Incorporation of N-acetyl-[14C]D-glucosamine and [3H]L-leucine by isolated pig gastric mucosal cells

Abstract

Glycoprotein and protein production of isolated pig gastric mucosal cells were determined by the incorporation of N-acetyl-[14C]D-glucosamine ([14C]GlcNAc) and [3H]L-leucine ([3H]Leu) into acid-insoluble macromolecules (AIM). In four cell fractions (F1-F4), obtained by counterflow centrifugation, specific [14C]GlcNAc incorporation was greatest in the mucous cell-enriched F2. Tracer incorporation by F2 cells, proceeded linearly up to 20 h, was inhibited by cycloheximide or incubation at 0 degree C, and enhanced by PGE2 1 mumol/l. Gel chromatography of released AIM revealed that PGE2-stimulated [14C]GlcNAc incorporation was predominantly directed into high molecular weight (2 X 10(6) daltons) glycoproteins, whereas [3H]Leu incorporation was mainly related to proteins of albumin-like molecular weight. We conclude that incorporation of [14C]GlcNAc by enriched pig gastric mucous cells (F2), further analyzed by gel chromatography, is a suitable probe to study the production of high molecular weight gastric mucous glycoproteins in vitro.