Biosynthesis of proteins by human gastric mucosa in vitro

Abstract

Incorporation of L-[U-14C]leucine and of D[U-14C]glucose into proteins of fresh human gastric mucosa in vitro was studied after incubation of homogenized tissue and of intact mucosal pieces. CsCl centrifugation was used to separate high-density mucus glycoproteins from other mucosal proteins, and the macromolecular nature of radioactive mucosal glycoprotein fractions was confirmed by SDS/polyacrylamide-gel electrophoresis and autoradiography of the polyacrylamide gels. In all experiments a substantial proportion of total incorporated radioactivity was associated with gastric-mucosal glycoprotein fractions (CsCl fraction L3), indicating their biosynthesis. Radioactivity of these fractions was shown to co-chromatograph with carbohydrates when fractionated either directly or after reduction and alkylation (1) Sephadex G-200 chromatography in the excluded fractions and (2) by DEAE-cellulose ion-exchange chromatography. On incubation of intact mucosa, the major portion of radioactivity associated with the glycoprotein fractions of both leucine- and glucose-labelled specimens was secreted into the mucosal media during the course of the experiment. It is suggested that biosynthesis of mucus in vivo by gastric mucosa may be associated with rapid secretion of the synthesized macromolecules into the lumen of the stomach and that investigations of the metabolic processes within the mucosa should consider the products of secretion of the tissue. Incorporation of L-[U-14C]leucine implies biosynthesis of the polypeptide components of the macromolecules.

Regulation of mucus secretion by cells isolated from the rat gastric mucosa.

“…Release of labelled material of lower molecular weight into the incubation medium was found by Roomi, Laburthe, Fleming, Crowther & Forstner (1984) using rat small intestinal slices to study goblet cell mucus secretion, by Slomiany et al (1987) with cultured scrapings of rat gastric mucosa, by Spohn & McColl (1987) with human antral mucosa, and by Seidler et al (1988) using organ culture of pieces of rabbit gastric mucosa. The more intact preparations seemed to release comparatively less lower and more higher molecular weight material.…”

The Effect of O-Acetylsalicylic Acid on Lipid Synthesis by Guinea Pig Gastric Mucosa in Vitro

“…Cholesterol spots, located with bromthymol blue (0.04% (w/v) in 0.01 M KOH) and identified by co-chromatography with cholesterol marker, were scraped into vials, mixed with scintillator solution, and counted (15). The level of cholesterol in these samples was too low for quantitative estimation.…”

“…Methyl esters of fatty acids were extracted from other products of methanolysis with hexane, and the hexane extracts separated by thin-layer chromatography on Silica gel G (Analtech Uniplate, 250 ym thick) in hexane-ether (85:15 (v/v)). Methyl ester bands, which were identified by co-chromatography with methyl heneicosanoate, were scraped into vials, mixed with scintillator solution, and counted for radioactivity (15). Recovery of total activity from the plates was 81.9 k 18.6% (12 samples).…”