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
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.