Cholecystokinin octa- and tetrapeptide degradation by synaptic membranes. II. Solubilization and separation of membrane-bound CCK-8 cleaving enzymes.
Solubilization of rat synaptic membranes by Triton X-100, followed by DEAE-cellulose chromatography allowed the identification of different CCK-8 cleaving enzymes. The first one (in the order of elution) removed the N-terminal aspartic acid residue of CCK-8 and was active on L-aspartic acid beta naphtylamide, suggesting that a corresponded to an aminopeptidase A. Two aminopeptidases of broad specificity hydrolyzed sequentially all the peptide bonds of CCK-8 as far as the release of free tryptophan. The removal of the sulfated tyrosine residue of CCK-8 occurred at a slower rate than that of the unsulfated residue. Another peptidase converted CCK-8 into its C-terminal heptapeptide. This enzyme had a lower affinity for the sulfated octapeptide in comparison with the unsulfated form (app Km of respectively 180 and 40 muM). The CCK-7 generating proteases displayed a moderate regional variation in five rat brain areas, with the highest activity in olfactory bulbs membranes and the lowest in cerebellar membranes. This distribution followed (with a lower amplitude) that of the CCK receptors.
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000302:isolation & purification; D000818:Animals; D002766:Cholecystokinin / Q000378:metabolism*; D002848:Chromatography, DEAE-Cellulose; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D017830:Octoxynol; D010446:Peptide Fragments / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000302:isolation & purification*; D011092:Polyethylene Glycols; D051381:Rats; D012844:Sincalide; D012995:Solubility; D013570:Synaptic Membranes / Q000201:enzymology*; D013574:Synaptosomes / Q000378:metabolism
D010446:Peptide Fragments; D011092:Polyethylene Glycols; D017830:Octoxynol; D002766:Cholecystokinin; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D012844:Sincalide