RESUMO
A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.
Assuntos
Asterina/enzimologia , Catepsina D/isolamento & purificação , Catepsina D/metabolismo , Neuraminidase/isolamento & purificação , Sequência de Aminoácidos , Animais , Catepsina D/química , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Dados de Sequência Molecular , Neuraminidase/metabolismo , Inibidores de Proteases/farmacologia , Homologia de Sequência de AminoácidosRESUMO
We previously reported that immature starfish oocytes contain a novel 530-kDa proteasome-associating complex PC500 [previously named PC530; E. Tanaka, M. Takagi Sawada, C. Morinaga, H. Yokosawa, H. Sawada, Isolation and characterization of a novel 530-kDa protein complex (PC 530) capable of associating with the 20S proteasome from star fish oocytes, Arch. Biochem. Biophys. 374 (2000) 181-188]. In the present study, in order to obtain an insight into the biological function of this complex, we investigated the effects of anti-PC500 monoclonal antibodies on oocyte maturation of the starfish Asterina pectinifera. A monoclonal antibody 7C5 strongly inhibited germinal vesicle breakdown (GVBD) in a concentration-dependent manner. In contrast to the inhibitory effect of the 7C5 antibody on GVBD, no inhibition of egg cleavage was observed in a 7C5-antibody-microinjected single blastomere in a 2-cell stage embryo. These results indicate that PC500 plays a key role in starfish oocyte maturation in a meiosis-specific manner.
Assuntos
Anticorpos Monoclonais/química , Oócitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Eletroforese em Gel Bidimensional , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/metabolismo , Complexos Multiproteicos/química , Complexo de Endopeptidases do Proteassoma/química , Estrelas-do-Mar , Ubiquitina/químicaRESUMO
Fertilization and gametogenesis are key events in sexual reproduction. Our recent studies, together with several reports by other authors, demonstrated that the extracellular ubiquitin-proteasome system plays a role in fertilization and gametogenesis in addition to the traditional intracellular ubiquitin-proteasome system. Here, we summarize our recent results showing the importance of the extracellular ubiquitin-proteasome system in the sperm penetration through the vitelline coat of the egg during ascidian fertilization, together with our recent reports implicating the participation of a novel proteasome-associating complex PC530 in starfish oocyte maturation. We also describe the results by other authors showing the participation of the ubiquitin system both in the elimination of defective sperm in epididymis and in the elimination of paternal mitochondria in fertilized eggs. These are evidence of non-traditional extracellular functions of the ubiquitin system.
