Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens)
Autor(a) principal: | |
---|---|
Data de Publicação: | 2017 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/13364 |
Resumo: | Acetylcholinesterase (AChE) (EC 3.1.1.7) is the enzyme responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine leading to the release of acetate and choline. In insects, acetylcholine is one of the most important neurotransmitters, and inhibition of AChE produces a generalized synaptic collapse causing the insect death. AChE is, thus, an important target for the development of new insecticides. In this context, this work describes the results obtained in the production of the active recombinant enzyme, expressed in Pichia pastoris and obtained through the gene coding for AChE of A. sexdens, cloned in the pPICZα-A vector. By sequencing the positive clone the sequence of the recombinant AChE was obtained and, by homology, a three-dimensional model was constructed and validated. In addition, this work presents, for the first time, the isolation, purification and biochemical characterization of two native AChE from Atta sexdens (AsAChE-A and AsAChE-B). The kinetic constants were determined using the colorimetric method with the enzymes in solution and acetylthiocholine as the substrate. The isolated enzymes were immobilized on fused silica capillary to produce AsAChE-A and AsAChE-B IMER. IMER activities were measured by quantification of the hydrolysis product of acetylcholine by zonal bioaffinity chromatography with mass spectrometry detection (LC-MS) to produce Michaelis-Menten constants. The differences in affinity for the substrates used: acetylthiocholine (colorimetric method) and acetylcholine (LC-MS) reveal AsAChE-B as the enzyme with the highest affinity for the natural substrate, a result confirmed, also, by measuring choline production with the enzyme in solution. The screening assay was validated using tacrine and galantamine as standard inhibitors of AChE, and the IMERs produced were used for screening inhibitors in a synthetic hydantoin (n = 12) collection. The results presented in this work show the importance of the use of acetylcholine (natural substrate) in AChE activity and inhibition assays. |
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Santos, Adriana Miranda dosSouza, Dulce Helena Ferreira dehttp://lattes.cnpq.br/3428955299526003http://lattes.cnpq.br/6845476344714243869845c0-9a86-4182-8788-5d4526867fd12020-10-22T20:03:32Z2020-10-22T20:03:32Z2017-09-29SANTOS, Adriana Miranda dos. Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens). 2017. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2017. Disponível em: https://repositorio.ufscar.br/handle/ufscar/13364.https://repositorio.ufscar.br/handle/ufscar/13364Acetylcholinesterase (AChE) (EC 3.1.1.7) is the enzyme responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine leading to the release of acetate and choline. In insects, acetylcholine is one of the most important neurotransmitters, and inhibition of AChE produces a generalized synaptic collapse causing the insect death. AChE is, thus, an important target for the development of new insecticides. In this context, this work describes the results obtained in the production of the active recombinant enzyme, expressed in Pichia pastoris and obtained through the gene coding for AChE of A. sexdens, cloned in the pPICZα-A vector. By sequencing the positive clone the sequence of the recombinant AChE was obtained and, by homology, a three-dimensional model was constructed and validated. In addition, this work presents, for the first time, the isolation, purification and biochemical characterization of two native AChE from Atta sexdens (AsAChE-A and AsAChE-B). The kinetic constants were determined using the colorimetric method with the enzymes in solution and acetylthiocholine as the substrate. The isolated enzymes were immobilized on fused silica capillary to produce AsAChE-A and AsAChE-B IMER. IMER activities were measured by quantification of the hydrolysis product of acetylcholine by zonal bioaffinity chromatography with mass spectrometry detection (LC-MS) to produce Michaelis-Menten constants. The differences in affinity for the substrates used: acetylthiocholine (colorimetric method) and acetylcholine (LC-MS) reveal AsAChE-B as the enzyme with the highest affinity for the natural substrate, a result confirmed, also, by measuring choline production with the enzyme in solution. The screening assay was validated using tacrine and galantamine as standard inhibitors of AChE, and the IMERs produced were used for screening inhibitors in a synthetic hydantoin (n = 12) collection. The results presented in this work show the importance of the use of acetylcholine (natural substrate) in AChE activity and inhibition assays.Acetilcolinesterase (AChE) (EC 3.1.1.7) é a enzima responsável por catalisar a hidrólise do neurotransmissor acetilcolina levando a liberação de acetato e colina. Nos insetos, a acetilcolina é um dos mais importantes neurotransmissores, e a inibição da AChE produz um colapso sináptico generalizado que leva o inseto a morte. A AChE é, assim, um alvo importante para o desenvolvimento de novos inseticidas. Neste contexto, este