Effects of peptidase inhibitors on the [Met5]-enkephalin hydrolysis in ileal and striatal preparations of guinea-pig: almost complete protection of degradation by the combination of amastatin, captopril and thiorphan

Abstract

The contents of [Met5]-enkephalin and its four hydrolysis products, Tyr, Tyr-Gly, Tyr-Gly-Gly, and Tyr-Gly-Gly-Phe, in the sample obtained after enkephalin was incubated with tissues in either the absence or the presence of the peptidase inhibitor were estimated by high performance liquid chromatography combined with electrochemical detection in order to elucidate the effect of the peptidase inhibitor on enkephalin hydrolysis. Free Tyr was the major degradative product in the absence of the peptidase inhibitor, while the major hydrolysis product was the Tyr-Gly-Gly fragment in the presence of amastatin in both total homogenates and membrane fractions prepared from either the myenteric plexus-longitudinal muscle strip of guinea-pig ileum or the striatum of guinea-pig brain. Additionally, captopril significantly decreased the generation of Tyr-Gly-Gly in the presence of amastatin in both ileal and striatal membrane fractions. Moreover, thiorphan significantly prevented the formation of Tyr-Gly in the presence of both amastatin and captopril in both membrane preparations. Finally, when [Met5]-enkephalin was incubated with either an ileal or a striatal membrane fraction for 60 min at 37 degrees C in the presence of three peptidase inhibitors, amastatin, captopril, and thiorphan, approximately 98 or 94%, respectively, of enkephalin remained intact. The results indicated that [Met5]-enkephalin was almost exclusively hydrolyzed by three distinct enzymes, amastatin-sensitive aminopeptidase, captopril-sensitive peptidyl dipeptidase A, and thiorphan-sensitive endopeptidase-24.11 in both ileal and striatal membrane fractions.