The use of galactosyltransferase to probe nitrocellulose-immobilized glycoproteins for nonreducing terminal N-acetylglucosamine residues.
We report the use of UDPgalactose:N-acetyl-D-glucosaminyl-glycopeptide 4-beta-D-galactosyl-transferase (EC 2.4.1.38), purified from bovine milk, to detect nonreducing terminal N-acetylglucosamine residues on glycoproteins immobilized on nitrocellulose by electrophoretic transfer from sodium dodecyl sulfate-polyacrylamide gels. Soluble galactosyltransferase incorporates radiolabeled galactose from the substrate UDP-[6-3H]galactose into the appropriate immobilized acceptor with high specificity. Incorporation is proportional to substrate amount and is saturable with time. The kinetics of labeling are independent of substrate amount. Half-maximal incorporation occurs by 4 h and saturation occurs by 16 h. We have used galactosyltransferase as a probe (i) to verify the presence of nonreducing terminal N-acetylglucosamine residues in bovine rod outer segment membrane rhodopsin and in several glycoproteins in F9 murine teratocarcinoma cells and (ii) to detect previously reported endo-beta-N-acetylglucosaminidase activity in a commercial preparation of endoglycosidase F.
D000117:Acetylglucosamine / Q000032:analysis*; D000118:Acetylglucosaminidase / Q000032:analysis; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003101:Collodion; D005700:Galactosyltransferases; D005944:Glucosamine / Q000031:analogs & derivatives*; D006023:Glycoproteins / Q000032:analysis*; D006026:Glycoside Hydrolases / Q000032:analysis; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D051379:Mice; D008892:Milk / Q000201:enzymology; D009363:Neoplasm Proteins / Q000032:analysis; D010084:Oxidation-Reduction; D012243:Rhodopsin / Q000032:analysis; D012995:Solubility; D013724:Teratoma
D006023:Glycoproteins; D009363:Neoplasm Proteins; D003101:Collodion; D012243:Rhodopsin; D005700:Galactosyltransferases; D006026:Glycoside Hydrolases; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D005944:Glucosamine; D000117:Acetylglucosamine