Topology and some properties of the renal brush border membrane-bound peptidase(s) participating in the metabolism of S-carbamidomethyl glutathione.

Topological features and some properties of the membrane-bound peptidase(s) participating in the metabolism of a glutathione S-conjugate in the kidney were studied. S-Carbamidomethyl glutathione, a model compound for glutathione S-conjugate, was demonstrated to be sequentially hydrolyzed by gamma-glutamyltransferase (5-glutamyl)-peptide:amino-acid 5-glutamyltransferase; EC 2.3.2.2) and peptidase(s) bound to rat renal brush border membrane vesicles. Hydrolysis of S-carbamidomethyl cysteinylglycine was found to be inhibited by 1,10-o-phenanthroline, suggesting a participation of a metal-requiring peptidase in this process. The hydrolytic activity of the membranous peptidase was markedly depressed by cysteinylglycine S-acetyldextran polymer (molecular weight, 500 000), a nonpermeating derivative for cysteinylglycine. Papain treatment of brush border membrane vesicles resulted in the solubilization of most hydrolytic activity toward S-carbamidomethyl cysteinylglycine. Amino-peptidase M was also solubilized from the membrane and the increase in the specific activity of this enzyme in the papain-soluble fraction was in parallel within that of the peptidase activity for hydrolysis of S-carbamidomethyl cysteinylglycine. The hydrolytic activity of purified brush border membrane vesicles toward S-carbamidomethyl glutathione was fully reconstituted by the combined use of purified gamma-glutamyltransferase and aminopeptidase M. These findings indicated that, as in the case of the cleavage of gamma-glutamyl linkage of glutathione and related compounds, hydrolysis of the S-substituted cysteinylglycine occurred exclusively on the lumenal surface of renal brush border membrane as catalyzed mainly by aminopeptidase M.