Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

Autor Puccia, Rosana Autor UNIFESP Google Scholar
Juliano, Maria Aparecida Autor UNIFESP Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Travassos, Luiz Rodolpho Autor UNIFESP Google Scholar
Carmona, Adriana Karaoglanovic Autor UNIFESP Google Scholar
Instituição A01
Universidade Federal de São Paulo (UNIFESP)
Resumo We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.
Palavra-chave P. brasiliensis
serine-thiol proteinase
SDS-PAGE
fluorescent-quenched peptides
Idioma Inglês
Data de publicação 1999-05-01
Publicado em Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 5, p. 645-649, 1999.
ISSN 0100-879X (Sherpa/Romeo, fator de impacto)
Publicador Associação Brasileira de Divulgação Científica
Extensão 645-649
Fonte http://dx.doi.org/10.1590/S0100-879X1999000500019
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:000080489600019
SciELO S0100-879X1999000500019 (estatísticas na SciELO)
Endereço permanente http://repositorio.unifesp.br/handle/11600/777

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