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

Animals Catalysis Chromatography, High Pressure Liquid / methods Guanylate Kinases / metabolism Kinetics Phosphorylation Plasmodium vivax / enzymology
Guanosine monophosphate kinase Guanylate kinase HPLC Nucleotide Sigmoidal kinetics Substrate inhibition

Journal

Analytical biochemistry
ISSN: 1096-0309
Titre abrégé: Anal Biochem
Pays: United States
ID NLM: 0370535

Informations de publication

Date de publication:
15 06 2019
Historique:
received: 23 12 2018
revised: 18 03 2019
accepted: 29 03 2019
pubmed: 4 4 2019
medline: 18 2 2020
entrez: 4 4 2019
Statut: ppublish

Résumé

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

Identifiants

DOI: 10.1016/j.ab.2019.03.022 PMID: 30943378 PMC: PMC6494078
pubmed: 30943378
pii: S0003-2697(18)31276-4
doi: 10.1016/j.ab.2019.03.022
pmc: PMC6494078
pii:
doi:

Substances chimiques

Guanylate Kinases EC 2.7.4.8

Types de publication

Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Langues

eng

Sous-ensembles de citation

IM

Pagination

63-69

Subventions

Organisme : NIGMS NIH HHS
ID : P50 GM064655
Pays : United States

Informations de copyright

Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.

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Auteurs

Liliana Pedro (L)

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.

Megan Cross (M)

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.

Andreas Hofmann (A)

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.

Tin Mak (T)

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia.

Ronald J Quinn (RJ)

Griffith Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia. Electronic address: r.quinn@griffith.edu.au.

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Classifications MeSH

questionsmedicales.fr › Eucaryotes › Animaux › Development of an HPLC-based guanosine...
questionsmedicales.fr › Phénomènes chimiques › Catalyse › Development of an HPLC-based guanosine...
questionsmedicales.fr › Techniques d'investigation › Techniques de chimie analytique › Chromatographie › Chromatographie en phase liquide › Chromatographie en phase liquide à haute performance › Development of an HPLC-based guanosine...
questionsmedicales.fr › Enzymes et coenzymes › Enzymes › Transferases › Phosphotransferases › Phosphotransferases (Phosphate Group Acceptor) › Guanylate kinase › Development of an HPLC-based guanosine...
questionsmedicales.fr › Phénomènes chimiques › Phénomènes biochimiques › Cinétique › Development of an HPLC-based guanosine...
questionsmedicales.fr › Métabolisme › Phosphorylation › Development of an HPLC-based guanosine...
questionsmedicales.fr › Eucaryotes › Alveolata › Apicomplexa › Haemosporida › Plasmodium › Plasmodium vivax › Development of an HPLC-based guanosine...