Determination of K_i for 2-BDB-TcAMP inhibition of cAS-PDE. (A) The initial rate of hydrolysis of cAMP was determined in triplicate and in the presence of varying concentrations of the substrate and inhibitor. The concentrations of 2-BDB-TcAMP used were 0 μmol/L (•), 5 μmol/L (▪), 10 μmol/L (▴), and 20 μmol/L (▾). The data was fit to the equation: 1/v = 1/V + K_m/V[S](1 + [I]/K_i ). (B) The slope of each line was plotted against 2-BDB-TcAMP concentrations according to the equation: slope = Km/V(1 + [I]/Ki) to give a K_i value of 5.5 ± 0.4 μmol/L (SEM).