Soybean seed galactinol synthase activity as determined by a novel colorimetric assay

Detalhes bibliográficos
Autor(a) principal: Ribeiro, Marluci
Data de Publicação: 2000
Outros Autores: Felix, Carlos R., Lozzi, Silene de Paulino
Tipo de documento: Artigo
Idioma: eng
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://dx.doi.org/10.1590/S0103-31312000000300004
https://locus.ufv.br//handle/123456789/27219
Resumo: Galactinol synthase (GS) is a key enzyme for the biosynthesis of raffinose oligosaccharides (RO) which are the flatulence factors present in soybean seeds and several other legumes. Understanding of soybean seed GS properties is, therefore, of biotechnological interest. The GS enzyme catalyses formation of galactinol and UDP from UDP-gal and myo-inositol. This enzyme is currently assayed by an isotopic method. We have then idealized a more convenient method for GS assay based on the indirect colorimetric determination of the UDP formed which is then hydrolyzed by exogenous apyrase and the resulting Pi quantified by a modification of the colorimetric method of Fiske & SubbaRow. The color developed is stable, and the method is suitable for detection of very low GS activity. The GS activity profiles of developing soybean seeds determined by the isotopic and the colorimetric methods are closely related. The GS enzyme was partially purified (46-fold) by treatment of seed extract with MnCl2, sequential chromatographies on DEAE-Sepharose, Phenyl-Sepharose CL-4B and Q-Sepharose columns. The crude and the partially purified enzyme showed maximum activity at pH 7.0 and 50 ºC. Dithiothreitol and MnCl2 enhanced considerably the activity of the partially purified enzyme. While UDP-glc could be hydrolyzed by the enzyme at a reative activity corresponding to 49% of that calculated for UDP-gal, UDP-man and sucrose were completely ineffective as alternative substrates.
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spelling Soybean seed galactinol synthase activity as determined by a novel colorimetric assayFlatulenceGalactinol synthaseColorimetric assayRaffinose oligosaccharidesFlatulênciaGalactinol sintaseEnsaio colorimétricoOligosacarídeos de rafinoseGalactinol synthase (GS) is a key enzyme for the biosynthesis of raffinose oligosaccharides (RO) which are the flatulence factors present in soybean seeds and several other legumes. Understanding of soybean seed GS properties is, therefore, of biotechnological interest. The GS enzyme catalyses formation of galactinol and UDP from UDP-gal and myo-inositol. This enzyme is currently assayed by an isotopic method. We have then idealized a more convenient method for GS assay based on the indirect colorimetric determination of the UDP formed which is then hydrolyzed by exogenous apyrase and the resulting Pi quantified by a modification of the colorimetric method of Fiske & SubbaRow. The color developed is stable, and the method is suitable for detection of very low GS activity. The GS activity profiles of developing soybean seeds determined by the isotopic and the colorimetric methods are closely related. The GS enzyme was partially purified (46-fold) by treatment of seed extract with MnCl2, sequential chromatographies on DEAE-Sepharose, Phenyl-Sepharose CL-4B and Q-Sepharose columns. The crude and the partially purified enzyme showed maximum activity at pH 7.0 and 50 ºC. Dithiothreitol and MnCl2 enhanced considerably the activity of the partially purified enzyme. While UDP-glc could be hydrolyzed by the enzyme at a reative activity corresponding to 49% of that calculated for UDP-gal, UDP-man and sucrose were completely ineffective as alternative substrates.Galactinol sintase (GS) é a enzima-chave para a biossíntese de oligosacarídeos de rafinose (RO), que são os fatores antinutricionais causadores de flatulência, os quais estão presentes em sementes de soja e em outros legumes. A GS catalisa a formação de galactinol e UDP a partir de UDP-gal e mioinositol. A atividade dessa enzima é determinada atualmente pelo método radioisotópico que, apesar de adequado tecnicamente, apresenta vários inconvenientes, tais como a necessidade de substrato de alto custo, bem como de cuidados adicionais e serviços especializados para descarte dos resíduos radioativos. Assim, desenvolveu-se um método colorimétrico alternativo ao método radioisotópico, baseado na determinação colorimétrica indireta do UDP formado pela hidrólise enzimática (apirase) desse nucleotídeo e determinação do Pi resultante pelo método de Fiske & SubbaRow, com modificações. A cor desenvolvida é estável e o método é sensível para detecção de quantidades nanomolares de Pi. Os perfis de atividade da GS em sementes de soja em diferentes fases de desenvolvimento, determinados pelos métodos colorimétrico e radioisotópico, são semelhantes. Adicionalmente, a GS de sementes de soja foi purificada (46-vezes) por tratamento do extrato das sementes com MnCl2, e uma seqüência de cromatografias em colunas de DEAE-Sepharose, Phenyl-Sepharose CL-4B e Q-Sepharose. As atividades de GS no extrato bruto e na amostra