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 neurula embryos of Xenopus laevis, since this enzyme was thought to be involved 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 carboxymethyl cellulose, gel filtration on Sephadex G-75 and affinity chromatography on concanavalin A-agarose. The purified enzyme had a molecular weight of 30 kDa according to both SDS-PAGE and Sephadex G-75 gel-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 leupeptin and E-64. The NH2-terminal amino acid sequence of the enzyme was similar 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 concentration in test solutions was 0.2 M. It was also confirmed that cysteine proteinase inhibitors inhibited yolk-lysis by the enzyme.
Yoshizaki
N
N
Department of Biological Diversity and Resources, Faculty of Agriculture, Gifu University, Japan. pflayer@cc.gifu-u.ac.jp
Moriyama
A
A
Yonezawa
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002403:Cathepsins / Q000737:chemistry*; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003208:Concanavalin A; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004530:Egg Yolk / Q000502:physiology; D004625:Embryo, Nonmammalian / Q000201:enzymology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009420:Nervous System / Q000196:embryology* / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014982:Xenopus laevis / Q000196:embryology*
D003208:Concanavalin A; D002403:Cathepsins; D003546:Cysteine Endopeptidases