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.