Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases

Autor Medeiros, M.a.s. Google Scholar
França, M.s.f. Google Scholar
Boileau, G. Google Scholar
Juliano, Luiz Autor UNIFESP Google Scholar
Carvalho, K.m. Google Scholar
Instituição Universidade Federal do Ceará
Université de Montréal
Universidade Federal de São Paulo (UNIFESP)
Resumo Two intramolecularly quenched fluorogenic peptides containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-<!-- $MVD$:face(Times) -->DArg-Arg-Leu-EDDnp (Abz-<!-- $MVD$:face(Times) -->DRRL-EDDnp) and Abz-<!-- $MVD$:face(Times) -->DArg-Arg-Phe-EDDnp (Abz-<!-- $MVD$:face(Times) -->DRRF-EDDnp), were selectively hydrolyzed by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC 3.4.24.11) at the Arg-Leu and Arg-Phe bonds, respectively. The kinetic parameters for the NEP-catalyzed hydrolysis of Abz-<!-- $MVD$:face(Times) -->DRRL-EDDnp and Abz-<!-- $MVD$:face(Times) -->DRRF-EDDnp were Km = 2.8 µM, kcat = 5.3 min-1, kcat/Km = 2 min-1 µM-1 and Km = 5.0 µM, kcat = 7.0 min-1, kcat/Km = 1.4 min-1 µM-1, respectively. The high specificity of these substrates was demonstrated by their resistance to hydrolysis by metalloproteases [thermolysin (EC 3.4.24.2), angiotensin-converting enzyme (ACE; EC 3.4.24.15)], serineproteases [trypsin (EC 3.4.21.4), <!-- $MVD$:face(Symbol) -->a-chymotrypsin (EC 3.4.21.1)] and proteases present in tissue homogenates from kidney, lung, brain and testis. The blocked amino- and carboxyl-terminal amino acids protected these substrates against the action of aminopeptidases, carboxypeptidases and ACE. Furthermore, <!-- $MVD$:face(Times) -->DR amino acids ensured total protection of Abz-<!-- $MVD$:face(Times) -->DRRL-EDDnp and Abz-<!-- $MVD$:face(Times) -->DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by <!-- $MVD$:face(Symbol) -->a-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations
Palavra-chave neutral endopeptidase
enkephalinase
neprilysin
fluorogenic substrates
phosphoramidon
Idioma Inglês
Data de publicação 1997-10-01
Publicado em Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 30, n. 10, p. 1157-1162, 1997.
ISSN 0100-879X (Sherpa/Romeo, fator de impacto)
Publicador Associação Brasileira de Divulgação Científica
Extensão 1157-1162
Fonte http://dx.doi.org/10.1590/S0100-879X1997001000003
Direito de acesso Acesso aberto Open Access
Tipo Artigo
Web of Science WOS:A1997YA76600003
SciELO S0100-879X1997001000003 (estatísticas na SciELO)
Endereço permanente http://repositorio.unifesp.br/handle/11600/538

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