Ehrlich ascites tumor cell UDP-Gal:N-acetyl-D-glucosamine beta(1,4)-galactosyltransferase. Purification, characterization, and topography of the acceptor-binding site.
A UDP-Gal:N-acetylglucosamine beta(1,4)-galactosyltransferase which catalyzes the synthesis of beta-D-Gal(1,4)-D-GlcNAc units has been purified 17,560-fold from Ehrlich tumor cells to apparent electrophoretic homogeneity. The enzyme appears to be a monomeric protein with Mr = 56,000-58,000. Enzymatic activity requires the presence of MnCl2, is stimulated by detergent, and exhibits a pH optimum at 6.9. The Km values for GlcNAc and UDP-Gal are 1.89 and 0.046 mM, respectively. The Ehrlich cell beta-galactosyltransferase acts efficiently on glycoproteins and glycolipids terminating in GlcNAc, but is inactive toward glycoconjugates possessing terminal GalNAc units. The oligosaccharides beta-D-GlcNAc(1,3)-D-Gal and beta-D-GlcNAc(1,3)[beta-D-GlcNAc(1,6)]-D-Gal are good acceptors for the beta-galactosyltransferase from Ehrlich cells, suggesting that the enzyme may participate in the biosynthesis of i/I structures. In addition, other linear and branched sugars presenting GlcNAc residues at their nonreducing termini also act as acceptors for the enzyme. The activity of Ehrlich cell beta-galactosyltransferase both in the presence and absence of alpha-lactalbumin has been studied using a series of derivatives of Glc and GlcNAc which were substituted at various positions of the pyranose ring. This study has provided a map of the molecular contacts necessary for enzymatic activity in the presence and in the absence of alpha-lactalbumin.
D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002236:Carbohydrate Conformation; D002286:Carcinoma, Ehrlich Tumor / Q000201:enzymology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006017:Glycolipids / Q000378:metabolism; D006023:Glycoproteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007768:Lactalbumin / Q000494:pharmacology; D007788:Lactose Synthase / Q000302:isolation & purification / Q000378:metabolism; D008345:Manganese / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009237:N-Acetyllactosamine Synthase / Q000302:isolation & purification / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D013379:Substrate Specificity; D014531:Uridine Diphosphate Galactose / Q000378:metabolism
D006017:Glycolipids; D006023:Glycoproteins; D014531:Uridine Diphosphate Galactose; D011092:Polyethylene Glycols; D008345:Manganese; D017830:Octoxynol; D007768:Lactalbumin; D007788:Lactose Synthase; D009237:N-Acetyllactosamine Synthase; D000117:Acetylglucosamine