Kinetics of dephosphorylation. (A) Representative Western blot of GC dephosphorylation at different resact concentrations. The amount of GC was adjusted to 120 nM. Sperm were stimulated with 2.5, 25, 250, or 500 nM resact. The time lapse in seconds after resact application is indicated above the respective lanes. The molecular mass of a protein size marker is shown on the right. (B) Fraction of dephosphorylated and phosphorylated GC versus time after stimulation with 250 nM resact. Inset shows the dephosphorylation reaction during the first 200 ms. Data shown are from a single representative experiment out of 12 from six different animals. Lines represent exponential fits. (C) Time course of dephosphorylation for resact concentrations from 50 to 500 nM. The time constant (τdephos = 133 ms) was obtained by using a global fit that includes ligand binding and unbinding. Each data point represents the mean ± SD of at least three experiments. The inset shows the dephosphorylation reaction during the first 2 s. (D) Comparison of the time course of relative turnover number and dephosphorylation. Both time courses were normalized according to the minimum–maximum method. The data shown are from a single representative experiment out of 12 from six different animals. (E) Amount of GCdephos after 3 min of incubation with different resact concentrations. Total GC concentration was adjusted to 120 nM. Each data point represents the mean ± SD. Number of experiments is given in parentheses. Lines represent exponential fits in B, D, and E.
