Figure 4.

Synergetic inhibition of rhodopsin kinase by recoverin and calmodulin. Rhodopsin kinase activity was measured as a function of Ca²⁺-sensor protein or free Ca²⁺ concentration by in vitro phosphorylation assay. (A) Inhibition of RK by increasing concentrations of either calmodulin (○), recoverin (□), or their mixture in the 1:1 ratio (▲) at saturating free Ca²⁺ concentration (200 μM). Fitting the data to the Hill equation as described in Section “Materials and Methods” resulted in IC₅₀ = 14.1 ± 0.9 μM, n = 1.8 ± 0.2 for calmodulin, IC₅₀ = 5.8 ± 0.7 μM, n = 1.6 ± 0.3 for recoverin, and IC₅₀ = 4.6 ± 0.2 μM, n = 2.2 ± 0.2 for calmodulin/recoverin 1:1 mixture. (B) Inhibition of RK activity by either calmodulin (○), recoverin (□), or their 1:1 mixture (▲) at different free Ca²⁺ concentrations. Concentration of each Ca²⁺-sensing protein including their mixture was 30 μM. Fitting the data to the Hill equation as described under Section “Materials and Methods” resulted in values of IC₅₀ = 0.36 ± 0.03 μM, n = 1.53 ± 0.13 for calmodulin, IC₅₀ = 1.36 ± 0.13 μM, n = 1.66 ± 0.23 for recoverin, and IC₅₀ = 1.07 ± 0.15 μM, n = 1.2 ± 0.1 for their 1:1 mixture. The fitted curves are drawn solid for calmodulin or recoverin data sets, and dashed for their mixture. Data points represent mean ± SE of the mean from three independent experiments.