Biosynthesis of blood-group I and i substances. Specificity of bovine colostrum beta-N-acetyl-D-glucosaminide beta 1 leads to 4 galactosyltransferase.

Highly purified bovine colostrum beta-N-acetylglucosaminide beta 1 leads to 4 galactosyltransferase was used to investigate the galactosylation of the synthetic, branched trisaccharide GlcNAcbeta 1 leads to 3(GlcNAcbeta 1 leads to 6)Gal, which is the branching point in blood-group I antigenic structures. Two galactose residues could readily be incorporated from UDP-galactose into the trisaccharide, yielding a pentasaccharide with the following structure: Galbeta 1 leads to 4GlcNAcbeta 1 leads to 3(Galbeta 1 leads to 4GlcNAcbeta 1 leads to 6)Gal. From a partially completed incubation an intermediate tetrasaccharide was isolated, the structure of which was investigated by use of an acetolysis method, involving high-pressure liquid chromatography and double labelling techniques. It appeared that this intermediate consisted for more than 95% of one of two possible structures: GlcNAcbeta 1 leads to 3(Galbeta 1 leads to 4GlcNAcbeta 1 leads to 6)Gal. This reveals that the enzymatic galactosylation of the trisaccharide proceeds in a highly preferred order, in which the 1 leads to 6-linked N-acetylglucosamine residue is galactosylated first and thus that the galactosyltransferase displays a high degree of 'branch specificity'. Kinetic data suggest that galactosylation of the 1 leads to 6-linked N-acetylglucosamine in the trisaccharide enhances the acceptor properties of the 1 leads to 3-linked N-acetylglucosamine residue.