A specific binding of the cholecystokinin-releasing peptide (monitor peptide) to isolated rat small-intestinal cells.
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.
Yamanishi
R
R
Department of Food Science and Technology, Faculty of Agriculture, Kyoto University, Japan.
Kotera
J
J
Fushiki
T
T
Soneda
T
T
Saitoh
T
T
Oomori
T
T
Satoh
T
T
Sugimoto
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins; D006133:Growth Substances; D006801:Humans; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D007413:Intestinal Mucosa / Q000378:metabolism; D007421:Intestine, Small / Q000378:metabolism*; D007457:Iodine Radioisotopes; D007583:Jejunum / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D016296:Mutagenesis; D051381:Rats; D017208:Rats, Wistar; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000494:pharmacology
D002352:Carrier Proteins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D007457:Iodine Radioisotopes; D011994:Recombinant Proteins; C486642:SPINK1 protein, human; C504705:Spink1 protein, rat; D014361:Trypsin Inhibitors; D014359:Trypsin Inhibitor, Kazal Pancreatic