Figure 3.

Quantitative phosphorylation assays show a strong Ca²⁺ activation of CPK6 activity for physiological [Ca²⁺] elevations. A, To determine proper conditions, the time-dependent phosphorylation of Syntide-2 (400 µm) by CPK6 (75 nm) was analyzed by measuring the incorporation of ³²P into the substrate (in counts per minute [cpm]). B, A time point in the linear range of product phosphorylation in A (4.5 min) was chosen to determine CPK6 activities at defined free Ca²⁺ concentrations. CPK6 activity is strongly dependent on the free Ca²⁺ concentration (fit parameters: K_A = 508 nm; Hill coefficient = 1.8 ± 0.2 se; R² = 0.98). Error bars represent sd (n = 3 experiments). See Supplemental Text S1 for a detailed description of the method used.