Assessment of the role of “enkephalinase” in cholecystokinin inactivation

Abstract

Cholecystokinin octapeptide and thé C-terminal tetrapeptide are hydrolysed by a highly purified preparation of “enkephalinase” (EC 3.4.24.11). In both cases the Asp-PheNH, bond is hydrolysed and the Gly 4-Trp 5 bond of the octapeptide is also cleaved, though more slowly. Evaluated from the appearance of Phe-NH 2, the K m for the hydrolysis of the octapeptide by the purified peptidase is 57 μ M and that for the tetrapeptide 65 μM. The apparent affinities of these peptides for the enzyme in striatal membranes are similar. The importance of this hydrolysis in the inactivation of endogenous Cholecystokinin was assessed by studying the fate of Cholecystokinin immunoreactivity released from slices of rat cerebral cortex and striatum by depolarization with potassium. In the absence of any peptidase inhibitor only 16% of the peptide released from the tissue was recovered in immunoreactive form in the medium, indicating that endogenous Cholecystokinin octapeptide is, like other neuropeptides, rapidly and extensively hydrolysed following release. Selective inhibition of “enkephalinase” by Thiorphan ( dl-3-mercapto-2-benzyl-propanoyl glycine) did not significantly alter the recovery from slices of cerebral cortex and had only a very slight effect in the case of striatal slices. This suggests that, while Cholecystokinin octapeptide is a substrate for “enkephalinase”, this enzyme plays a less important (if any) role in the inactivation of endogenous Cholecystokinin than for the opioid peptides.