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

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dc.contributor.authorCarvalho, K. M.
dc.contributor.authorBoileau, G.
dc.contributor.authorCamargo, ACM
dc.contributor.authorJuliano, L.
dc.contributor.institutionUNIV MONTREAL
dc.contributor.institutionINST BUTANTA
dc.contributor.institutionUniversidade Federal de São Paulo (UNIFESP)
dc.date.accessioned2016-01-24T11:40:32Z
dc.date.available2016-01-24T11:40:32Z
dc.date.issued1996-06-01
dc.description.abstractAn 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 3.4.24.11) 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.en
dc.description.affiliationUNIV MONTREAL,FAC MED,DEPT BIOCHIM,MONTREAL,PQ H3C 3J7,CANADA
dc.description.affiliationINST BUTANTA,BR-05503900 São Paulo,BRAZIL
dc.description.affiliationESCOLA PAULISTA MED,DEPT BIOFIS,BR-04044 São Paulo,BRAZIL
dc.description.affiliationUnifespESCOLA PAULISTA MED,DEPT BIOFIS,BR-04044 São Paulo,BRAZIL
dc.description.sourceWeb of Science
dc.format.extent167-173
dc.identifierhttp://dx.doi.org/10.1006/abio.1996.0224
dc.identifier.citationAnalytical Biochemistry. San Diego: Academic Press Inc Jnl-comp Subscriptions, v. 237, n. 2, p. 167-173, 1996.
dc.identifier.doi10.1006/abio.1996.0224
dc.identifier.issn0003-2697
dc.identifier.urihttp://repositorio.unifesp.br/handle/11600/25597
dc.identifier.wosWOS:A1996UP62500001
dc.language.isoeng
dc.publisherAcademic Press Inc Jnl-comp Subscriptions
dc.relation.ispartofAnalytical Biochemistry
dc.rightsinfo:eu-repo/semantics/restrictedAccess
dc.titleA highly selective assay for neutral endopeptidase based on the cleavage of a fluorogenic substrate related to Leu-enkephalinen
dc.typeinfo:eu-repo/semantics/article
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