Purification and characterization of a protease from Xenopus embryos

Abstract

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.Nearly all proteins in animal cells are continuously degraded to their constituent amino acids and this process contributes in an important way to the control of the levels of many enzymes and functional factors [l -31. Despite the importance of this process, the exact identification of the proteolytic enzymes that catalyze the degradation of proteins in vivo remains obscure. Animal cells contain many different proteases and there are both lysosomal and non-lysosomal pathways of protein degradation [4-61. Two of the major types of pathways for the degradation of proteins are clearly distinguishable, but many features of both pathways are not well understood. In general, the lysosomal proteases are small (20 -40 kDa) and they are maximally active under acidic conditions.Recent studies have indicated that the degradation of many intracellular proteins also occurs in the cytoplasm [7, 81. Although the mechanism of selection of substrates for degradation in the cytoplasm have not yet been established, many non-lysosomal proteases have been purified and characterized. For example, ATP-dependent proteases, multicatalytic proteases, and Ca2 +-dependent proteases have been found in animal cells as non-lysosomal proteases [4 -61.It has been reported that proteases play an important role in embryonic development [9 -121. We reported previously that a protease inhibitor, antipain, inhibits the incorporation of [3H]uridine into RNA in Xenopus embryos [13] and that the level of the proteolytic activity which is sensitive to antipain increased by a factor of several thousand during development up to the tail-bud stage [14]. In this report, we describe the purification of the antipain-sensitive enzyme and some of its properties. MATERIALS AND METHODS Chemicals Preparation ojeggs and embryosXenopus embryos obtained after mating were dejellied and allowed to develop to the tail-bud stage at room temperature. The embryos were immersed in cold acetone and homogenized in a glass homogenizer. The homogenate was centrifuged at 3000 r...

Effects on properties of a thiol protease from Xenopus embryos of changes in substrate and assay conditions.

“…Although the contribution of degradation by lysosomal enzymes to yolk resorption is also recognized, relatively little is known about this process (Decroly et at., 1979;Wall and Meleka 1985). We have purified a protease from Xenopus embryos that is activated at the early stages of embryogenesis (Miyata and Kihara, 1989). The purified protease has characteristics of an acid thiol protease.…”