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A ph sensitive colorometric assay for the high-throughput screening of enzyme inhibitors and substrates: A case study using kinases

Academic Article
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Overview

related to degree

  • Chapman, Eli, Ph.D. in Chemistry, Scripps Research , Transferred from Columbia University 1998 - 2002

authors

  • Chapman, Eli
  • Wong, Chi-Huey

publication date

  • March 2002

journal

  • Bioorganic & Medicinal Chemistry  Journal

abstract

  • We have developed an uncoupled, pH sensitive kinase assay that can be used for high-throughput screening of potential inhibitors or for determining substrate specificity. Kinases catalyze the transfer of a gamma-phosphoryl group from ATP to an appropriate hydroxyl acceptor with the release of a proton. This assay is based on the detection of this proton using an appropriately matched buffer/indicator system. The assay was used to measure the activity of four readily available kinases, including hexokinase, glucokinase, glycerokinase, and pyruvate kinase, which was run in the reverse direction. We also went on to screen a small series of mono- and diphosphonucleotides for inhibition of hexokinase as well as a modest set of potential hexokinase substrates. We determined sucrose to be a modest substrate for hexokinase with a K(m) of 1.8 +/- 0.2 mM, a k(cat) of 142 +/- 3 min(-1), and a V(max) that is 15% of that for glucose. Given the importance of kinases in a diverse array of biological functions and disease states, there is a need for a simple, rapid assay system. We feel this assay will lend itself well to meet this end. This method should be applicable to many other enzymatic reactions which involve a change in pH.

subject areas

  • Adenosine Triphosphate
  • Animals
  • Carbohydrate Metabolism
  • Color
  • Drug Evaluation, Preclinical
  • Enzyme Inhibitors
  • Humans
  • Hydrogen-Ion Concentration
  • Kinetics
  • Nucleotides
  • Phosphotransferases
  • Spectrum Analysis
  • Substrate Specificity
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Identity

International Standard Serial Number (ISSN)

  • 0968-0896

Digital Object Identifier (DOI)

  • 10.1016/s0968-0896(01)00306-6

PubMed ID

  • 11814841
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Additional Document Info

start page

  • 551

end page

  • 555

volume

  • 10

issue

  • 3

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