EXPERIMENTAL FIGURE 12-29 Synthesis of ATP by ATP synthase depends on a pH gradient across the membrane. Isolated chloroplast thylakoid vesicles containing ATP synthase (F₀F₁ particles) were equilibrated in the dark with a buffered solution at pH 4.0. When the pH in the thylakoid lumen reached 4.0, the vesicles were rapidly mixed with a solution at pH 8.0 containing ADP and P_i. A burst of ATP synthesis accompanied the transmembrane movement of protons driven by the 10,000-fold H⁺ concentration gradient (10⁻⁴ M versus 10⁻⁸ M). In similar experiments using “inside-out” preparations of mitochondrial membrane vesicles, an artificially generated membrane electric potential also resulted in ATP synthesis.