptor substrate, we have determined the experimental conditions (pH 5.0, 4 mM Cd2+) in which only the beta-3-galactosyltransferase activity is detectable. The reaction product has been characterized by chemical methods and glycosidase studies. Under these experimental conditions, some of the enzyme properties have been further investigated. Apparent Km values are for UDP-galactose, 0.170 mM; for lactose, 242 mM; and for lactosylceramide, 2.5 mM. Acceptor specificity studies suggest that the beta-3-galactosyltransferase is specific for terminal Gal beta 1-4Glc(NAc) residues and responsible for elongation of oligosaccharide chains in glycolipids. Competition studies with lactose and N-acetylgalactosamine as acceptor substrates indicate that the transferase described here can be distinguished from the UDP-galactose:2-acetamide-2-deoxy-D-galactose beta-3-galactosyltransferase and therefore represents a novel enzyme capable of synthesizing unusual carbohydrate structures similar to those which accumulate in certain neurological diseases.