Fig. 4.

Activation of autophosphorylation of CaMKII by WT and mutant CaMs. (A) Reactions were performed with 10 μM CaM and 0.28 μM CaMKII subunits for 1 min in the absence (−) or presence (+) of 300 μM Ca²⁺, as described in Methods. The positions of α and β subunits of CaMKII are indicated. (B) Dependence of autophosphorylation on the concentration of mutant CaMs. Autophosphorylation was performed as in A, except reactions were performed for 30 sec. To determine the K_act values reported in Table 2, the data were normalized to the level of autophosphorylation in saturating Ca²⁺/CaM for 1 min and fit with a model assuming binding of 1 CaM to 1 catalytic subunit. (C) Dependence of autophosphorylation on Ca²⁺ in the presence of WT and mutant CaMs. Reactions were performed as in A, except with 5 μM CaM, and quantified as in B. (D) Dependence of autophosphorylation on CaM concentration in the presence of WT and mutant CaMs. Reactions were performed as in A, except with 12 μM Ca²⁺, and quantified as in B. Mean ± SD of four experiments. WT (▾), CaM-C^WT (●), and CaM-N^WT (■).