| Assay Method Information | |
| | Enzymatic Assay |
| Description: | Enzymatic activity of the isolated catalytic domains of wild type PDE5A1 and its deletion mutants was assayed by using [3H]cGMP as substrate in a reaction mixture. After the reaction was terminated, the radioactivity of unreacted [3H]cGMP in the supernatant was measured by a liquid scintillation counter. The turnover rate was measured at nine concentrations of cGMP and controlled at hydrolysis of 15-40% substrate. Each measurement was repeated three times. For measurement of IC50 values, 10 concentrations of inhibitors were used at a substrate concentration that was one-tenth of the Km value and an enzyme concentration that hydrolyzed 50% of substrate. |
| Affinity data for this assay | |
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