Inactivation of cholecystokinin octapeptide in vitro involves a metalloendopeptidase (EC 18.104.22.168) also called enkephalinase that inactivated the peptide both by a sequential pathway of hydrolysis (removal of Phe-NH2 followed by cleavage of Trp-Met-Asp) and by an endopeptidase action (production of the tetrapeptides). As enkephalinase cleaved CCK-8 at the Gly4-Trp5, Trp5-Met6 and Asp7-Phe8 bonds, we investigated the stability of analogues having: (1) substitutions of l amino acids by a d stereoisomer, (2) a substitution of Asp7 by a β Ala residue and (3) modifications of the Trp residue obtained by replacing the nitrogen atom in the indol ring by either an oxygen ([Bfa5]CCK-8) or a sulphur atom ([Bta5]CCK-8). Among these different CCK derivatives, [βAla7], [dMet6] and [dTrp5]CCK-8 were not hydrolyzed by enkephalinase: [dAlad]CCK-8 was rapidly cleaved by the enzyme. [Bta5] and [Bfa5]CCK-8 did not prove to be quite resistant; however the C-terminal tetrapeptides having the same modifications on the Trp residue were not cleaved although they interacted with the enzyme binding site. The stability and biological activity of the peptidase-resistant analogues of CCK-8 remain to be determined in vivo.
ASJC Scopus subject areas
- Cellular and Molecular Neuroscience
- Cell Biology