Galactosyltransferase Activities In Ehrlich Ascites Tumor Cells.

Abstract

Oligosaccharides covalently bound to Synsorb beads (Chembiomed Ltd., Alberta, Canada) have been utilized as probes for the detection, purification, and characterization of glycosyltransferases in Ehrlich ascites tumor cells. Appropriate conditions have been developed to assay for transferase activities by determining the incorporation of radioactive sugars into the oligosaccharide-Synsorb beads. The nature of the anomeric linkage formed in the enzymatic reaction was assessed by digestion with various exoglycosidases. Additionally, a permethylation procedure has been devised to investigate the intersugar linkage generated upon incubation of ('14)C -(beta)-D-Gal-(1,4)-(beta)-D-GlcNAc-Synsorb beads with UDP-Gal, or UDP-GlcNAc, in the presence of microsomal extracts of Ehrlich tumor cells. In this fashion, Ehrlich cells have been shown to contain an (alpha)(1,3)-galactosyltransferase and a (beta)(1,3)-N-acetylglucosaminyltransferase which may be responsible for the presence of (alpha)-D-galactosyl-terminated poly-N-acetyllactosaminoglycan structures on the plasma membrane of Ehrlich cells. A separate (beta)-N-acetylglucosaminyltransferase activity which utilized (beta)-D-GlcNAc(1,3)-(beta)-D-Gal-(1,4)-(beta)-D-GlcNAc-Synsorb as acceptor was found in Ehrlich cell microsomes, and was postulated to initiate the synthesis of blood group I antigenic structures. A (beta)-D-Gal-(1,4)-D-GlcNAc (alpha)(1,3)-galactosyltransferase and a GlcNAc (beta)(1,4)-galactosyltransferase have been purified 205,000- and 17,560-fold, respectively, to apparent homogeneity from Lubrol PX extracts of Ehrlich tumor cell microsomal membranes. The two transferases were separated from contaminating proteins by affinity chromatography on (alpha)-lactalbumin-Sepharose and (beta)GlcNAc-Synsorb, and purified by adsorption to UDP-hexanolamine-Sepharose. The subunit molecular weights of the (alpha)- and (beta)-galactosyltransferases were 80,000 and 56,000, respectively. Both enzymes required manganese and detergent for catalytic activity. N-Acetyllactosamine and a variety of glycoconjugates terminated in this disaccharide all served as acceptors for the Ehrlich cell (alpha)-galactosyltransferase. (alpha)-Lactalbumin modified the acceptor specificity of the Ehrlich tumor cell (beta)-galactosyltransferase by decreasing the Michaelis constant for glucose with a concomitant increase in the K(,m) for N-acetylglucosamine. Molecules of Glc and GlcNAc that had been substituted at various positions of the pyranose ring were tested for their ability to serve as acceptors for the Ehrlich cell (beta)-galactosyltransferase, both in the presence and in the absence of (alpha)-lactalbumin. This study allowed a determination of the molecular contacts between the (beta)-galactosyltransferase and its acceptor substrate necessary for enzymatic activity.