FIGURE 2.

Biochemical properties of Ins4A protein. A, basal (gray bars) and receptor-mediated (dark gray bars) nucleotide exchange rates for wild type and 4-alanine insertion (Ins4A) mutations in Gα_i1 proteins. Nucleotide exchange was monitored by measuring the enhancement in intrinsic tryptophan (Trp²¹¹) fluorescence (λ_ex = 290 nm, λ_em = 340 nm) as a function of time after the addition of GTPγS (32). The data were normalized to the baseline and maximum fluorescence and then fit to the exponential association equation (y = y_max × (1 − e^−kt)) to calculate the rate constant (k). Data were collected at 21 °C for 90 min. Results represent the mean ± S.E. (error bars) values of at least three independent experiments. B, membrane binding of wild type and mutant Gα_i1 proteins. The assay was performed as described under “Experimental Procedures.” Dark, from dark sample; Light, from light activated sample; GTPγS or GDP, from light-activated and nucleotide-incubated samples. S, supernatant; P, pellet. C, densitometric quantification of supernatant from light supernatant samples. Each sample from SDS-PAGE (b) was evaluated by comparison of the amount of Gα_i1 subunits in pellet (P) or supernatant (S) to the total amount of Gα_i1 subunits (P + S) in both treatments and expressed as a percentage of the total Gα_i1 protein. Data represent the average of three independent experiments. D, concentration-response curves of Meta-rhodopsin II (MII) signal stabilized by WT Gα_i1 (black square) and Ins4A (black circle). E, concentration-response curves of MII signal stabilized by WT Gα_i1 (black square) and Ins4A (black circle) in the presence of 0.5 mm GDP. The EC₅₀ value of WT Gα_i1 and Ins4A protein for rhodopsin in ROS membranes was 9.43 ± 0.13 and 0.99 ± 0.02 μm, respectively. Solid curves are best fits from a four-parameter logistic equation. Results are mean ± S.E. from of at least three independent experiments.