Purification and characterization of cathepsin B from hepatopancreas of carp Cyprinus carpio

Cathepsin B was purified from the crude extract of carp hepatopancreas by a modified method, and the specific activity increased about 3,400-fold with a 17% recovery. The purified enzyme gave a single protein band on Native PAGE, whereas two bands with molecular weights of 30,000 (single chain) and 26,000 (heavy chain) migrated on SDS-PAGE. The enzyme potently hydrolyzed Z-Arg-Arg-MCA and Z-Phe-Arg-MCA but lacked the ability to hydrolyze most of the other MCA substrates. The kinetic constants of the enzyme with two Z-blocked substrates revealed that V-max and K-cat Values of Z-Phe-Arg-MCA were much higher than Z-Arg-Arg-MCA, while their K-m values were approximate. The optimum hydrolysis pH and temperature of the enzyme for these two substrates were determined to be pH 6.0 and 45 degrees C, respectively. A Variety of protease inhibitors such as E-64, DTNB, antipain, leupeptin, chymostatin, TLCK and TPCK and metal compounds such as CuCl2, CdCl2, HgCl2, and Zn(CH3COO)(2) were able to significantly inactivate the enzyme. In contrast, cysteine and 2-mercaptoethanol activated the enzyme, with cysteine being more effective for activation than 2-mercaptoethanol over the tested concentrations. (C) 1997 Elsevier Science Inc.