sincalide and 1-10-phenanthroline

A cholecystokinin octapeptide (CCK-8)-degrading peptidase was purified from rat liver cytosol by heat precipitation of other proteins followed by gel filtration, ion exchange chromatography and preparative gel electrophoresis, using a silicate binding assay to quantitate the degradation of radiolabeled CCK-8. The purified peptidase (M(r) approximately 60,000) had a pH optimum of 6.0; its activity was inhibited by EDTA and 1, 10-phenanthroline but not by phosphoramidon, calpain inhibitor I, bestatin or bacitracin. CCK-8 peptidase rapidly degraded radiolabeled Met-enkephalin as well as 125I-CCK-8, but not a series of other unrelated peptides. Unlabeled Leu-enkephalin, beta-casomorphin and neurotensin competitively inhibited the degradation of 125I-CCK-8, suggesting that these opioids are also substrates for the enzyme. These data suggest that this protein is a novel hepatic enzyme which may play a role in the degradation of neuropeptides.

Topics: Animals; Cytosol; Edetic Acid; Endopeptidases; Hydrogen-Ion Concentration; Liver; Phenanthrolines; Protease Inhibitors; Rats; Rats, Sprague-Dawley; Sincalide; Substrate Specificity

Inhibitors of metalloendopeptidases interfere with events involving Ca2(+)-dependent membrane fusion in a number of cell types. The divalent ion chelating agent 1,10-phenanthroline inhibited pancreatic amylase secretion stimulated by carbachol, cholecystokinin-octapeptide (CCK-8), or bombesin, but detailed studies indicated that this is unlikely to be a result of inhibition of metalloendopeptidase activity. The binding of [3H]N-methylscopolamine to pancreatic acini was reduced by 1,10-phenanthroline and this would explain the marked inhibition of carbachol-induced amylase secretion by the chelating agent. CCK-8-stimulated hydrolysis of phosphatidylinositol-4,5-bisphosphate was reduced by 1,10-phenanthroline while the binding of CCK-8 to acini was not affected. This inhibition of hydrolysis would explain the inhibition of CCK-8- and bombesin-induced amylase secretion. The metalloendopeptidase substrate carbobenzoxyglycylphenylalanylamide did not affect bombesin-stimulated amylase secretion. Amylase secretion evoked by treating pancreatic acini with the ionophore A23187 or dibutyryl-cyclic AMP was not reduced by 1,10-phenanthroline, indicating a lack of involvement of metalloendopeptidases in the process of exocytosis in this cell type.

Topics: Amylases; Animals; Bombesin; Bucladesine; Calcimycin; Carbachol; Dipeptides; Metalloendopeptidases; Mice; N-Methylscopolamine; Pancreas; Phenanthrolines; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylinositols; Receptors, Muscarinic; Scopolamine Derivatives; Sincalide