GC is inactivated by dephosphorylation. (A and B) Western blots showing dephosphorylation of GC induced by intracellular alkalization. pH_i was elevated by different concentrations of NH₄Cl (A) or TMA (B). The concentrations of the base and incubation times (minutes) are indicated above the respective lanes. The molecular masses of two protein size markers are shown on the left. (C) Representative Western blot of dephosphorylation by alkaline pH. (right) First, GC was dephosphorylated by incubation of sperm with 122 mM TMA for 15 min. Subsequently, sperm were stimulated with 250 nM resact. The time lapse after resact stimulation (seconds) is indicated above the respective lines. The molecular masses of two protein size markers are shown on the left. The left shows control dephosphorylation after resact stimulation in the absence of TMA. (D) Time course of cGMP synthesis in the presence of 2 mM IBMX after alkaline dephosphorylation. Control without TMA preincubation is shown. Lines represent exponential fits. Each data point represents the mean ± SD of three experiments based on duplicates.