Figure 4.

Effects of adrenodoxin on CYP11B2 inhibitor binding and inhibition.A, spectral shifts for LCI699 binding to CYP11B2 alone compared with LCI699 binding to the adrenodoxin–CYP11B2 fusion protein. For each experiment a CYP11B2 concentration of 0.2 μM was used. Data are shown in technical triplicates and were analyzed using the tight-binding or Morrison equation to determine the dissociation constants (B). C, CYP11B2 11-deoxycorticosterone 11β-hydroxylase activity was measured with increasing LCI699 inhibitor concentration in the presence 1-fold, 10-fold, and 40-fold adrenodoxin (46). For experiments with 1-fold adrenodoxin, a CYP11B2 concentration of 0.2 μM was used. For experiments with 10-fold and 40-fold adrenodoxin, a CYP11B2 reduced concentration of 0.05 μM was used to prevent substrate depletion. Data were measured in technical triplicates and fit to the dose–response inhibitor (four-parameter) equation to determine the half-maximal inhibitory concentration (IC₅₀) (D).