Figure 5.

Threshold concentrations of wt Rac and O₂⁻for the initiation of NOX2 autoactivation. NOX2 O₂⁻ production in a cell-free system that contains the NOX2 phox complex and inactive wt Rac with various trigger amounts of active wt Rac or O₂⁻ was measured by monitoring oxidation of cytochrome c. Wt Rac activation that corresponds to NOX2 O₂⁻ production also was measured by using mant-GTP as described in the Experimental procedures section. The O₂⁻ production and wt Rac activity-change profiles over time were fit to the autoactivation equation (see the Supporting information section, red lines), which gave the k_auto values for the NOX2 O₂⁻ production as well as wt Rac activation that was triggered by the various fractions of active wt Rac or O₂⁻. A1, the k_auto values for NOX2 O₂⁻ production with <2, 2, 5, and 10 mol % active wt Rac trigger in the cell-free NOX2 system are, respectively, >15,000, 781, 509, and 383 s. A2, the corresponding k_auto values of the wt Rac activation, are, respectively, >15,000, 790, 511, and 389 s. B1, the k_auto values for the NOX2 O₂⁻ production with <2, 2, 5, and 10 mol % of the trigger O₂⁻ are, respectively, >15,000, 740, 445, and 369 s. B2, the corresponding k_auto values of the wt Rac activation, are, respectively, >15,000, 753, 467, and 344 s. C, the threshold concentrations of the active wt Rac and O₂⁻ to trigger NOX2 autoactivation were determined by titration of the NOX2 phox complex containing inactive wt Rac in the cell-free system with titrants. The titrants were various concentrations of the active wt Rac (◯) and O₂⁻ (●). The threshold concentrations of the active wt Rac and O₂⁻ triggers for NOX2 autoactivation were determined, respectively, to be ∼2.0 and 2.5 mol % of active wt Rac with inactive wt Rac. NOX2, NADPH oxidase 2.