13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin.

As a prelude to studies using bovine N-acetylglucosaminide-beta-(1 leads to 4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched D-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of D-galactose from UDP-D-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-D-glucosyl group and exhibits selectivity towards saccharide chains containing D-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-D-glucose and D-mannose in the carbohydrate chains of hen ovalbumin. Uniformly 13C-enriched D-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The 13C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of D-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to D-galactosyl groups attached to 2-acetamido-2-deoxy-D-glucose respectively linked beta-(1 leads to 4) and beta-(1 leads to 2), to D-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of D-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-D-glucosyl group to the penultimate D-mannosyl residue.