An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Autores
Yep, Alejandra; Bejar, Clarisa M.; Ballicora, Miguel A.; Dubay, Jennifer R.; Iglesias, Alberto Alvaro; Preiss, Jack
Año de publicación
2004
Idioma
inglés
Tipo de recurso
artículo
Estado
versión publicada
Descripción
Adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5′-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [ 14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[ 14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S0.5 determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.
Fil: Yep, Alejandra. Michigan State University; Estados Unidos
Fil: Bejar, Clarisa M.. Michigan State University; Estados Unidos
Fil: Ballicora, Miguel A.. Michigan State University; Estados Unidos
Fil: Dubay, Jennifer R.. Michigan State University; Estados Unidos
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Preiss, Jack. Michigan State University; Estados Unidos
Materia
ADP-GLUCOSE
ADP-GLUCOSE PYROPHOSPHORYLASE
GLYCOGEN SYNTHASE
POLYSACCHARIDE PRECIPITATION
Nivel de accesibilidad
acceso abierto
Condiciones de uso
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
Repositorio
CONICET Digital (CONICET)
Institución
Consejo Nacional de Investigaciones Científicas y Técnicas
OAI Identificador
oai:ri.conicet.gov.ar:11336/85713
Acceder
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oai_identifier_str oai:ri.conicet.gov.ar:11336/85713
network_acronym_str CONICETDig
repository_id_str 3498
network_name_str CONICET Digital (CONICET)
spelling An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthaseYep, AlejandraBejar, Clarisa M.Ballicora, Miguel A.Dubay, Jennifer R.Iglesias, Alberto AlvaroPreiss, JackADP-GLUCOSEADP-GLUCOSE PYROPHOSPHORYLASEGLYCOGEN SYNTHASEPOLYSACCHARIDE PRECIPITATIONhttps://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1Adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5′-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [ 14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[ 14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S0.5 determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.Fil: Yep, Alejandra. Michigan State University; Estados UnidosFil: Bejar, Clarisa M.. Michigan State University; Estados UnidosFil: Ballicora, Miguel A.. Michigan State University; Estados UnidosFil: Dubay, Jennifer R.. Michigan State University; Estados UnidosFil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; ArgentinaFil: Preiss, Jack. Michigan State University; Estados UnidosAcademic Press Inc Elsevier Science2004-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/85713Yep, Alejandra; Bejar, Clarisa M.; Ballicora, Miguel A.; Dubay, Jennifer R.; Iglesias, Alberto Alvaro; et al.; An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase; Academic Press Inc Elsevier Science; Analytical Biochemistry; 324; 1; 1-2004; 52-590003-2697CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/doi/10.1016/j.ab.2003.09.024info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-sa/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-03T10:07:37Zoai:ri.conicet.gov.ar:11336/85713instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-03 10:07:38.094CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse
dc.title.none.fl_str_mv An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
title An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
spellingShingle An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
Yep, Alejandra
ADP-GLUCOSE
ADP-GLUCOSE PYROPHOSPHORYLASE
GLYCOGEN SYNTHASE
POLYSACCHARIDE PRECIPITATION
title_short An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
title_full An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
title_fullStr An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
title_full_unstemmed An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
title_sort An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase
dc.creator.none.fl_str_mv Yep, Alejandra
Bejar, Clarisa M.
Ballicora, Miguel A.
Dubay, Jennifer R.
Iglesias, Alberto Alvaro
Preiss, Jack
author Yep, Alejandra
author_facet Yep, Alejandra
Bejar, Clarisa M.
Ballicora, Miguel A.
Dubay, Jennifer R.
Iglesias, Alberto Alvaro
Preiss, Jack
author_role author
author2 Bejar, Clarisa M.
Ballicora, Miguel A.
Dubay, Jennifer R.
Iglesias, Alberto Alvaro
Preiss, Jack
author2_role author
author
author
author
author
dc.subject.none.fl_str_mv ADP-GLUCOSE
ADP-GLUCOSE PYROPHOSPHORYLASE
GLYCOGEN SYNTHASE
POLYSACCHARIDE PRECIPITATION
topic ADP-GLUCOSE
ADP-GLUCOSE PYROPHOSPHORYLASE
GLYCOGEN SYNTHASE
POLYSACCHARIDE PRECIPITATION
purl_subject.fl_str_mv https://purl.org/becyt/ford/1.6
https://purl.org/becyt/ford/1
dc.description.none.fl_txt_mv Adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5′-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [ 14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[ 14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S0.5 determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.
Fil: Yep, Alejandra. Michigan State University; Estados Unidos
Fil: Bejar, Clarisa M.. Michigan State University; Estados Unidos
Fil: Ballicora, Miguel A.. Michigan State University; Estados Unidos
Fil: Dubay, Jennifer R.. Michigan State University; Estados Unidos
Fil: Iglesias, Alberto Alvaro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Agrobiotecnología del Litoral. Universidad Nacional del Litoral. Instituto de Agrobiotecnología del Litoral; Argentina
Fil: Preiss, Jack. Michigan State University; Estados Unidos
description Adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5′-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [ 14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[ 14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S0.5 determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.
publishDate 2004
dc.date.none.fl_str_mv 2004-01
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
http://purl.org/coar/resource_type/c_6501
info:ar-repo/semantics/articulo
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/11336/85713
Yep, Alejandra; Bejar, Clarisa M.; Ballicora, Miguel A.; Dubay, Jennifer R.; Iglesias, Alberto Alvaro; et al.; An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase; Academic Press Inc Elsevier Science; Analytical Biochemistry; 324; 1; 1-2004; 52-59
0003-2697
CONICET Digital
CONICET
url http://hdl.handle.net/11336/85713
identifier_str_mv Yep, Alejandra; Bejar, Clarisa M.; Ballicora, Miguel A.; Dubay, Jennifer R.; Iglesias, Alberto Alvaro; et al.; An assay for adenosine 5′-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase; Academic Press Inc Elsevier Science; Analytical Biochemistry; 324; 1; 1-2004; 52-59
0003-2697
CONICET Digital
CONICET
dc.language.none.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv info:eu-repo/semantics/altIdentifier/doi/10.1016/j.ab.2003.09.024
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
eu_rights_str_mv openAccess
rights_invalid_str_mv https://creativecommons.org/licenses/by-nc-sa/2.5/ar/
dc.format.none.fl_str_mv application/pdf
application/pdf
dc.publisher.none.fl_str_mv Academic Press Inc Elsevier Science
publisher.none.fl_str_mv Academic Press Inc Elsevier Science
dc.source.none.fl_str_mv reponame:CONICET Digital (CONICET)
instname:Consejo Nacional de Investigaciones Científicas y Técnicas
reponame_str CONICET Digital (CONICET)
collection CONICET Digital (CONICET)
instname_str Consejo Nacional de Investigaciones Científicas y Técnicas
repository.name.fl_str_mv CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas
repository.mail.fl_str_mv dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar
_version_ 1842270010194526208
score 13.13397

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