PURIFICATION AND PROPERTIES OF EMBRYONIC CYSTEINE PROTEINASE WHICH PARTICIPATES IN YOLK-LYSIS OF XENOPUS-LAEVIS

The study reported here aimed to purify a cysteine proteinase from neu rula embryos of Xenopus lacvis, since this enzyme was thought to be in volved in yolk-lysis in developing embryos. The purification procedure consisted of fractionation of an embryonic extract by means of 30-90% ammonium sulfate, chromatography on diethylaminoethyl cellulose and c arboxymethyl cellulose, gel filtration on Sephadex G-75 and affinity c hromatography on concanavalin A-agarose. The purified enzyme had a mol ecular weight of 30 kDa according to both SDS-PAGE and Sephadex G-75 g el-filtration and an optimum pH of 5.5, and it preferentially cleaved the synthetic substrate, Z-Phe-Arg-MCA. Its activity was inhibited by Z-Phe-Phe-CHN2, a specific cathepsin L inhibitor, as well as by leupep tin and E-64. The NH2-terminal amino acid sequence of the enzyme was s imilar to that of chicken cathepsin B. These characteristics indicate that the purified enzyme is a member of the cysteine proteinase family . The antibody raised against the purified enzyme specifically stained a 30 kDa protein of neurula embryo extracts on immunoblot tests. The enzyme effectively digested Xenopus yolk proteins when the NaCl concen tration in test solutions was 0.2 M. It was also confirmed that cystei ne proteinase inhibitors inhibited yolk-lysis by the enzyme. (C) 1998 Elsevier Science Inc. All rights reserved.