trabalho descreve os resultados obtidos na produção da enzima recombinante ativa, expressa em Pichia pastoris e obtida através do gene codificante para AChE de A. sexdens, clonado no vetor pPICZα-A. Através do sequenciamento do clone positivo a sequência da AChE recombinante foi obtida e, por método de modelagem por homologia, um modelo tridimensional foi construído e validado. Ademais, este trabalho apresenta de forma inédita, o isolamento, purificação e caracterização bioquímica de duas AChE nativas de Atta sexdens (AsAChE-A e AsAChE-B). As constantes cinéticas foram determinadas usando o método colorimétrico com as enzimas em solução e acetiltiocolina como substrato. Além disso, as enzimas isoladas foram imobilizadas em capilar de sílica fundida para produzir AsAChE-A e AsAChE-B IMER. As atividades dos IMER foram mensuradas através da quantificação do produto de hidrólise da acetilcolina por cromatografia zonal de bioafinidade, com detecção por espectrometria de massas (LC-MS), para o cálculo das constantes de Michaelis-Menten. As diferenças de afinidade pelos substratos usados: acetiltiocolina (método colorimétrico) e acetilcolina (LC-MS) revelam a AsAChE-B como a enzima de maior afinidade pelo substrato natural (acetilcolina), resultado confirmado, também, mensurando a produção de colina com a enzima em solução. Além disso, foi realizado um ensaio de triagem que foi validado usando tacrina e galantamina como inibidores padrão de AChE e os IMER produzidos foram usados para triagem de inibidores em uma coleção sintética de hidantoínas (n= 12). Os resultados apresentados neste trabalho mostram a importância do uso da acetilcolina, substrato natural, nos ensaios de atividade e inibição de AChE.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP 2012/16811-5porUniversidade Federal de São CarlosCâmpus São CarlosPrograma de Pós-Graduação em Química - PPGQUFSCarAttribution-NonCommercial-NoDerivs 3.0 Brazilhttp://creativecommons.org/licenses/by-nc-nd/3.0/br/info:eu-repo/semantics/openAccessAcetilcolinesteraseAtta sexdensImobilização em CapilarInibidoresCIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULARCIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOSCIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIACIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULASCIENCIAS EXATAS E DA TERRA::QUIMICACIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOSEstudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens)Biochemistry and kinetic studies of acetylcholinesterase of leaf-cutting antsinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis600600d64acd2a-121f-4d93-b884-274d4b53bf99reponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINALTese_versão_completa.pdfTese_versão_completa.pdfTeseapplication/pdf25261471https://repositorio.ufscar.br/bitstream/ufscar/13364/1/Tese_vers%c3%a3o_completa.pdf23fd436742e90aa04688c7a8db345bc9MD51carta-comprovante_homologacao.pdfcarta-comprovante_homologacao.pdfCarta comprovante assinada pelo orientadorapplication/pdf231729https://repositorio.ufscar.br/bitstream/ufscar/13364/3/carta-comprovante_homologacao.pdf35916084851c39769ba96ac53faf3ea1MD53CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8811https://repositorio.ufscar.br/bitstream/ufscar/13364/4/license_rdfe39d27027a6cc9cb039ad269a5db8e34MD54TEXTTese_versão_completa.pdf.txtTese_versão_completa.pdf.txtExtracted texttext/plain205187https://repositorio.ufscar.br/bitstream/ufscar/13364/5/Tese_vers%c3%a3o_completa.pdf.txt62d08e0823b0e6a05c00954343d584b2MD55carta-comprovante_homologacao.pdf.txtcarta-comprovante_homologacao.pdf.txtExtracted texttext/plain1101https://repositorio.ufscar.br/bitstream/ufscar/13364/7/carta-comprovante_homologacao.pdf.txtbbea63bcbe949afd2b4671f557152656MD57THUMBNAILTese_versão_completa.pdf.jpgTese_versão_completa.pdf.jpgIM Thumbnailimage/jpeg8693https://repositorio.ufscar.br/bitstream/ufscar/13364/6/Tese_vers%c3%a3o_completa.pdf.jpgf82442218bfd5d866fd9820c890b2dbaMD56carta-comprovante_homologacao.pdf.jpgcarta-comprovante_homologacao.pdf.jpgIM Thumbnailimage/jpeg10831https://repositorio.ufscar.br/bitstream/ufscar/13364/8/carta-comprovante_homologacao.pdf.jpg94c7812ae7980aca12040175b34316abMD58ufscar/133642023-09-18 18:32:03.197oai:repositorio.ufscar.br:ufscar/13364Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:32:03Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) |
dc.title.alternative.eng.fl_str_mv |
Biochemistry and kinetic studies of acetylcholinesterase of leaf-cutting ants |
title |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) |
spellingShingle |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) Santos, Adriana Miranda dos Acetilcolinesterase Atta sexdens Imobilização em Capilar Inibidores CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOS CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS CIENCIAS EXATAS E DA TERRA::QUIMICA CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS |
title_short |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) |
title_full |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) |
title_fullStr |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) |
title_full_unstemmed |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) |
title_sort |
Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens) |
author |
Santos, Adriana Miranda dos |
author_facet |
Santos, Adriana Miranda dos |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/6845476344714243 |
dc.contributor.author.fl_str_mv |
Santos, Adriana Miranda dos |
dc.contributor.advisor1.fl_str_mv |
Souza, Dulce Helena Ferreira de |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/3428955299526003 |
dc.contributor.authorID.fl_str_mv |
869845c0-9a86-4182-8788-5d4526867fd1 |
contributor_str_mv |
Souza, Dulce Helena Ferreira de |
dc.subject.por.fl_str_mv |
Acetilcolinesterase Atta sexdens Imobilização em Capilar Inibidores |
topic |
Acetilcolinesterase Atta sexdens Imobilização em Capilar Inibidores CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOS CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS CIENCIAS EXATAS E DA TERRA::QUIMICA CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS |
dc.subject.cnpq.fl_str_mv |
CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOLOGIA MOLECULAR CIENCIAS BIOLOGICAS::BIOQUIMICA::BIOQUIMICA DOS MICROORGANISMOS CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA CIENCIAS BIOLOGICAS::BIOQUIMICA::QUIMICA DE MACROMOLECULAS CIENCIAS EXATAS E DA TERRA::QUIMICA CIENCIAS BIOLOGICAS::GENETICA::GENETICA MOLECULAR E DE MICROORGANISMOS |
description |
Acetylcholinesterase (AChE) (EC 3.1.1.7) is the enzyme responsible for catalyzing the hydrolysis of the neurotransmitter acetylcholine leading to the release of acetate and choline. In insects, acetylcholine is one of the most important neurotransmitters, and inhibition of AChE produces a generalized synaptic collapse causing the insect death. AChE is, thus, an important target for the development of new insecticides. In this context, this work describes the results obtained in the production of the active recombinant enzyme, expressed in Pichia pastoris and obtained through the gene coding for AChE of A. sexdens, cloned in the pPICZα-A vector. By sequencing the positive clone the sequence of the recombinant AChE was obtained and, by homology, a three-dimensional model was constructed and validated. In addition, this work presents, for the first time, the isolation, purification and biochemical characterization of two native AChE from Atta sexdens (AsAChE-A and AsAChE-B). The kinetic constants were determined using the colorimetric method with the enzymes in solution and acetylthiocholine as the substrate. The isolated enzymes were immobilized on fused silica capillary to produce AsAChE-A and AsAChE-B IMER. IMER activities were measured by quantification of the hydrolysis product of acetylcholine by zonal bioaffinity chromatography with mass spectrometry detection (LC-MS) to produce Michaelis-Menten constants. The differences in affinity for the substrates used: acetylthiocholine (colorimetric method) and acetylcholine (LC-MS) reveal AsAChE-B as the enzyme with the highest affinity for the natural substrate, a result confirmed, also, by measuring choline production with the enzyme in solution. The screening assay was validated using tacrine and galantamine as standard inhibitors of AChE, and the IMERs produced were used for screening inhibitors in a synthetic hydantoin (n = 12) collection. The results presented in this work show the importance of the use of acetylcholine (natural substrate) in AChE activity and inhibition assays. |
publishDate |
2017 |
dc.date.issued.fl_str_mv |
2017-09-29 |
dc.date.accessioned.fl_str_mv |
2020-10-22T20:03:32Z |
dc.date.available.fl_str_mv |
2020-10-22T20:03:32Z |
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info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/doctoralThesis |
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doctoralThesis |
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publishedVersion |
dc.identifier.citation.fl_str_mv |
SANTOS, Adriana Miranda dos. Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens). 2017. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2017. Disponível em: https://repositorio.ufscar.br/handle/ufscar/13364. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/13364 |
identifier_str_mv |
SANTOS, Adriana Miranda dos. Estudos bioquímicos e cinéticos de acetilcolinesterases de formigas cortadeiras (Atta sexdens). 2017. Tese (Doutorado em Química) – Universidade Federal de São Carlos, São Carlos, 2017. Disponível em: https://repositorio.ufscar.br/handle/ufscar/13364. |
url |
https://repositorio.ufscar.br/handle/ufscar/13364 |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ info:eu-repo/semantics/openAccess |
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Attribution-NonCommercial-NoDerivs 3.0 Brazil http://creativecommons.org/licenses/by-nc-nd/3.0/br/ |
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openAccess |
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Universidade Federal de São Carlos Câmpus São Carlos |
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Programa de Pós-Graduação em Química - PPGQ |
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UFSCar |
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Universidade Federal de São Carlos Câmpus São Carlos |
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