A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay

In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5'-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to th...

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Detalles Bibliográficos
Autores principales: Zhang, Lianwen, Ren, Feifei, Li, Jing, Ma, Xiaofeng, Wang, Peng
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Methodology
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056017/
https://www.ncbi.nlm.nih.gov/pubmed/19957065
http://dx.doi.org/10.1007/s12575-009-9016-x
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author Zhang, Lianwen
Ren, Feifei
Li, Jing
Ma, Xiaofeng
Wang, Peng
author_facet Zhang, Lianwen
Ren, Feifei
Li, Jing
Ma, Xiaofeng
Wang, Peng
author_sort Zhang, Lianwen
collection PubMed
description In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5'-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E(m) = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT.
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spelling pubmed-30560172011-03-15 A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay Zhang, Lianwen Ren, Feifei Li, Jing Ma, Xiaofeng Wang, Peng Biol Proced Online Methodology In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5'-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E(m) = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT. BioMed Central 2009-12-03 /pmc/articles/PMC3056017/ /pubmed/19957065 http://dx.doi.org/10.1007/s12575-009-9016-x Text en Copyright ©2009 Zhang et al. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology
Zhang, Lianwen
Ren, Feifei
Li, Jing
Ma, Xiaofeng
Wang, Peng
A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay
title A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay
title_full A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay
title_fullStr A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay
title_full_unstemmed A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay
title_short A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay
title_sort modified coupled enzyme method for o-linked glcnac transferase activity assay
topic Methodology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056017/
https://www.ncbi.nlm.nih.gov/pubmed/19957065
http://dx.doi.org/10.1007/s12575-009-9016-x
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