PURIFICATION AND CHARACTERIZATION OF A UDP-GAL-BETA-D-GAL(1,4)-D-GLCNAC ALPHA-(1,3)-GALACTOSYLTRANSFERASE FROM EHRLICH ASCITES TUMOR-CELLS

Abstract

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 .mu.M 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.