Role of galactosyl-transferases in rat gastric epithelial glycoprotein synthesis

Two galactosyl-transferases have been found in the Golgi-enriched sub-cellular fractions derived from rat gastric mucosa. One incorporates galactose into ovomucoid at optimal pH 6.8. The reaction can be completely inhibited by acetylglucosamine. The apparent Km for UDPgalactose is 0.024 mM. The other galactosyl-transferase incorporates galactose into desialated ovine submaxillary mucin at optimal pH 7.5 and the transfer cannot be inhibited by acetylglucosamine. The apparent Km for UDPgalactose is 0.191 mM. Both enzymes require Mn2+ and Triton X-100 for optimal galactose incorporation. The enzymes could be separated by polyacrylamide gel electrophoresis. Incorporation into endogenous glycoprotein was studied in conditions optimal for the two galactosyl-transferases: (1) at pH 6.8, using Mes as buffer system, and (2) at pH 7.5, using Tris-HCl in the presence of an inhibitory excess of acetylglucosamine. In both cases, most radioactive galactose is incorporated into macromolecules, which could be identified as epithelial glycoprotein. Endogenous incorporation in the presence of excess acetylglucosamine results in the formation of a substantial amount of a disaccharide (probably galactose-β-(1–3)acetylgalactosamine), whereas upon incorporation at pH 6.8 almost no disaccharide is formed. Quantitative immunoprecipitaton experiments with specific antibodies to the endogenous product, labelled by [3H]galactose in the presence of varying amounts of desialated ovine submaxillary mucin and/or acetylglucosamine, indicated that other galactosyl-transferases are involved in the biosynthesis of epithelial glycoprotein

Differential modulation of enterocyte-like Caco-2 cells after exposure to short-chain fatty acids

The response of intestinal epithelial cells to short-chain fatty acids, which are increasingly used as food additives, was investigated. Human small intestinal epithelial cell model Caco-2 cells were exposed to formate, propionate and butyrate to assess their effect on cellular growth, metabolism, differentiation and protection against bacteria. The Caco-2 cells were entirely grown in the different short-chain fatty acids and respective growth patterns were determined. Differentiated cells were exposed to 0-20 mM short-chain fatty acids for 48 h and changes in DNA, RNA, (glyco)protein syntheses, sucrase isomaltase activity, transepithelial electrical resistance and protection against Salmonella enteritidis were measured. The short-chain fatty acids, altered linearly and differentially the growth pattern ranging from stimulation by formate to inhibition by butyrate. Formate inhibited cellular metabolism. Low concentrations of up to 5 mM propionate and 2 mM butyrate stimulated metabolism, while higher doses were inhibitory. Formate had no effect on sucrase isomaltase enzyme activity and transepithelial electrical resistance, whereas propionate and butyrate increased these markers of differentiation. Infection with S. enteritidis did not benefit from the short-chain fatty acid-induced transepithelial electrical resistance. It is concluded that formate, propionate and butyrate selectively and differentially modulate growth characteristics, cellular metabolism, sucrase isomaltase activity and transepithelial electrical resistance in a concentration- and carbon atom-related fashion. The short-chain fatty acid-induced transepithelial electrical resistance does not confer protection against S. enteritidis