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Cited by 28Biochemical and Molecular ResearchProtein Kinase Regulation and GTPase SignalingAdenosine and Purinergic Signaling

Phosphatase-Coupled Universal Kinase Assay and Kinetics for First-Order-Rate Coupling Reaction

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Wu, Zhengliang L.·2011·PLoS ONE
DOI 10.1371/journal.pone.0023172PMC PMC3154929
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Abstract

Kinases use adenosine-5'-triphosphate (ATP) as the donor substrate and generate adenosine-5'-diphosphate (ADP) as a product. An ADP-based phosphatase-coupled kinase assay is described here. In this assay, CD39L2, a nucleotidase, is added into a kinase reaction to hydrolyze ADP to AMP and phosphate. The phosphate is subsequently detected using malachite green phosphate-detection reagents. As ADP hydrolysis by CD39L2 displays a first-order rate constant, relatively simple equations are derived to calculate the coupling rate and the lagging time of the coupling reaction, allowing one to obtain kinase kinetic parameters without the completion of the coupling reaction. ATP inhibition of CD39L2-catalyzed ADP hydrolysis is also determined for correction of the kinetic data. As examples, human glucokinase, P. chrysogenum APS kinase and human ERK1, kinases specific for sugar, nucleotide and protein respectively, are assayed. To assess the compatibility of the method for high-throughput assays, Z' factors >0.5 are also obtained for the three kinases.

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  • The assay is based on the detection of inorganic phosphate, which is released during the sugar transfer reaction and subsequent hydrolysis of UDP catalyzed by a phosphatase.

    “The assay is based on the detection of inorganic phosphate, which is released during the sugar transfer reaction and subsequent hydrolysis of UDP catalyzed by a phosphatase.”

    From High level in vivo mucin-type glycosylation in Escherichia coli (2018) · cites this paper as [19]

    The Introduction and Discussion describe detecting inorganic phosphate released by a phosphatase-coupled assay. But this paper’s assay measures phosphate released from ADP produced by kinase reactions, not from UDP following a sugar-transfer reaction. The Introduction mentions a glycosyltransferase assay only as a similar assay. (LLM, conf=0.90)

    Experimental — accuracy not yet measured

  • The released phosphate is optically quantified using malachite green reagents.

    “The released phosphate is optically quantified using malachite green reagents [19].”

    From High level in vivo mucin-type glycosylation in Escherichia coli (2018) · cites this paper as [19]

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Research works printed but not linked: 36

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Authors
Wu, Z. L.
Affiliations
R&D Systems (United States) (US)
Corresponding author
Wu, Zhengliang L.
ORCID
Wu, Zhengliang L.
Venue
PLoS ONE
Publisher
Public Library of Science (PLoS)
Published
2011-08-11
Volume
6
Issue
8
Pages
e23172
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0023172
PMC
PMC3154929
OpenAlex
W1989517450
Cited by
28
References
1
Citations by year
2026: 2 · 2025: 1 · 2023: 2 · 2020: 2 · 2019: 2 · 2018: 4 · 2017: 3 · 2016: 3 · 2015: 3 · 2014: 4 · 2012: 2
FWCI
0.78
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
Yale University
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Biochemical and Molecular Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Physiology › Adenosine and Purinergic Signaling
Keywords
ADP hydrolysis, nucleotidase, kinase kinetics, time delay, ATP inhibition, glucokinase, ERK1, Z' factor
MeSH terms
Adenosine Diphosphate, Adenosine Triphosphatases, Adenosine Triphosphate, Animals, Glucokinase, Humans, Hydrolysis, Kinetics, Penicillium, Phosphoric Monoester Hydrolases, Phosphotransferases, Recombinant Proteins, Substrate Specificity, Time Factors, Phosphotransferases (Alcohol Group Acceptor), Extracellular Signal-Regulated MAP Kinases, Mice, Biocatalysis, Enzyme Assays

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