Figure 4.

Impact of constitutively active human IF₁on ATP hydrolysis and ATP synthesis by bovine SMPs.A, ATP hydrolysis inhibited by increasing concentrations of constitutively active human IF₁(1–81)-H49K. (), no inhibitor; (—), plus 1 μM oligomycin. IF₁ at various concentrations; (), 0.05 μM; (), 0.1 μM; (), 0.2 μM; (), 0.4 μM; (), 0.8 μM; (), 1.6 μM. Measurements were made in triplicate, and the traces represent the average values corrected to an initial value of zero, and ± SD shown with bounding dashed lines (File S15). B–D, ATP synthesis by bovine SMPs coupled to NADH oxidation, in the presence or absence of human IF₁(1–81)-H49K. ATP synthesis was initiated by the addition of NADH (0.2 mM). In (B) and (C), ATP synthesis was measured with a luminescence continuous real-time assay, and in (D), by a quench assay with luciferase-luciferin reagent. In (B), the concentrations of human IF₁(1–81)-H49K were as follows: (), no inhibitor; (), 0.2 μM; (), 1 μM; (), 5 μM; (), 10 μM; (), 20 μM. A concentration of 20 μM IF₁ provides a molar excess of ca. 1600:1 with respect to the quantity of ATP synthase. Background luminescence levels were established for ten measurement cycles. N = 4 wells, and data points correspond to the average signal ± SD (File S16). In (C), the luminescence signal was calibrated with 2 μM ATP before the addition of NADH, and the background signal before addition of ATP was subtracted. (), no inhibitor; (), 5 μM IF₁(1–81)-H49K. The data points are the average signal ± SD, n = 4 wells (File S17). D, ATP synthesis determined with the quench assay. (), no inhibitor; (), 5 μM IF₁(1–81)-H49K, corresponding to an IF₁:ATP synthase molar ratio of ca. 700:1. Data points are the average signal ± SD, n = 3 (File S18). In (C), a linear regression () was applied to the initial rate (25 s) of ATP synthesis in the presence of IF₁ H49K, and in (D) to the rate over 240 s. The rates of ATP synthesis (magenta) in μmol min⁻¹ mg⁻¹ were calculated from the slopes. SMP, submitochondrial particle.