Purification and characterization of cathepsin B from ordinary muscle of common mackerel Scomber japonicus.

Abstract

Cathepsin B (EC 3.4.22.1) was purified from the ordinary muscle of common mackerel by ammonium sulfate fractionation and successive chromatographies on Sephadex G-75, DEAE-Sepharose, CM-Sepharose, Q-Sepharose, and Sephacryl S-100 HR.The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 23, 000 by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 HR. The optimal pH of the enzyme for the hydrolysis of Z-Arg-Arg-NMec was 5.5. The enzyme was activated by sulfhydryl compounds, such as 2-mercapto-ethanol, cysteine, dithiothreitol, and glutathione. Among the compounds, cysteine was the most effective. The enzyme was moderately inhibited by pCMB and NEM, and strongly inhibited by TLCK, TPCK, antipain, leupeptin, E-64, Cu2+, and He2+. The enzyme hydrolyzed Z-Phe-Arg-NMec and Bz-Arg-NNap, but not Arg-NMec or Leu-NNap. The ratio of hydrolyzing activity against Z-Phe-Arg-NMec and Z-Arg-Arg-NMec was about 1:0.7.