Z phe leu oh

Peptidase activity of each purified recombinant CPY was assayed by incubating with N‐benzyloxycarbonyl‐l‐phenylalanyl‐l‐leucine (Z‐Phe‐Leu‐OH; Bachem, Bubendorf, Switzerland) at pH 7.0 and room temperature. l‐Leucine, a hydrolysis product, was measured by amino acid analysis using the LaChromUltra U‐HPLC system (Hitachi High‐Technologies, Tokyo, Japan). Anilidase activity of each CPY was determined spectrophotometrically 17. The release of p‐nitroaniline from N‐benzoyl‐l‐tyrosine p‐nitroanilide (Bz‐Tyr‐pNA; Sigma‐Aldrich, St. Louis, MO, USA) at pH 7.0 and room temperature was monitored at 410 nm using a model U‐2000A spectrophotometer (Hitachi High‐Technologies).

The following reagents, substrates, and protease inhibitors were utilized in this research: Bz-Arg-Gly-Phe-Phe-Pro-4M2NA, Leu-AMC, pepstatin A, bestatin, RKLLW-NH2, human Hb, and fluorescamine from Sigma-Aldrich. Z-Phe-Arg-AMC and Z-Phe-Leu-OH from Bachem, Z-Ala-Ala-Asn-AMC, CA-074, and legumain inhibitor1 from MedChemExpress. E-64 and EDTA from Merck. PMSF and EDTA-free protease inhibitor cocktail from Roche (Merck). The stock solutions of these products were prepared, following the instructions provided by the respective providers.