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.