Abstract
Biosynthetic incorporation of labeled amino acids into the major gastrins in rat antrum, Component I, gastrin 34 ("big" gastrin), and gastrin 17-like peptides ("little" gastrin) was demonstrated after in vitro incubation of antral mucosal slices. The antral gastrin synthesis was stimulated by fundectomy, which ablates gastric acid secretion and thereby increases the gastrin concentration in plasma 20-fold. Tyrosine O-sulfation of gastrin was also demonstrated by incorporation of [35S]sulfate. Sulfate-labeled peptides precipitated by gastrin antisera coeluted with sulfated gastrin immunoreactivity on ion-exchange chromatography. Aryl-sulfatase treatment removed the [35S]sulfate from labeled gastrin and resulted in a change in elution pattern of the immunoreactivity to that of the unsulfated gastrins. The presence of 35S-labeled tyrosine O-sulfate residues was directly demonstrated by two-dimensional thin-layer electrophoresis after alkaline hydrolysis of [35S]sulfate-labeled gastrin. All the antral gastrins incorporated [35S]sulfate.
Highlights
Biosynthetic incorporation of labeled amino acids cursors of gastrin would not be immunoprecipitated. after into the major gastrins in rat antrum, Component I, radiolabeling
The antral gastrinsynthesis was stimulated by fundectomy, which ablates gastric acid secretion and thereby increases the concentration in plasma20-fold
Tyrosine 0-sulfation of gastrin was demonstrated by incorporation of [36S]sulfate.Sulfate-labeledpeptidesprecipitated by gastrin antisera coeluted with sulfated gastrinimmunoreactivity on ion-exchange chromatography
Summary
Essential for the biological activity [5].Gastrin is tyrosine 0-sulfated [6], a post-translational modification which otherwise occurs only in CCK’ [7] andLeu-enkephalin [8]among regulatory peptides. Gel Filtration Chromatography-The peptide extract was applied to a Sephadex G-50 superfine column (10 X 1000 mm) eluted with 0.25 M ammonium bicarbonate with 0.01% bovine serum albumin at 5 ml/h, and 10-min fractions were collected. One-minute fractionswere collected and assayed for 36S radioactivity by liquid scintillation and for gastrin by radioimmunoassay after removing the acetic acid by lyophilization. Reverse-phase Chromatography-Extracts of antral slices were pooled, lyophilized, and applied to a column (8X 250 mm) ofCle nucleosil silica, which was eluted with a gradient of acetonitrile in 0.045% trifluoroacetic acid a t room temperature and a flow rate of 1 ml rnin". Thin-layer Electrophoresis-Sulfated peptides identified by gel filtration were dried and dissolved in saturated barium hydroxide, evacuated, incubated for 24 h at 110 "C, neutralized with sulfuric acid, and centrifuged. The ionic strength of the incubation mixture was reduced by dilution with water and thesample was applied to a Mono Q ion-exchange column
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