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Characterization of Cysteine Proteases Functioning in Degradation of Dynorphin in Neuroblastoma Cells: Evidence for the Presence of a Novel Enzyme with Strict Specificity Toward Paired Basic Residues
Author(s) -
Satoh Mitsuo,
Yokosawa Hideyoshi,
Ishii Shinichi
Publication year - 1989
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1989.tb10898.x
Subject(s) - leupeptin , antipain , chemistry , cysteine , affinity chromatography , size exclusion chromatography , proteases , trypsin , biochemistry , sepharose , enzyme , cysteine protease , protease , chromatography
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 and p ‐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 by p ‐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 Arg 6 ‐Arg 7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1–17) at the Lys 11 ‐Leu 12 bond and the Leu 12 ‐Lys 13 bond 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 were examined as substrates for the enzyme.