Open AccessPurification and characterization of a protease from Xenopus embryos
Author(s) -
MIYATA Shohei,
YOSHIDA Yoichi,
KIHARA Hirozi K.
Publication year - 1989
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1989.tb15176.x
Subject(s) - leupeptin , pepstatin , iodoacetic acid , protease , biochemistry , chemistry , phenylmethylsulfonyl fluoride , antipain , proteolytic enzymes , chromatography , sephadex , polyacrylamide gel electrophoresis , dithiothreitol , molecular mass , size exclusion chromatography , xenopus , enzyme , gene
A proteolytic enzyme was purified from Xenopus embryos. The purification procedure consisted of fractionation of an extract of embryos with acetone, gel filtration of Sephadex G‐75 and chromatography on carboxymethyl‐cellulose and hydroxylapatite. The preparation of enzyme appeared to be homogeneous as judged by electrophoresis in polyacrylamide gels. This protease had a molecular mass of 43–44 kDa and was composed of two subunits with molecular masses of 30 kDa and 13 kDa. The optimal pH of the reaction catalysed by the protease was approximately 4.0. This proteolytic activity was inhibited by antipain, leupeptin and iodoacetic acid; it was not affected by phenylmethylsulfonyl fluoride and pepstatin; and it was enhanced by dithiothreitol. In the presence of RNA, the optimal pH was shifted from pH 4.0 to pH 4.5. The protease was activated by addition of total RNA from Xenopus embryos, by poly(rU) or poly(rG). In contrast, after addition of tRNA or poly(rC), no activation of the protease was observed.