Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates
Autor(a) principal: | |
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Data de Publicação: | 1998 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNIFESP |
Texto Completo: | http://repositorio.unifesp.br/handle/11600/25941 http://dx.doi.org/10.1074/jbc.273.35.22672 |
Resumo: | In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. in this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (k(cat)/K-m) between 9.4 x 10(4) M-1 s(-1)(Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s(-1) (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s(-1) (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s(-1) (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited k(cat) of less than 0.05 s(-1). Substitution of ornithine for Lys at the P4 position did not significantly affect the k(cat) but increased the K-m 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1, These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters. |
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Johanning, K.Juliano, Maria Aparecida [UNIFESP]Juliano, Luiz [UNIFESP]Lazure, C.Lamango, N. S.Steiner, D. F.Lindberg, ILouisiana State UnivUniversidade Federal de São Paulo (UNIFESP)Inst Rech Clin MontrealUniv Chicago2016-01-24T12:30:38Z2016-01-24T12:30:38Z1998-08-28Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 273, n. 35, p. 22672-22680, 1998.0021-9258http://repositorio.unifesp.br/handle/11600/25941http://dx.doi.org/10.1074/jbc.273.35.2267210.1074/jbc.273.35.22672WOS:000075616600073In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. in this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (k(cat)/K-m) between 9.4 x 10(4) M-1 s(-1)(Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s(-1) (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s(-1) (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s(-1) (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited k(cat) of less than 0.05 s(-1). Substitution of ornithine for Lys at the P4 position did not significantly affect the k(cat) but increased the K-m 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1, These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters.Louisiana State Univ, Med Ctr, Sch Med, Dept Biochem & Mol Biol, New Orleans, LA 70112 USAEscola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilInst Rech Clin Montreal, Neuropeptide Struct & Metab Lab, Quebec City, PQ 2W 1R7, CanadaUniv Chicago, Sch Med, Howard Hughes Med Inst, Chicago, IL 60637 USAUniv Chicago, Sch Med, Dept Biochem, Chicago, IL 60637 USAEscola Paulista Med, Dept Biophys, BR-04044020 São Paulo, BrazilWeb of Science22672-22680engAmer Soc Biochemistry Molecular Biology IncJournal of Biological ChemistrySpecificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substratesinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP11600/259412023-02-15 09:10:17.695metadata only accessoai:repositorio.unifesp.br:11600/25941Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestopendoar:34652023-02-15T12:10:17Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false |
dc.title.en.fl_str_mv |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates |
title |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates |
spellingShingle |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates Johanning, K. |
title_short |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates |
title_full |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates |
title_fullStr |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates |
title_full_unstemmed |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates |
title_sort |
Specificity of prohormone convertase 2 on proenkephalin and proenkephalin-related substrates |
author |
Johanning, K. |
author_facet |
Johanning, K. Juliano, Maria Aparecida [UNIFESP] Juliano, Luiz [UNIFESP] Lazure, C. Lamango, N. S. Steiner, D. F. Lindberg, I |
author_role |
author |
author2 |
Juliano, Maria Aparecida [UNIFESP] Juliano, Luiz [UNIFESP] Lazure, C. Lamango, N. S. Steiner, D. F. Lindberg, I |
author2_role |
author author author author author author |
dc.contributor.institution.none.fl_str_mv |
Louisiana State Univ Universidade Federal de São Paulo (UNIFESP) Inst Rech Clin Montreal Univ Chicago |
dc.contributor.author.fl_str_mv |
Johanning, K. Juliano, Maria Aparecida [UNIFESP] Juliano, Luiz [UNIFESP] Lazure, C. Lamango, N. S. Steiner, D. F. Lindberg, I |
description |
In the central and peripheral nervous systems, the neuropeptide precursor proenkephalin must be endoproteolytically cleaved by enzymes known as prohormone convertases 1 and 2 (PC1 and PC2) to generate opioid-active enkephalins. in this study, we have investigated the specificity of recombinant mouse PC2 for proenkephalin-related internally quenched (IQ) peptides, for methylcoumarin amide-based fluorogenic peptides, and for recombinant rat proenkephalin. IQ peptides exhibited specificity constants (k(cat)/K-m) between 9.4 x 10(4) M-1 s(-1)(Abz-Val-Pro-Arg-Met-Glu-Lys-Arg-Tyr-Gly-Gly-Phe-Met-Gln-EDDnp; where Abz is ortho-aminobenzoic acid and EDDnp is N-(2,4-dinitrophenyl)ethylenediamine)) and 0.24 x 10(4) M-1 s(-1) (Abz-Tyr-Gly-Gly-Phe-Met-Arg-Arg-Val-Gly-Arg-Pro-Glu-EDDnp), with the peptide B to Met-enk-Arg-Phe cleavage preferred (Met-enk is met-enkephalin). Fluorogenic substrates with P1, P2, and P4 basic amino acids were hydrolyzed with specificity constants ranging between 2.0 x 10(3) M-1 s(-1) (Ac-Orn-Ser-Lys-Arg-MCA; where MCA is methylcoumarin amide) and 1.8 x 10(4) M-1 s(-1) (<Glu-Arg-Thr-Lys-Arg-MCA; where <Glu is pyroglutamic acid). Substrates containing only a single basic residue were not appreciably hydrolyzed, and substrates lacking a P4 Arg exhibited k(cat) of less than 0.05 s(-1). Substitution of ornithine for Lys at the P4 position did not significantly affect the k(cat) but increased the K-m 2-fold. Data from both sets of fluorogenic substrates supported the contribution of a P4 Arg to PC2 preference. Analysis of proenkephalin reaction products using immunoblotting and gel permeation chromatography demonstrated that PC2 can directly cleave proenkephalin and that the generation of small opioid peptides from intermediates is mediated almost entirely by PC2 rather than by PC1, These results are in accord with the analysis of PC2 knock-out brains, in which the amounts of three mature enkephalins were depleted by more than three-quarters. |
publishDate |
1998 |
dc.date.issued.fl_str_mv |
1998-08-28 |
dc.date.accessioned.fl_str_mv |
2016-01-24T12:30:38Z |
dc.date.available.fl_str_mv |
2016-01-24T12:30:38Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 273, n. 35, p. 22672-22680, 1998. |
dc.identifier.uri.fl_str_mv |
http://repositorio.unifesp.br/handle/11600/25941 http://dx.doi.org/10.1074/jbc.273.35.22672 |
dc.identifier.issn.none.fl_str_mv |
0021-9258 |
dc.identifier.doi.none.fl_str_mv |
10.1074/jbc.273.35.22672 |
dc.identifier.wos.none.fl_str_mv |
WOS:000075616600073 |
identifier_str_mv |
Journal of Biological Chemistry. Bethesda: Amer Soc Biochemistry Molecular Biology Inc, v. 273, n. 35, p. 22672-22680, 1998. 0021-9258 10.1074/jbc.273.35.22672 WOS:000075616600073 |
url |
http://repositorio.unifesp.br/handle/11600/25941 http://dx.doi.org/10.1074/jbc.273.35.22672 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.none.fl_str_mv |
Journal of Biological Chemistry |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
22672-22680 |
dc.publisher.none.fl_str_mv |
Amer Soc Biochemistry Molecular Biology Inc |
publisher.none.fl_str_mv |
Amer Soc Biochemistry Molecular Biology Inc |
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 |
|
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1802764191257329664 |