Bioluminescence method for direct measurement of phosphodiesterase activity q

Antoine Younès, Yevgeniya O. Lukyanenko, Alexey E. Lyashkov, Edward Lakatta, Steven J. Sollott

Research output: Contribution to journalArticle

Abstract

We have adapted bioluminescence methods to be able to measure phosphodiesterase (PDE) activity in a one-step technique. The method employs a four-enzyme system (PDE, adenylate kinase (AK) using excess CTP instead of ATP as substrate, pyruvate kinase (PK), and firefly luciferase) to generate ATP, with measurement of the concomitant luciferase-light emission. Since AK, PK, and luciferase reactions are coupled to recur in a cyclic manner, AMP recycling maintains a constant rate of ATP formation, proportional to the steady-state AMP concentration. The cycle can be initiated by the PDE reaction that yields AMP. As long as the PDE reaction is rate limiting, the system is effectively at steady state and the bioluminescence kinetics progresses at a constant rate proportional to the PDE activity. In the absence of cAMP and PDE, low concentrations of AMP trigger the AMP cycling, which allows standardizing the system. The sensitivity of the method enables detection of

Original languageEnglish (US)
Pages (from-to)36-40
Number of pages5
JournalAnalytical Biochemistry
Volume417
Issue number1
DOIs
StatePublished - Oct 2011
Externally publishedYes

Fingerprint

Bioluminescence
Phosphoric Diester Hydrolases
Adenosine Monophosphate
Adenylate Kinase
Pyruvate Kinase
Adenosine Triphosphate
Luciferases
Firefly Luciferases
Cytidine Triphosphate
Light emission
Recycling
Cyclic AMP
Reaction rates
Light
Kinetics
Substrates
Enzymes

Keywords

  • Assay
  • Bioluminescence
  • Phosphodiesterase

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology

Cite this

Bioluminescence method for direct measurement of phosphodiesterase activity q. / Younès, Antoine; Lukyanenko, Yevgeniya O.; Lyashkov, Alexey E.; Lakatta, Edward; Sollott, Steven J.

In: Analytical Biochemistry, Vol. 417, No. 1, 10.2011, p. 36-40.

Research output: Contribution to journalArticle

Younès, Antoine ; Lukyanenko, Yevgeniya O. ; Lyashkov, Alexey E. ; Lakatta, Edward ; Sollott, Steven J. / Bioluminescence method for direct measurement of phosphodiesterase activity q. In: Analytical Biochemistry. 2011 ; Vol. 417, No. 1. pp. 36-40.
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