title: A bioluminescence method for direct measurement of phosphodiesterase activity.
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 <1 μU (pmol/min) of PDE activity in cell extracts containing 0.25-10 μg protein. Assays utilizing pure enzyme showed that 0.2 mM IBMX completely inhibited PDE activity. This single-step enzyme- and substrate-coupled cyclic-reaction system yields a simplified, sensitive, reproducible, and accurate method for quantifying PDE activities in small biological samples.
authors.affiliation: Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Intramural Research Program, National Institutes of Health, Baltimore, MD 21224, USA.
authors.forename: Antoine
authors.initials: A
authors.lastname: Younès
authors.forename: Yevgeniya O
authors.initials: YO
authors.lastname: Lukyanenko
authors.forename: Alexey E
authors.initials: AE
authors.lastname: Lyashkov
authors.forename: Edward G
authors.initials: EG
authors.lastname: Lakatta
authors.forename: Steven J
authors.initials: SJ
authors.lastname: Sollott
mesh_terms: D015056:1-Methyl-3-isobutylxanthine / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D057075:Enzyme Assays / Q000379:methods*; D049454:Firefly Luciferin / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008156:Luciferases / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D032383:Myocytes, Cardiac / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D011817:Rabbits; D012015:Reference Standards
chemical_list: D000249:Adenosine Monophosphate; D049454:Firefly Luciferin; D000255:Adenosine Triphosphate; D008156:Luciferases; D010727:Phosphoric Diester Hydrolases; D015056:1-Methyl-3-isobutylxanthine