FIGURE 4.

Measurement of Δp in SMPs hydrolyzing ATP and evaluation of ΔpH. A, the rate of NADH:fumarate oxidoreduction, recorded during ATP hydrolysis (1 mm ATP+MgSO₄) to generate Δp and with 400 μm KCN to inhibit respiration depends on the difference between the NAD⁺ and fumarate potentials, ΔE = −0.335 V + 0.020 − RT/2F·ln{([NADH][fumarate])/([NAD⁺][succinate])}, set using [NADH] = 0.1 mm, [NAD⁺] = 1 mm, [succinate] = 0.5 mm, and [fumarate] = 0.025–40 mm. The rate is 0 when −2ΔE = 4Δp. When −2ΔE is close to 4Δp, the data vary linearly with potential, but at high and low potential the rates are determined kinetically; the points with arrows are in principal at infinite potential ([NADH] = [fumarate] = 0 shown at 0.11 V, [NAD⁺] = [succinate] = 0 shown at 0.18 V). Similar titrations varying the succinate, NAD⁺, and NADH concentrations gave, overall, Δp = 0.162 ± 0.005 V. B, the fluorescence of ACMA (500 nm) is quenched upon the addition of 100 μm ATP+MgSO₄ to SMPs (112 μg ml⁻¹) in varying KCl concentrations. 2 nm nigericin (a K⁺/H⁺ exchanger) or 20 μg ml⁻¹ gramicidin (a cation transporter) abolish the quenching effect. Note that the ACMA fluorescence is affected by ATP (the final value varies). See “Experimental Procedures” for conditions.