Specific fluorogenic substrates for neprilysin (neutral endopeptidase, EC 3.4.24.11) which are highly resistant to serine- and metalloproteases
Autor(a) principal: | |
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Data de Publicação: | 1997 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Brazilian Journal of Medical and Biological Research |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001000003 |
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") --><FONT FACE="Symbol">a</font>-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") --><FONT FACE="Symbol">a</font>-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparations |
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oai:scielo:S0100-879X1997001000003 |
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Brazilian Journal of Medical and Biological Research |
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|
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") --><FONT FACE="Symbol">a</font>-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") --><FONT FACE="Symbol">a</font>-chymotrypsin. The high specifity of these substrates suggests their use for specific NEP assays in crude enzyme preparationsAssociação Brasileira de Divulgação Científica1997-10-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001000003Brazilian Journal of Medical and Biological Research v.30 n.10 1997reponame:Brazilian Journal of Medical and Biological Researchinstname:Associação Brasileira de Divulgação Científica (ABDC)instacron:ABDC10.1590/S0100-879X1997001000003info:eu-repo/semantics/openAccessMedeiros,M.A.S.França,M.S.F.Boileau,G.Juliano,L.Carvalho,K.M.eng1998-10-07T00:00:00Zoai:scielo:S0100-879X1997001000003Revistahttps://www.bjournal.org/https://old.scielo.br/oai/scielo-oai.phpbjournal@terra.com.br||bjournal@terra.com.br1414-431X0100-879Xopendoar:1998-10-07T00:00Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC)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,L. Carvalho,K.M. |
author_role |
author |
author2 |
França,M.S.F. Boileau,G. Juliano,L. Carvalho,K.M. |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Medeiros,M.A.S. França,M.S.F. Boileau,G. Juliano,L. 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") --><FONT FACE="Symbol">a</font>-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") --><FONT FACE="Symbol">a</font>-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 |
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://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001000003 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0100-879X1997001000003 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S0100-879X1997001000003 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
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 |
Brazilian Journal of Medical and Biological Research v.30 n.10 1997 reponame:Brazilian Journal of Medical and Biological Research instname:Associação Brasileira de Divulgação Científica (ABDC) instacron:ABDC |
instname_str |
Associação Brasileira de Divulgação Científica (ABDC) |
instacron_str |
ABDC |
institution |
ABDC |
reponame_str |
Brazilian Journal of Medical and Biological Research |
collection |
Brazilian Journal of Medical and Biological Research |
repository.name.fl_str_mv |
Brazilian Journal of Medical and Biological Research - Associação Brasileira de Divulgação Científica (ABDC) |
repository.mail.fl_str_mv |
bjournal@terra.com.br||bjournal@terra.com.br |
_version_ |
1754302928823255040 |