Fig. 10. Time dependence and substrate dependence of CaM phosphorylation by CaPK-2. (A) Phosphorylation by CaPK-2 was performed with 2 µg CaM and 20 units of kinase in the presence of Ca²⁺. The same volume of reaction mixture was withdrawn at indicated time points, and the reaction was stopped by precipitating the protein with 15% TCA. (B) The amount of CaM for phosphorylation by 20 units of kinase was increased as indicated. The reaction was performed for 20 minutes at room temperature. Autophosphorylation of CaPK-2 (M_r 50,000) and CaM (M_r 17,000) phosphorylation are indicated by arrows. (C) The radioactivity incorporated into CaM was quantified from the dried gel using a Betagen detector system (Intelligenetics, Mountainview, CA) and plotted with time.