Structure of the Disaccharide Chain of Galactosyl‐ N ‐acetylgalactosaminyl‐protein Synthesized in vitro

In order to obtain a [ 14 C]galactosyl‐ N ‐acetylgalactosaminyl‐protein which would be useful as an acceptor in studies on the specificity of glycosyltransferases, a porcine submaxillary gland microsomal galactosyltransferase preparation was used for the galactosylation in vitro of N ‐acetylgalactos‐aminyl‐protein (desialylated ovine submaxillary mucin). The newly formed oligosaccharide unit was obtained as a reduced disaccharide after alkaline borohydride treatment of the [ 14 C]galactosyl‐ N ‐acetylgalactosaminyl‐protein product and as glycopeptides by proteolytic digestion of the glyco‐protein. The reduced disaccharide consisted of equimolar amounts of galactose and N ‐acetylgalactosaminitol and was characterized by thin‐layer chromatography, high‐voltage electrophoresis and gas‐liquid chromatography. Periodate oxidation studies on the reduced disaccharide revealed that [ 14 C]galactose was linked to position C‐3 on the N ‐acetylgalactosaminyl residue. Digestion of the reduced disaccharide and the glycopeptides with galactosidases gave equivocal results as to the anomeric configuration of the [ 14 C]galactose residue. Nuclear magnetic resonance of the reduced disaccharide, however, definitely indicated that the configuration was β. The specificity of the porcine submaxillary gland galactosyltransferase thus can be defined as a uridine diphosphogalactose: α‐ d ‐ N ‐acetylgalactosaminyl‐protein β→ 3 transferase activity.