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

• Published in: Biochimica et biophysica acta Vol. 675; no. 3-4; pp. 379 - 385
• Main Authors: Okajima, Kenji, Inoue, Masayasu, Morino, Yoshimasa
• Format: Journal Article
• Language: English
• Published: Netherlands 17.07.1981
• Subjects: Aminopeptidases - metabolism; CD13 Antigens; Cell Fractionation; Diazooxonorleucine - pharmacology; Dipeptidases - metabolism; gamma-Glutamyltransferase - metabolism; Glutathione - analogs & derivatives; Glutathione - metabolism; Kidney - enzymology; Membranes - enzymology; Microsomes - enzymology; Microvilli - enzymology; Peptide Hydrolases - metabolism; Phenanthrolines - pharmacology
• ISSN: 0304-4165; 0006-3002
• DOI: 10.1016/0304-4165(81)90029-5

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.