Purification and characterization of a UDP-Gal:beta-D-Gal(1,4)-D-GlcNAc alpha(1,3)-galactosyltransferase from Ehrlich ascites tumor cells.
A UDP-Gal:N-acetyllactosaminide alpha (1,3)-galactosyltransferase from Ehrlich ascites tumor cells has been purified over 200,000-fold to apparent electrophoretic homogeneity. The purified enzyme transfers D-galactosyl groups from UDP-Gal to beta-D-Gal-(1,4)-D-GlcNAc in alpha-linkage. The apparent Km values for donor and acceptor substrates are 12.6 microM and 1.15 mM, respectively. The trisaccharides beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)- or (1,6)-D-Man exhibit a Km 5-fold lower than that of N-acetyllactosamine, and an even more pronounced effect is observed with the biantennary pentasaccharide beta-D-Gal(1,4)-beta-D-GlcNAc(1,2)-[beta-D-Gal(1, 4)-beta-D-GlcNAc-(1,6)]-D-Man (Km 0.10 mM). The transferase shows a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions with an apparent subunit molecular weight of 80,000, exhibits a pH optimum at 6.2, and requires Mn2+ ions and detergent for enzymatic activity. Specificity studies using immobilized oligosaccharides show that the minimum acceptor structure for the alpha-galactosyltransferase is N-acetyllactosamine. The narrow specificity of the alpha-galactosyltransferase is indicated by the fact that lactose, beta-D-Gal(1,3)-D-GlcNAc, and beta-D-Gal(1,4)-[alpha-L-Fuc(1,3)]-D-GlcNAc are very poor acceptors. The enzyme differs from the blood-group B-specified galactosyltransferase in that the sequence alpha-L-Fuc(1,2)-beta-D-Gal(1,4)-D-GlcNAc is not an acceptor. Oligosaccharides, glycoproteins, glycolipids, and glycosaminoglycans containing the terminal nonreducing N-acetyllactosamine unit all serve as acceptors for the enzyme.
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002846:Chromatography, Affinity; D003902:Detergents / Q000494:pharmacology; D005700:Galactosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006026:Glycoside Hydrolases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D051379:Mice; D017830:Octoxynol; D009844:Oligosaccharides; D011092:Polyethylene Glycols / Q000494:pharmacology; D013347:Subcellular Fractions / Q000201:enzymology; D013379:Substrate Specificity
D003902:Detergents; D009844:Oligosaccharides; D011092:Polyethylene Glycols; D017830:Octoxynol; D005700:Galactosyltransferases; C038887:N-acetyllactosamine alpha-D-galactosyltransferase; D006026:Glycoside Hydrolases