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Diphosphosinositol Polyphosphates and Energy Metabolism: Assay for ATP/ADP Ratio

  • Andreas Nagel
  • Christopher J. Barker
  • Per-Olof Berggren
  • Christopher IlliesEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 645)

Abstract

Several inositol compounds undergo rapid cycles of phosphorylation and dephosphorylation. These cycles are dependent on ATP and energy metabolism. Therefore, interfering with the cellular energy metabolism can change the concentration of rapidly turning over inositols. Many pharmacological inhibitors, apart from their intended action, also affect the energy metabolism of the cells and lower ATP. This can unspecifically influence rapidly turning over inositol phosphates. Thus, the ATP concentration should be checked when reduced inositol phosphates are observed after application of pharmacological inhibitors.

A luminescence-based assay for the measurement of ATP and ADP is described. ATP is measured luminometrically using firefly luciferase. Detection of ADP is performed in a two-step enzymatic procedure: (1) The sample ATP is degraded to AMP and (2) ADP is phosphorylated to ATP, which can then be measured luminometrically. This method gives a better signal-to-noise ratio than other methods that do not degrade the sample ATP, but convert ADP directly to ATP and then measure the sum of ATP plus ADP.

Key words:

ATP/ADP ratio Firefly luciferase Luminescence ATP-sulfurylase HIT-T15 Diphos­phoinositol pentakisphosphate 

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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Andreas Nagel
    • 1
  • Christopher J. Barker
    • 1
  • Per-Olof Berggren
    • 1
  • Christopher Illies
    • 1
    Email author
  1. 1.The Rolf Luft Research Center for Diabetes and EndocrinologyKarolinska InstitutetStockholmSwedene

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