A specific binding of the cholecystokinin-releasing peptide (monitor peptide) to isolated rat small-intestinal cells

Abstract

A specific binding of the cholecystokinin (CCK)-releasing peptide (monitor peptide) to isolated rat jejunal mucosal cells was investigated. The 125I-labelled purified monitor peptide bound to the rat jejunal cells, and a large excess amount of the non-labelled monitor peptide inhibited the binding. The binding was completed within 60 min at 37 degrees C. The optimum pH for the binding was 8-9. A Scatchard plot of the specific binding was linear, and the dissociation constant was 50 nM. The density of the monitor-peptide-binding sites was high in duodenum but low in ileal and absent in colonic mucosa. A recombinant monitor peptide and four kinds of point mutants of it were prepared. The binding of the mutant monitor peptides to the cells indicated that only a trypsin inhibitor of the mutants could bind to the mucosal cells. Human pancreatic secretory trypsin inhibitor inhibited the specific binding, but other trypsin inhibitors, i.e. bovine basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, egg-white trypsin inhibitor, leupeptin, antipain and FOY-305, did not affect the specific binding at all. These findings suggested that the specific binding site for the monitor peptide on the jejunal mucosal cells has a trypsin-like specificity, exhibiting a special specificity for the pancreatic-secretory-trypsin-inhibitor family. Autoradiography of an affinity-cross-linked complex of the 125I-labelled intact monitor peptide and the binding site suggested that its molecular mass was 33 kDa or 53 kDa in the presence or absence of 2-mercaptoethanol respectively.

The Bifunctional Rat Pancreatic Secretory Trypsin Inhibitor/Monitor Peptide Provides Protection against Premature Activation of Pancreatic Juice

“…10 -8 M was obtained with preparations of sucrose-gradient-purified membranes from the small intestine. The same value has been reported when whole cells, scraped from the mucosa, were used [30]. It is clear that both approaches, binding to cells or membrane preparations, give a crude estimate of the binding constant.…”

Regulation of Pancreatic Secretion

“…276,277 Monitor peptide is produced by pancreatic acinar cells and is secreted into the pancreatic duct where it eventually reaches the duodenum. 281 Specific monitor peptide binding was demonstrated to be reversible, temperature-and pH-dependent, and consistent with receptor binding. Because monitor peptide is secreted in pancreatic juice, the levels in the intestine are not elevated unless pancreatic secretion is stimulated.…”

Recognition System for Dietary Fatty Acids in the Rat Small Intestine

“…This peptide has specifically stimulated the Ca 2 + efflux from isolated intestinal cells. 16) Next, the influx of 45Ca 2 + into isolated intestinal cells when incubated with the other sample solutions was examined. With reference of the time-course when incubated with a linoleic acid solution, the influx after 10 min of incubation was determined.…”