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: Revista Brasileira de Fisiologia Vegetal (Online)
Texto Completo: http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-31312000000300004
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 oligosaccharidesGalactinol 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.Sociedade Brasileira de Fisiologia Vegetal2000-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-31312000000300004Revista Brasileira de Fisiologia Vegetal v.12 n.3 2000reponame:Revista Brasileira de Fisiologia Vegetal (Online)instname:Sociedade Brasileira de Fisiologia Vegetal (SBFV)instacron:SBFV10.1590/S0103-31312000000300004info:eu-repo/semantics/openAccessRIBEIRO,MARLUCIFELIX,CARLOS R.LOZZI,SILENE DE PAULINOeng2003-06-11T00:00:00Zoai:scielo:S0103-31312000000300004Revistahttps://www.scielo.br/j/rbfv/ONGhttps://old.scielo.br/oai/scielo-oai.phppmazza@unicamp.br1806-93550103-3131opendoar:2003-06-11T00:00Revista Brasileira de Fisiologia Vegetal (Online) - Sociedade Brasileira de Fisiologia Vegetal (SBFV)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
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
topic Flatulence
galactinol synthase
colorimetric assay
raffinose oligosaccharides
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 2000-01-01
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=S0103-31312000000300004
url http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0103-31312000000300004
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S0103-31312000000300004
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
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dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Sociedade Brasileira de Fisiologia Vegetal
publisher.none.fl_str_mv Sociedade Brasileira de Fisiologia Vegetal
dc.source.none.fl_str_mv Revista Brasileira de Fisiologia Vegetal v.12 n.3 2000
reponame:Revista Brasileira de Fisiologia Vegetal (Online)
instname:Sociedade Brasileira de Fisiologia Vegetal (SBFV)
instacron:SBFV
instname_str Sociedade Brasileira de Fisiologia Vegetal (SBFV)
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reponame_str Revista Brasileira de Fisiologia Vegetal (Online)
collection Revista Brasileira de Fisiologia Vegetal (Online)
repository.name.fl_str_mv Revista Brasileira de Fisiologia Vegetal (Online) - Sociedade Brasileira de Fisiologia Vegetal (SBFV)
repository.mail.fl_str_mv pmazza@unicamp.br
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