FIGURE 3.
Calcineurin inhibition by CN585 is reversible. A, a preincubated mixture of 15 nm calcineurin and 40 μm CN585 in assay buffer containing 100 nm calmodulin was used to examine the reversibility of inhibition in a dialysis experiments. The mixture and a non-inhibited calcineurin control were placed in a 3-kDa MicroDialyzer and dialyzed. Initial rates of calcineurin activity of the dialyzed enzyme or CN585-enzyme complex were determined using RII phosphopeptide substrate. B, additionally, initial rates of 4–28 nm calcineurin preincubated with calmodulin, 1–10 μm CN585, and 33P-labeled RII phosphopeptide were measured at 30 °C. Vmax was plotted versus the concentration of calcineurin. In the case of a reversible inhibitor, all straight lines have a common point of intersection. The data shown are the means ± S.D. of three independent experiments.