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Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate.

08:00 EDT 31st March 2019 | BioPortfolio

Summary of "Development of an HPLC-based guanosine monophosphate kinase assay and application to Plasmodium vivax guanylate."

The development of a high-performance liquid chromatography (HPLC)-based method, for guanosine monophosphate kinase activity assays, is presented. The method uses the intrinsic UV absorption (at 260 nm) of substrates and products of the enzymatic reaction (GMP, ATP, ADP and GDP) to unambiguously determine percent conversion of substrate into product. It uses a commercially available C18 column which can separate reaction samples by elution under isocratic conditions in 12 min per run. The kinetics of the forward reaction catalyzed by Plasmodium vivax guanylate kinase (PvGK), a potential drug target against malaria, was determined. The relative concentrations of the two substrates (GMP and ATP) have a distinct effect on reaction velocity. Kinetic analyses showed the PvGK-catalyzed reaction to be associated with atypical kinetics, where substrate inhibition kinetics and non-Michaelis-Menten (sigmoidal) kinetics were found with respect to GMP and ATP, respectively. Additionally, the method was used in inhibition assays to screen twenty fragment-like compounds. The assays were robust and reproducible, with a signal window of 3.8 and a Z' factor of 0.6. For the best inhibitor, an IC curve was generated.

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This article was published in the following journal.

Name: Analytical biochemistry
ISSN: 1096-0309
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Medical and Biotech [MESH] Definitions

A guanine nucleotide containing one phosphate group esterified to the sugar moiety and found widely in nature.

Guanosine 5'-monophosphate. A guanine nucleotide containing one phosphate group esterified to the sugar moiety and found widely in nature.

A 150-kDa MAP kinase kinase kinase that may play a role in the induction of APOPTOSIS. It has specificity for MAP KINASE KINASE 3; MAP KINASE KINASE 4; and MAP KINASE KINASE 6.

Thiamine dihydrogen phosphate ester. The monophosphate ester of thiamine. Synonyms: monophosphothiamine; vitamin B1 monophosphate.

A 70-kDa MAP kinase kinase kinase with specificity for MAP KINASE KINASE 5. It is activated during the cellular response to GROWTH FACTORS, oxidative stress, and hyperosmotic conditions.

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