Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases
Braz. j. med. biol. res
; 30(10): 1157-62, Oct. 1997. ilus, tab
Artigo
em Inglês
| LILACS
| ID: lil-201531
Biblioteca responsável:
BR1.1
ABSTRACT
Two intramolecularly quenched fluorogenic peptides containing oaminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid rsidues, Abz-Darg-Arg-Leu-EDDnp (Abz-DRRL-EDDnp) and Abz-DArg-Arg-Phe-EDDnp (Abz-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-DRRL-EDDnp and Abz-DRRF-EDDnp were Km = 2.8muM, Kcat = 5.3 min-1, Kcat/Km = 2 min-1 muM-1 and Km = 5.0 muM, Kcat = 7.0 min-1, Kcat/Km = 1.4 min-1 muM-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), alpha-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, DR amino acids ensured total protection of Abz-DRRL-EDDnp and Abz-DRRF-EDDnp against the action of thermolysin and trypsin. Leu-EDDnp and Phe-EDDnp were resistant to hydrolysis by alpha-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations.
Texto completo:
Disponível
Coleções:
Bases de dados internacionais
Base de dados:
LILACS
Assunto principal:
Técnicas In Vitro
/
Neprilisina
/
Substratos para Tratamento Biológico
/
Metaloproteases
/
Serina Proteases
Limite:
Animais
Idioma:
Inglês
Revista:
Braz. j. med. biol. res
Assunto da revista:
Biologia
/
Medicina
Ano de publicação:
1997
Tipo de documento:
Artigo