Figure 5.

Impact of phosphomimetic mutant forms of human IF₁on the hydrolytic and synthetic activities of bovine ATP synthase.A–D, the impacts on ATP hydrolysis. E–H, the impacts on ATP synthesis. The following inhibitors were employed. In (A) and (E), IF₁(1–81)-S14D-Y33W; in (B) and (F), IF₁(1–81)-S14D-Y33W-H49K; in (C) and (G), IF₁(1–81)-S14E-Y33W; in (D) and (H), IF₁(1–81)-S14E-Y33W-H49K. In (A–D), inhibition of ATP hydrolysis by bovine SMPs by increasing concentrations of IF₁. (), 0.05 μM; (), 0.1 μM; (), 0.2 μM; (), 0.4 μM; (), 0.8 μM; (), 1.6 μM. () and (—), controls with no inhibitor and 1 μM oligomycin, respectively. Measurements were made in triplicate, and traces correspond to the average values corrected to an initial value of zero, with ± SD shown as bounding dashed lines (Files S19–S22). In (E–H), ATP synthesis by bovine heart SMPs coupled to NADH oxidation, measured by a luminescence continuous real-time assay and with a luciferase-luciferin reagent. The luminescence signal was calibrated with 2 μM ATP, and the background signal before addition of ATP was subtracted. ATP synthesis was initiated by the addition of NADH (0.2 mM). (), no inhibitor; (), 0.5 μM IF₁; (), 5 μM IF₁. A concentration of 5 μM IF₁ provides a molar ratio of ca. 400:1 with respect to the ATP synthase. Data points are the average signal ± SD, n = 4 wells (Files S23–S26). SMP, submitochondrial particle.