Figure 5.

Phosphorylation rate is not affected by Ca²⁺ concentration. (A) Kinetics of Rho* phosphorylation were analyzed at 30 nM (○) and 1 μM (•) [Ca²⁺]_free. Both experiments were done in parallel; only the [Ca²⁺]_free in the incubation buffer was different. After incubation of retina punches with α-toxin and the addition of [γ-³²P]ATP in the dark, the phosphorylation reaction was started at t = 0 by a flash. At the end of the reaction (Inset), punches were solubilized and Rho was affinity-purified. (a) Coomassie staining, (b) Western blot probed with a second anti-Rho mAb, and (c) autoradiogram. Only the retina treated with α-toxin showed Rho radioactive phosphorylation (t = 60 min). (B) Western blot analysis of trephine punches. Before Rho purification, solubilized punches incubated with or without α-toxin and at low or high [Ca²⁺]_free were analyzed for recoverin or RK content. (a) Coomassie staining, (b) Western blots probed with a polyclonal antibody against recoverin, and (c) Western blots probed with a mAb against RK.