Glycoprotein Biosynthesis in Small Intestinal Mucosa

An active multiglycosyltransferase system involved in the transfer of N-acetylgalactosamine, N-acetylglucosamine, and galactose to various glycoprotein acceptors was found in the rat small intestinal mucosa. The present studies are concerned with the subcellular localization of these glycosyltransferases. Satisfactory isolation of smooth and rough surfaced microsomes from the intestinal mucosa was obtained by modifying the method of Dallner (Acfa Pafhol. Microbial. Stand Suppl., 166, 1 (1963)). Kinetic studies, performed to establish optimum conditions for quantitative assays of the glycosyltransferases, were linear with respect to enzyme concentration and time. Smooth surfaced microsomes were the main submicrosomal loci for the glycosyltransferases; for example 95 % of total microsomal polypeptidyl:N-acetylgalactosaminyltransferase activity was found in the smooth surfaced microsomes. The results of alkaline borohydride treatment and acid hydrolysis indicated that N-acetylgalactosamine was incorporated into the heat-treated rough surfaced microsomal acceptors primarily at the protein to carbohydrate linkages while in the case of the heat-treated smooth surfacedmicrosomal acceptors, this sugar was probably added to positions more distal in the polysaccharide moiety. Immunodiffusion showed that only the smooth surfaced microsomes contained a component immunologically identical with purified rat small intestinal mucin. These results suggest that both stepwise glycosylation and probably the attainment of an immunological identity of rat small intestinal mucin occur in the smooth surfaced microsomes.