Soybean seed galactinol synthase activity as determined by a novel colorimetric assay
Autor(a) principal: | |
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Data de Publicação: | 2000 |
Outros Autores: | , |
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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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 |
eu_rights_str_mv |
openAccess |
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) |
instacron_str |
SBFV |
institution |
SBFV |
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 |
_version_ |
1754820904236351488 |