PURIFICATION OF URIDINE DIPHOSPHATE-GALACTOSE-GLUCOSYL CERAMIDE, BETA-1-4 GALACTOSYLTRANSFERASE FROM HUMAN KIDNEY

A galactosyltransferase that transfers galactose from UDP-galactose to glucosylceramide was purified 440-fold to apparent homogeneity from n ormal human kidney "buffy coat" preparation employing detergent extrac tion, ultrafiltration, and Sepharose Q column chromatography. On reduc ing and nonreducing gels, the enzyme resolved into two bands with appa rent molecular weights on the order of 60,000 and 58,000, respectively . The activity of the enzyme was also associated with these two bands following separation on polyacrylamide gels. Analytical isoelectric fo cusing revealed that the pI of this enzyme is approximately 4.55. Prod uct characterization and substrate specificity studies employing chrom atography, enzymatic digestion with various glycosidases, and use of a variety of glycosphingolipid substrates revealed that the major produ ct synthesized by this enzyme was Cer1-1-beta-Glc4-1Gal, and Cer1-1-be ta-Glc was the preferred substrate. Digestion of the 60- and 58-kDa pr oteins with Staphylococcus aureus (V-8) protease revealed at least six peptides having identical electrophoretic migration. This finding sug gests that the two proteins may be related to each other. Western immu noblot assays revealed that the antibody against UDP-galactose:GlcCer, beta-1-4 galactosyltransferase (GalT-2) but not galactosyltransferase UDP-Gal:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyltrans ferase (EC 2.4.1.38) (B-GT) immunoprecipitated (recognized) the kidney GalT-2. In contrast, antibody against B-GT did not immunoprecipitate GalT-2. Thus our data indicate that GalT-2 and B-GT are two distinct e nzymes. The availability of the enzyme GalT-2 and corresponding antibo dy will allow functional studies in the near future.