Abstract:Two dynorphin‐degrading cysteine proteases, I and II, were extracted with Triton X‐100 from neuroblastoma cell membrane, isolated from accompanying dynorphin‐degrading trypsin‐like enzyme by affinity chromatography on columns of soybean trypsin inhibitor‐immobilized Sepharose andp‐mercuribenzoate–Sepharose, and separated by ion‐exchange chromatography on diethylaminoethyl (DEAE)‐cellulose and TSK gel DEAE‐5PW columns. Cysteine protease II was purified further by hydroxyapatite chromatography and gel filtration. The molecular weights of cysteine proteases I and II were estimated to be 100,000 and 70,000, respectively, by gel filtration. Both of the enzymes were inhibited byp‐chloromercuribenzoate,N‐ethylmaleimide, and high‐molecular‐weight kininogen, but not or only slightly inhibited by diisopropylphosphorofluoridate, antipain, leupeptin, E‐64, calpain inhibitor, and phosphoramidon. Cysteine protease I cleaved dynorphin(1–17) at the Arg6‐Arg7bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1–17) at the Lys11‐Leu12bond and the Leu12‐Lys13bond with the optimum pH values of 8.0 and 6.0, respectively. These bonds corresponded to those that had been proposed as the initial sites of degradation by neuroblastoma cell membrane. Cysteine protease I was further found to show strict specificity toward the Arg‐Arg doublet, when susceptibilities of various peptides containing paired basic residues w