| Assay Method Information | |
| | PDE3 Assay |
| Description: | Evaluation of the effects of compounds on the activity of the human phosphodiesterase-3A is quantified by measuring the formation of 5′AMP from cAMP using a human recombinant enzyme expressed in a clonal isolate of Spodoptera frugiperda cells (Sf9) cells.The test compound, reference compound or water (control) are added to a buffer containing 40 mM tris(hydroxymethyl)aminomethane (Tris)/HCl (pH 7.4) and 8 mM MgCl2, 450 nMcAMP and 0.25 μCi [3H]cAMP.Thereafter, the reaction is initiated by addition of the enzyme (about 1 U) and the mixture is incubated for 20 min at 22° C.For basal control measurements, the enzyme is omitted from the reaction mixture. Following incubation SPA beads are added. After 30 min at 22° C. under shaking, the amount of [3H]+ 5′AMP is quantified with a scintillation counter (Topcount, Packard).The results are expressed as a percent inhibition of the control enzyme activity. The standard inhibitory reference compound is milrinone (CAS number 78415-72-2), which is tested in each experiment at several concentrations to obtain an inhibition curve from which its IC50 value is calculated. |
| Affinity data for this assay | |
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