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.