Incorporation of N-Acetyl-[14C]D-Glucosamine and [3H]L-Leucine by Isolated Pig Gastric Mucosal Cells

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Abstract
Glycoprotein and protein production of isolated pig gastric mucosal cells were determined by the incorporation of N-acetyl-[14C]D-glucosamine ([14C]G1cNAc) and [3H]L-leucine ([3H]Leu) into acid-insoluble macromolecules (AIM). In four cell fractions (F1-F4), obtained by counterflow centrifugation, specific [14C]G1cNAc incorporation was greatest in the mucous cell-enriched F2. Tracer incorporation by F2 cells, proceeded lineraly up to 20 h, was inhibited by cycloheximide or incubation at 0 ° C, and enhanced by PGE2 1 μmol/l. Gel chromatography of released AIM revealed that PGE2-stimulated [14C]GlcNAc incorporation was predominantly directed into high molecular weight (2 × 106 daltons) glycoproteins, whereas [3H]Leu incorporation was mainly related to proteins of albumin-like molecular weight. We conclude that incorporation of [14C]G1cNAc 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.