Specific binding and degradation of somatostatin by membrane vesicles from pig gut.
Plasma membrane vesicles were prepared from the basolateral face of pig small intestinal epithelial cells and were enriched in the activity of Na+-K+-ATPase (9-fold relative to the cell homogenate) and ranged in size from 0.15 to 0.40 micron diam. Incubation of somatostatin-14 and [125I-Tyr11]-somatostatin-14 with the vesicles at 37 degrees C resulted in rapid proteolytic degradation of the peptides. Metabolites were isolated by reverse-phase high-performance liquid chromatography and identified by amino acid composition. Cleavages between Ala1-Gly2, Phe6-Phe7, Phe7-Trp8, and Thr10-Phe11 were observed, indicative of aminopeptidase and endopeptidase action. Degradation was inhibited by 1,10-phenanthroline and by bacitracin, and in the presence of these inhibitors and at 21 degrees C binding of [125I-Tyr11]somatostatin-14 to the vesicles was observed. Binding was inhibited in a concentration-dependent manner by somatostatin-14 (half-maximal inhibition at 2.0 +/- 0.1 nM) and by somatostatin-28 (0.8 +/- 0.1 nM) but not by structurally unrelated peptides. The rate of degradation of [125I-Leu8, D-Trp22, Tyr25]somatostatin-28 by basolateral membrane was less than 20 fold that of [125I-Tyr11]somatostatin-14 and a two- to three-fold enhanced binding to the vesicles was observed. Analysis of the inhibition of binding of this analogue by somatostatin-28 indicates the presence of single class of binding site with Kd = 1.3 +/- 0.3 nM. Rapid degradation but no specific binding of somatostatin-14 by brush-border membranes was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Weber
M
M
Cole
T
T
Conlon
J M
JM
D000596:Amino Acids / Q000378:metabolism; D000626:Aminopeptidases / Q000378:metabolism; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D010450:Endopeptidases / Q000378:metabolism; D004848:Epithelium; D007421:Intestine, Small / Q000378:metabolism*; D008854:Microscopy, Electron; D010446:Peptide Fragments / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013004:Somatostatin / Q000031:analogs & derivatives / Q000378:metabolism*; D053561:Somatostatin-28 / Q000031:analogs & derivatives; D013552:Swine; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D000596:Amino Acids; D010446:Peptide Fragments; D014660:Vasoactive Intestinal Peptide; D013004:Somatostatin; C032367:somatostatin, Tyr(11)-; D053561:Somatostatin-28; C045259:somatostatin 28, Leu(8)-Trp(22)-iodo-Tyr(25)-; D010450:Endopeptidases; D000626:Aminopeptidases; D000254:Sodium-Potassium-Exchanging ATPase