Purification and characterization of cathepsin B from carp ordinary muscle.

Cathepsin B [EC 3.4.22.1] was purified from carp Cyprinus carpio muscle by ammonium sulfate fractionation at pH 3.0, CM-cellulose chromatography, gel-filtration on Ultrogel AcA54, and DEAE-Sephacel chromatography. The purified enzyme gave a single protein band on polyacryl-amide gel electrophoresis, and its molecular weight was estimated to be 29, 000 by gel-filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The activity of the enzyme was maximal at pH 6.0 with benzoyl-DL-Arg β-naphthylamide as a substrate. The enzyme required a thiol compound (cysteine, dithiotheritol or glutathione) and a metalchelating reagent (EDTA, EGTA or citric acid) for the activation, but was inhibited by 2-mercaptoethanol even in the presence of both cysteine and EDTA. The enzyme was also inhibited by E-64, leupeptin, antipain, Hg2+, Cd2+, Cu2+ and Zn2+. These properties except the inhibition by 2-mercaptoethanol were similar to those of cathepsin B from other sources. The enzyme hydrolyzed various neuropeptides such as Leu-enkephalin, β-neoendorphin and α-neoendorphin. It appeared to act on these peptides mainly as a dipeptidylcarboxypeptidase, although not so rigorously. Besides, the enzyme hydrolyzed myosin heavy chain, actin, and troponin-T of carp muscle, but did not hydrolyze tropomyosin and troponin-C.