Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases
Autor(a) principal: | |
---|---|
Data de Publicação: | 1997 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://dx.doi.org/10.1590/S0100-879X1997001000003 http://repositorio.unifesp.br/handle/11600/538 |
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 |
id |
UFSP_66de82e1592f8b047f69fee154493b93 |
---|---|
oai_identifier_str |
oai:repositorio.unifesp.br/:11600/538 |
network_acronym_str |
UFSP |
network_name_str |
Repositório Institucional da UNIFESP |
repository_id_str |
3465 |
spelling |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteasesneutral endopeptidaseenkephalinaseneprilysinfluorogenic substratesphosphoramidonTwo 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 preparationsUniversidade Federal do CearáUniversité de MontréalUniversidade Federal de São Paulo (UNIFESP)UNIFESP, EPM, São Paulo, BrazilSciELOAssociação Brasileira de Divulgação CientíficaUniversidade Federal do CearáUniversité de MontréalUniversidade Federal de São Paulo (UNIFESP)Medeiros, M.a.s.França, M.s.f.Boileau, G.Juliano, Luiz [UNIFESP]Carvalho, K.m.2015-06-14T13:24:39Z2015-06-14T13:24:39Z1997-10-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion1157-1162application/pdfhttp://dx.doi.org/10.1590/S0100-879X1997001000003Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 30, n. 10, p. 1157-1162, 1997.10.1590/S0100-879X1997001000003S0100-879X1997001000003.pdf0100-879XS0100-879X1997001000003http://repositorio.unifesp.br/handle/11600/538WOS:A1997YA76600003engBrazilian Journal of Medical and Biological Researchinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-07-29T16:54:22Zoai:repositorio.unifesp.br/:11600/538Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-07-29T16:54:22Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.none.fl_str_mv |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases |
title |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases |
spellingShingle |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases Medeiros, M.a.s. neutral endopeptidase enkephalinase neprilysin fluorogenic substrates phosphoramidon |
title_short |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases |
title_full |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases |
title_fullStr |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases |
title_full_unstemmed |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases |
title_sort |
Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases |
author |
Medeiros, M.a.s. |
author_facet |
Medeiros, M.a.s. França, M.s.f. Boileau, G. Juliano, Luiz [UNIFESP] Carvalho, K.m. |
author_role |
author |
author2 |
França, M.s.f. Boileau, G. Juliano, Luiz [UNIFESP] Carvalho, K.m. |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade Federal do Ceará Université de Montréal Universidade Federal de São Paulo (UNIFESP) |
dc.contributor.author.fl_str_mv |
Medeiros, M.a.s. França, M.s.f. Boileau, G. Juliano, Luiz [UNIFESP] Carvalho, K.m. |
dc.subject.por.fl_str_mv |
neutral endopeptidase enkephalinase neprilysin fluorogenic substrates phosphoramidon |
topic |
neutral endopeptidase enkephalinase neprilysin fluorogenic substrates phosphoramidon |
description |
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 |
publishDate |
1997 |
dc.date.none.fl_str_mv |
1997-10-01 2015-06-14T13:24:39Z 2015-06-14T13:24:39Z |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1590/S0100-879X1997001000003 Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 30, n. 10, p. 1157-1162, 1997. 10.1590/S0100-879X1997001000003 S0100-879X1997001000003.pdf 0100-879X S0100-879X1997001000003 http://repositorio.unifesp.br/handle/11600/538 WOS:A1997YA76600003 |
url |
http://dx.doi.org/10.1590/S0100-879X1997001000003 http://repositorio.unifesp.br/handle/11600/538 |
identifier_str_mv |
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 30, n. 10, p. 1157-1162, 1997. 10.1590/S0100-879X1997001000003 S0100-879X1997001000003.pdf 0100-879X S0100-879X1997001000003 WOS:A1997YA76600003 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Brazilian Journal of Medical and Biological Research |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
1157-1162 application/pdf |
dc.publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica |
publisher.none.fl_str_mv |
Associação Brasileira de Divulgação Científica |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UNIFESP instname:Universidade Federal de São Paulo (UNIFESP) instacron:UNIFESP |
instname_str |
Universidade Federal de São Paulo (UNIFESP) |
instacron_str |
UNIFESP |
institution |
UNIFESP |
reponame_str |
Repositório Institucional da UNIFESP |
collection |
Repositório Institucional da UNIFESP |
repository.name.fl_str_mv |
Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP) |
repository.mail.fl_str_mv |
biblioteca.csp@unifesp.br |
_version_ |
1814268331320934400 |