Evaluation of nonradioactive cell-free cAMP assays for measuring in vitro phosphodiesterase activity.
Phosphodiesterases (PDE) are enzymes that catalyze the hydrolysis of cAMP/cGMP to 5'-AMP/GMP. In vitro assays have routinely assayed cAMP/cGMP levels as a direct indicator of PDE activity. Earlier PDE assays depended on radiometric detection of radiolabeled cAMP. Of late, nonradiometric cAMP detection systems have been developed that are cheaper and more amenable to high-throughput screening. Two such assays, namely the enzyme fragment complementation technology and homogeneous time-resolved fluorescence assays, are currently used for monitoring cAMP as a correlate for G-protein-coupled-receptor-induced cellular signaling events. Here, we have compared and validated both of these assays for the measurement of PDE4 enzyme activity in cell-free systems.
Eapen
Mathew S
MS
Department of Pharmacology, New Drug Discovery Research, Ranbaxy Laboratories Ltd., Gurgaon, Haryana, India.
Sodhi
Reena
R
Balakrishnan
Gayathri
G
Dastidar
Sunanda
S
Ray
Abhijit
A
Vijayakrishnan
Lalitha
L
D002474:Cell-Free System; D000242:Cyclic AMP / Q000378:metabolism*; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4 / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D017248:Enzyme Multiplied Immunoassay Technique; D015200:Fluoroimmunoassay; D007700:Kinetics; D057230:Limit of Detection; D049408:Luminescent Agents; D009994:Osmolar Concentration; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction; D013997:Time Factors
D049408:Luminescent Agents; D000242:Cyclic AMP; D010727:Phosphoric Diester Hydrolases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4