FIGURE 1.

RD3 binds both native isozymes of RetGC in mouse retinas and prevents mouse RetGC activation by GCAP1 and GCAP2. A and B, basal activities of RetGC1 (A) and RetGC2 (B) in ROS fractions isolated from their respective RetGC2^−/−GCAPs^−/− and RetGC1^−/−GCAPs^−/− retinas were assayed in the presence of recombinant mouse RD3 purified from E. coli; the data were fitted using the Synergy KaleidaGraph 4 software assuming the Hill function, a = a₀/(1 + ([RD3]/IC₅₀)^h), where a is the activity of RetGC, [RD3] is the concentration of RD3, IC₅₀ is the RD3 concentration that inhibited activation to half-maximal, and h is the Hill coefficient; the IC₅₀ and the Hill coefficient values from the fit were 10 ± 2 nm and 0.78 ± 0.01 (n = 4) (RetGC2^−/−GCAPs^−/−), and 17 ± 3 and 0.8 ± 0.08 (n = 3) (RetGC1^−/−GCAPs^−/−). C and D, ROS fraction isolated from the GCAPs^−/− knock-out mice was assayed in the presence of the indicated concentrations of recombinant mouse GCAP1 (C) or GCAP2 (D) in the absence (○,●) or in the presence of 15 nm (▵, ▴) or 45 nm (□, ■) mouse RD3; data are fitted assuming Michaelis-Menten hyperbolic function. Here and further, the data are mean average ± S.E. unless specified otherwise. See “Experimental Procedures” for other details.