Glucosyltransferase activity in calf pancreas microsomes. Formation of dolichyl D[14C]glucosyl phosphate and 14C-labeled lipid-linked oligosaccharides from UDP-D-[14C]glucose.

A Herscovics,B Bugge,R W Jeanloz

doi:10.1016/s0021-9258(17)40550-3

Abstract

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.

Highlights

Calf pancreas microsomes incorporated radioactively labeled n-glucose from DDP-o-glucose into products extracted with chloroform/methanol (2:1, v/v), chloroform/methanol/
We have shown that calf pancreas microsomes contain lipid-bound oligosaccharides consisting of n-mannose, n-glucose, and N-acetyl-n-glucosamine linked to
The results presented demonstrate that calf pancreas microsomes incubated with labeled UDP-n-glucose synthesize a labeled glucolipid with the properties of dolichyl n-glucosyl phosphate, and n-glucose-labeled lipid-bound oligosaccharides in which the oligosaccharides are slightly larger than the D

Summary

PROCEDURES

Materials -The source of chemicals is described in the preceding publication (1). In addition, UDP-n-[l-SHlglucose (4.85 Ci/mmol) and UDP-n-lU-14Clglucose (139 mCilmmo1) were purchased from New. Use of the Polytron homogenizer allowed the preparation of larger quantities of microsomes in a relatively short time These microsomes were much more active in the utilization of labeled UDP-n-glucose/mg of protein than those prepared by the previous method. Incubation with Labeled UDP-D-GLUCOSE -For standard conditions, microsomes were incubated for 30 min at 30” in a total volume of 0.5 ml containing 4 to 6 mg of protein/ml, TrisJmaleate buffer, pH. Incubation with Pancreatic Dolichyl D-[~4CIGlucosyl Phosphate - The chloroform/methanol extract obtained from incubation of total microsomes with UDP-n-[“C]glucose was used. It was necessary to let the mixture stand overnight at 4” during the first water extraction to allow sedimentation of the precipitate About 75% of the incorporated radioactivity was found in the chloroform/methanol/water extract after 30 min of incubation.

; , RESULTS

II I i6

DISCUSSION