A highly selective assay for neutral endopeptidase based on the cleavage of a fluorogenic substrate related to Leu-enkephalin

A highly selective assay for neutral endopeptidase based on the cleavage of a fluorogenic substrate related to Leu-enkephalin

Autor Carvalho, K. M. Google Scholar
Boileau, G. Google Scholar
Camargo, ACM Google Scholar
Juliano, L. Google Scholar
Universidade Federal de São Paulo (UNIFESP)
Resumo An intramolecularly quenched fluorogenic peptide structurally related to Leu-enkephalin, containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (EDDnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Gly-Gly-D-Phe-Leu-Arg-Arg-Val-EDDnp (Abz-GGDFLRRV-EDDnp), was selectively hydrolyzed at the Arg-Val bond by neutral endopeptidase (NEP, enkephalinase, neprilysin, EC with kinetic parameters (K-m = 3 mu M, k(cat) = 127 min(-1) and K-cat/K-m = 42 min(-1) mu M(-1)) similar to those of the Leu-enkephalin. the specificity of the NEP assay was demonstrated by incubating Abz-GGDFLRRV-EDDnp with a kidney homogenate or with crude membrane preparations of brain and lung: more than 95% of all products released were the complementary fragments Abz-GGDFLRR and V-EDDnp which were totally inhibited by 1 mu M thiorphan, a highly specific NEP inhibitor. the blocked amino- and carboxyl-terminal amino acids protected this substrate against the action of aminopeptidases as well as of carboxypeptidases. Furthermore, D-Phe amino acid also ensured a very good protection of Abz-GGDFLRRV-EDDnp against the action of other tissue endopeptidases distinct from NEP. A continuous fluorometric assay for only 5 min was sufficient to quantify the NEP activity with a minimum sensitivity of 5 ng of purified enzyme or the equivalent enzymatic activity in crude tissue preparations, Therefore, amounts as little as 0.5 ng of enzyme could be quantified employing longer times of incubation. (C) 1996 Academic Press, Inc.
Idioma Inglês
Data 1996-06-01
Publicado em Analytical Biochemistry. San Diego: Academic Press Inc Jnl-comp Subscriptions, v. 237, n. 2, p. 167-173, 1996.
ISSN 0003-2697 (Sherpa/Romeo, fator de impacto)
Editor Academic Press Inc Jnl-comp Subscriptions
Extensão 167-173
Fonte http://dx.doi.org/10.1006/abio.1996.0224
Direito de acesso Acesso restrito
Tipo Artigo
Web of Science WOS:A1996UP62500001
URI http://repositorio.unifesp.br/handle/11600/25597

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