LIPID INTERMEDIATES ARISING DURING GLYCOPROTEIN-BIOSYNTHESIS IN LIVER-MICROSOMES

Incubation of liver [pig, beef] microsomes with GDP[14C]mannose leads to the formation of lipid-linked derivatives of [14C]mannose, a dolichol phosphate monosaccharide and dolichol pyrophosphate oligosaccharides. Standard procedures for separating these 2 types of compounds from each other were deficient in that fractions thought to contain only dolichol pyrophosphate oligosaccharides are contaminated with dolichol phosphate mannose. A column chromatographic procedure which conveniently separates the products of an 8 min labeling experiment into 2 components is presented; dolichol phosphate [14C]mannose and a [14C]-mannose containing oligosaccharide which is also lipid bound. When this oligosaccharide is released from the lipid by hydrolysis and chromatographed on Sephadex G-50 or G-15 it gives a single peak with an indicated MW of 1100. When released oligosaccharide is chromatographed on concanavalin A Sepharose it is resolved into 2 peaks suggesting that there may be 2 oligosaccharides of approximately the same size but different structures. After brief periods of labeling with GDP[14C]mannose (5 s) an additional oligosaccharide of 3-4 sugar residues can be found in the dolichol pyrophosphate oligosaccharides fraction. Incubation of liver microsomes with UDP[14C]glucose or UDP[14C]galactose produces oligosaccharide components containing 7-8 sugar residues. Labeling of microsomes with UDP[14C]acetylglucosamine gives rise to 3 different components, including a lipid bound oligosaccharide containing 3-5 sugar residues.