Glucosyltransferase activity in calf pancreas microsomes. Formation of dolichyl D[14C]glucosyl phosphate and 14C-labeled lipid-linked oligosaccharides from UDP-D-[14C]glucose.
Calf pancreas microsomes incorporated radioactively labeled D-glucose from UDP-D-glucose into products extracted with chloroform/methanol (2:1, v/v), chloroform/methanol/water (10:102.5, v/v), and into the residual precipitate, with a pH optimum in Tris/maleate buffer of about 5.3. The chloroform/methanol extract contained a single 14C-labeled acidic product, which was identified as dolichyl beta-D-glucosyl phosphate. It was stable to mild alkali, yielded D-[14C]glucose upon mild acid hydrolysis, and a 14C-labeled compound with the chromatographic mobility of 1,6-anhydro-beta-D-glucopyranosyl upon hot alkali treatment. The [14C]glucolipid had the same chromatographic mobility as dolichyl beta-D-[14C]mannosyl phosphate, and its formation was stimulated by exogenous dolichyl phosphate. The chloroform/methanol/water extract contained radioactive lipid-bound oligosaccharides which were retained on DEAE-cellulose more strongly than dolichyl D-[14C]glucosyl phosphate. They were stable to mild alkali, but labile to acid and hot alkali. Acid treatment yielded a D-glucose-labeled oligosaccharide fraction which was shown by gel filtration to be slightly larger than most of the D-mannose-labeled oligosaccharides. About 80% of the radioactive D-glucose residues could be removed with alpha-glucosidase, but not with beta-glucosidase. Pancreatic dolichyl beta-D-[14C]glucosyl phosphate incubated with calf pancreas microsomes served as direct donor of D-glucosyl residues to lipid-bound oligosaccharides and to the precipitate. These oligosaccharides had the same size as those labeled from UDP-D-[14C]glucose, and the D-[14C]glucose residues could also be removed with alpha-glucosidase.
D000818:Animals; D002417:Cattle; D002854:Chromatography, Paper; D002855:Chromatography, Thin Layer; D004286:Dolichols / Q000378:metabolism; D005958:Glucosephosphates / Q000096:biosynthesis; D005964:Glucosyltransferases / Q000378:metabolism*; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D009844:Oligosaccharides / Q000096:biosynthesis; D010179:Pancreas / Q000201:enzymology*; D014532:Uridine Diphosphate Glucose / Q000378:metabolism
D004286:Dolichols; D005958:Glucosephosphates; D009844:Oligosaccharides; D005964:Glucosyltransferases; D014532:Uridine Diphosphate Glucose