A SPECIFIC BINDING OF THE CHOLECYSTOKININ-RELEASING PEPTIDE (MONITOR PEPTIDE) TO ISOLATED RAT SMALL-INTESTINAL CELLS

Citation
R. Yamanishi et al., A SPECIFIC BINDING OF THE CHOLECYSTOKININ-RELEASING PEPTIDE (MONITOR PEPTIDE) TO ISOLATED RAT SMALL-INTESTINAL CELLS, Biochemical journal, 291, 1993, pp. 57-63
Citations number
33
Journal title
ISSN journal
02646021
Volume
291
Year of publication
1993
Part
1
Pages
57 - 63
Database
ISI
SICI code
0264-6021(1993)291:<57:ASBOTC>2.0.ZU;2-R
Abstract
A specific binding of the cholecystokinin (CCK)-releasing peptide (mon itor peptide) to isolated rat jejunal mucosal cells was investigated. The I-125-labelled purified monitor peptide bound to the rat jejunal c ells, and a large excess amount of the non-labelled monitor peptide in hibited the binding. The binding.was completed within 60 min at 37-deg rees-C. The optimum pH for the binding was 8-9. A Scatchard plot of th e specific binding was linear, and the dissociation constant was 50 nM . The density of the monitor-peptide-binding sites was high in duodenu m but low in ileal and absent in colonic mucosa. A recombinant monitor peptide and four kinds of point mutants of it were prepared. The bind ing of the mutant monitor peptides to the cells indicated that only a trypsin inhibitor of the mutants could bind to the mucosal cells. Huma n pancreatic secretory trypsin inhibitor inhibited the specific bindin g, but other trypsin inhibitors, i.e. bovine basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, egg-white trypsin inhibitor, leu peptin, antipain and FOY-305, did not affect the specific binding at a ll. These findings suggested that the specific binding site for the mo nitor peptide on the jejunal mucosal cells has a trypsin-like specific ity, exhibiting an especial specificity for the pancreatic-secretory-t rypsin-inhibitor family. Autoradiography of an affinity-cross-linked c omplex of the I-125-labelled intact monitor peptide and the binding si te suggested that its molecular mass was 33 kDa or 53 kDa in the prese nce or absence of 2-mercaptoethanol respectively.