Figure 5. The kinetic parameters showed saturation with respect to cBid, but not Bax concentrations, and exhibited an inverse relationship between [cBid] and [Bax].

(A) Dependence of the rate constants on Bax concentration was linear. Some small deviations from linearity at low Bax concentrations probably reflect systematic error due to the need for long incubations under those conditions. (B) Dependence of the rate constants on cBid concentration was saturable and displayed an inverse relationship with the amount of Bax (lower EC₅₀ values at higher Bax concentration). The assay was performed as in Figure 2A,C. Data shown in (A) and (B) are representative of three independent experiments. Kinetic parameters were determined using equation 9; cBid dose-response data (B) were fit to hyperbolic curves (solid lines) using GraphPad Prizm software. (C) cBid-dependent dextran-Cascade Blue release induced by 70 nM (blue lines) and 210 nM (red lines) Bax at indicated concentrations of cBid. Bax was added at time zero; cBid was added 2 min prior to Bax. The data were used for quantification of kinetic parameters shown in (B). Dextran content is defined as the fluorescence signal at a given time point relative to the maximal fluorescence and quantified as described in Materials and Methods. See also Figure S5 demonstrating theoretical Bax dose-response curves. (D) Molecular scheme, modified from Figure 4B to include the data in (C) confirming that cBid activates Bax nonstoichiometrically, that is catalytically. Also, as k₁ shows the same dependencies on [cBid] and [Bax] as k₂, we infer that activated Bax enters into both tier I and tier II reactions. As k₁ and k2 are linearly related to [Bax], the entire process shows no cooperativity with regard to Bax, and we conclude that Bax promotes the Catalyst Assembly reaction but cannot be the monomer M. Activated Bax could enter into the tier II reaction II, either as a catalyst or a direct participant. Data are representative of three independent experiments.