The binding of glycoconjugates to human-milk D-galactosyltransferase.
Through the use of affinity chromatography, a homogeneous preparation of human beta(1 leads to 4)-D-galactosyltransferase (the A protein of lactose synthase) was obtained. The specificity of this protein for glycoconjugates was studied in the presence and absence of human alpha-lactalbumin. A kinetic analysis of the transfer of D-galactose to N-acetyl-D-glucosamine and the beta(1 leads to 4) linked N-acetylglucosamine oligomers, suggested that the active site region of the enzyme contains more than one binding site for acceptor moleucles. Furthermore, experiments with Na-acetylglucosamine-beta(1 leads to4)-N-acetylmuramic-pentapeptide isolated from Micrococcus luteus indicated that the presence of a peptide chain does not enhance enzymic activity, as compared with the corresponding free disaccharide. Similar results were obtained using ovalbumin and the ovalbumin glycopeptide (which have similar apparent Km values for A protein) as galactose acceptors. In contrast to its ability to inhibit N-acetyllactosamine production, alpha-lactalbumin did not inhibit the transfer of D-galactose to the N-acetylglucosamine oligomers or the glycopeptides. Although alpha-lactalbumin can switch the specificity of A protein from N-acetyl-D-glucosamine to D-glucose resulting in the production of lactose, no transfer of galactose was observed to beta(1 leads to 4)-linked glycose oligomers or to a collagen glycopeptide, D-glycopyranosyl-alpha(1 leads to 2)-D-galactopyranosyloxy-beta(1 leads to 5)-lysine. IT therefore appears that alpha-lactalbumin can only modify human A protein for monosaccharide acceptors.
D000117:Acetylglucosamine / Q000378:metabolism; D001665:Binding Sites; D005260:Female; D005690:Galactose / Q000378:metabolism; D006020:Glycopeptides / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D007768:Lactalbumin / Q000494:pharmacology; D007788:Lactose Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D008895:Milk, Human / Q000201:enzymology*; D009237:N-Acetyllactosamine Synthase / Q000378:metabolism; D009844:Oligosaccharides / Q000378:metabolism; D010047:Ovalbumin / Q000378:metabolism; D011247:Pregnancy; D011485:Protein Binding; D013329:Structure-Activity Relationship
D006020:Glycopeptides; D009844:Oligosaccharides; D010047:Ovalbumin; D007768:Lactalbumin; D007788:Lactose Synthase; D009237:N-Acetyllactosamine Synthase; D000117:Acetylglucosamine; D005690:Galactose