Hydrolysis of the C-terminal octapeptide of cholecystokinin by rat kidney membranes: Characterization of the cleavage by solubilized endopeptidase - 24.11

Abstract

Rat kidney membranes were solubilized by Triton X-100 and the CCK-8 degrading peptidases were resolved by chromatography on DEAE-cellulose. Four proteases were detected: two phosphoramidon-sensitive endopeptidases (EC 3.4.24.11), a bestatin-sensitive aminopeptidase and an unidentified enzyme. The pattern of cleavage of CCK-8 and shorter C-terminal fragments by endopeptidase 24.11 was investigated and indicated that the Gly 29-Trp 30-Met 31 and Asp 32-Phe 33 were scissile bonds. However, the cleavage pattern differed markedly from one CCK peptide to another: in the penta- and hexapeptide of CCK the bonds hydrolyzed were either Asp-Phe and Trp-Met or, Asp-Phe and Gly-Trp, respectively. The presence of the sulfate group on the tyrosine residue of CCK-8 influence markedly the nature of the major cleavage fragments produced by the endopeptidase. The major bonds cleaved were Asp-Phe, Trp-Met and Gly-Trp for unsulfated CCK-8, whilst for the sulfated octapeptide, the Trp-Met bond became a minor cleavage site.