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.
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 Arg6-Arg7 bond with the optimum pH of 8.0, whereas II cleaved dynorphin(1-17) at the Lys11-Leu12 bond and the Leu12-Lys13 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.
Satoh
M
M
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Yokosawa
H
H
Ishii
S
S
D000818:Animals; D002460:Cell Line; D002729:Chloromercuribenzoates / Q000494:pharmacology; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004399:Dynorphins / Q000378:metabolism* / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D051379:Mice; D008970:Molecular Weight; D009447:Neuroblastoma / Q000201:enzymology / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D011480:Protease Inhibitors; D020245:p-Chloromercuribenzoic Acid; D004399:Dynorphins; D003546:Cysteine Endopeptidases; D005033:Ethylmaleimide