parcialmente purificada foram máximas em pH 7.0 e 50 ºC. Ditiotreitol e MnCl2 aumentaram consideravelmente a atividade da enzima parcialmente purificada. Enquanto UDP-glc pode ser hidrolisado pela enzima com uma atividade relativa correspondendo a 49% da atividade contra UDP-gal, UDP-man e sacarose foram completamente ineficazes como substratos alternativos. Os valores de KM para conversão de UDP-gal e mio-inositol foram de 2,0 mM e 2,93 mM, respectivamente, determinados pelo método de Lineaweaver-Burk.Revista Brasileira de Fisiologia Vegetal2019-10-03T14:08:40Z2019-10-03T14:08:40Z2000info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlepdfapplication/pdf0103-3131http://dx.doi.org/10.1590/S0103-31312000000300004https://locus.ufv.br//handle/123456789/27219engv. 12, n. 03, p. 203- 212, 2000Ribeiro, MarluciFelix, Carlos R.Lozzi, Silene de Paulinoinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFV2024-07-12T08:39:25Zoai:locus.ufv.br:123456789/27219Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452024-07-12T08:39:25LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.none.fl_str_mv Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
title Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
spellingShingle Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
Ribeiro, Marluci
Flatulence
Galactinol synthase
Colorimetric assay
Raffinose oligosaccharides
Flatulência
Galactinol sintase
Ensaio colorimétrico
Oligosacarídeos de rafinose
title_short Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
title_full Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
title_fullStr Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
title_full_unstemmed Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
title_sort Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
author Ribeiro, Marluci
author_facet Ribeiro, Marluci
Felix, Carlos R.
Lozzi, Silene de Paulino
author_role author
author2 Felix, Carlos R.
Lozzi, Silene de Paulino
author2_role author
author
dc.contributor.author.fl_str_mv Ribeiro, Marluci
Felix, Carlos R.
Lozzi, Silene de Paulino
dc.subject.por.fl_str_mv Flatulence
Galactinol synthase
Colorimetric assay
Raffinose oligosaccharides
Flatulência
Galactinol sintase
Ensaio colorimétrico
Oligosacarídeos de rafinose
topic Flatulence
Galactinol synthase
Colorimetric assay
Raffinose oligosaccharides
Flatulência
Galactinol sintase
Ensaio colorimétrico
Oligosacarídeos de rafinose
description Galactinol synthase (GS) is a key enzyme for the biosynthesis of raffinose oligosaccharides (RO) which are the flatulence factors present in soybean seeds and several other legumes. Understanding of soybean seed GS properties is, therefore, of biotechnological interest. The GS enzyme catalyses formation of galactinol and UDP from UDP-gal and myo-inositol. This enzyme is currently assayed by an isotopic method. We have then idealized a more convenient method for GS assay based on the indirect colorimetric determination of the UDP formed which is then hydrolyzed by exogenous apyrase and the resulting Pi quantified by a modification of the colorimetric method of Fiske & SubbaRow. The color developed is stable, and the method is suitable for detection of very low GS activity. The GS activity profiles of developing soybean seeds determined by the isotopic and the colorimetric methods are closely related. The GS enzyme was partially purified (46-fold) by treatment of seed extract with MnCl2, sequential chromatographies on DEAE-Sepharose, Phenyl-Sepharose CL-4B and Q-Sepharose columns. The crude and the partially purified enzyme showed maximum activity at pH 7.0 and 50 ºC. Dithiothreitol and MnCl2 enhanced considerably the activity of the partially purified enzyme. While UDP-glc could be hydrolyzed by the enzyme at a reative activity corresponding to 49% of that calculated for UDP-gal, UDP-man and sucrose were completely ineffective as alternative substrates.
publishDate 2000
dc.date.none.fl_str_mv 2000
2019-10-03T14:08:40Z
2019-10-03T14:08:40Z
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.uri.fl_str_mv 0103-3131
http://dx.doi.org/10.1590/S0103-31312000000300004
https://locus.ufv.br//handle/123456789/27219
identifier_str_mv 0103-3131
url http://dx.doi.org/10.1590/S0103-31312000000300004
https://locus.ufv.br//handle/123456789/27219
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv v. 12, n. 03, p. 203- 212, 2000
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv pdf
application/pdf
dc.publisher.none.fl_str_mv Revista Brasileira de Fisiologia Vegetal
publisher.none.fl_str_mv Revista Brasileira de Fisiologia Vegetal
dc.source.none.fl_str_mv reponame:LOCUS Repositório Institucional da UFV
instname:Universidade Federal de Viçosa (UFV)
instacron:UFV
instname_str Universidade Federal de Viçosa (UFV)
instacron_str UFV
institution UFV
reponame_str LOCUS Repositório Institucional da UFV
collection LOCUS Repositório Institucional da UFV
repository.name.fl_str_mv LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)
repository.mail.fl_str_mv fabiojreis@ufv.br
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