Glycoprotein biosynthesis in intestinal epithelial cells during differentiation incorporation of [ 14C]mannose from GDP-[ 14C]mannose into dolichol derivatives

Abstract

Epithelial cells of the rat small intestine were collected as a gradient of villus to crypt cells. Homogenates of these cells incubated with GDP- D-[ 14C]mannose in the presence of MnCl 2 incorporated radioactivity into dolichyl mannosyl phosphate and a mixture of dolichyl pyrophosphate oligosaccharides varying in the size of their oligosaccharide moiety. The labeled oligosaccharides formed in villus cell homogenates appeared shorter than those formed in crypt cell homogenates. The addition of dolichyl phosphate greatly stimulated the synthesis of dolichyl mannosyl phosphate. The initial rate of synthesis of dolichyl mannosyl phosphate from GDP- D-[ 14C]mannose and exogenous dolichyl phosphate was highest in an intermediate cell fraction having a low specific activity of sucrase and alkaline phosphatase and an intermediate specific activity of thymidine kinase. To compare the rates of dolichyl mannosyl phosphate synthesis in the different cell fractions, it was essential to control degradation of GDP- D-[ 14]mannose by the addition of AMP to the incubation, since villus cells degraded GDP- D-[ 14C]mannose much faster than crypt cells.