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Figure 1 Cartoon of the structure and function of the P.modestum ATP synthase. Schematic view of the enzyme consisting of the F1 headpiece with the catalytic binding sites on the three  subunits, the F0 motor module with the Na+-binding sites on the c subunits and an Na+-conducting channel within the a subunit. The rotor (green) comprises subunits c9-12  and the stator (blue) comprises the ab2 33 assembly. The pathway of Na+ during ATP synthesis is from the periplasm through the a subunit stator channel onto an empty c subunit rotor site at the rotor–stator interface. The Na+ ion dissociates from this site into the cytoplasm after the rotor has turned. The positive stator charge (aR227) that contributes in the torque-generating mechanism through electrostatic attraction of negatively charged empty rotor sites is indicated in red.
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 | Figure 2 Generation of a membrane potential by the 'acid bath procedure'. The procedure involves two consecutive steps, the acidic and the basic stage. During the acidic stage, the proteoliposomes containing 5 mM K+-phosphate buffer, pH 7.2, are incubated with 100 mM succinate buffer, pH 5.0 (not shown). At this pH, 70.2% of the succinate is present as monoanion and 12.6% is in the undissociated form. As both succinate species are membrane permeable (see below), they will diffuse into the liposome interior in response to the concentration gradient. The membrane potential transiently generated by the uptake of the succinate monoanion collapses rapidly due to the proton permeability of the liposome membrane (see Figure 4). After 2 min at 25°C, the succinate has equilibrated between the outside and the inside, and the pH is 5.0 on both sides. The cartoon depicts the events occurring during the shift to the basic stage by 1:1 dilution into 100 mM glycylglycine buffer, pH 8.5. The pH jump shifts the external equilibrium towards the succinate dianion (99.9%), resulting in a large concentration gradient of the succinate monoanion across the membrane (1). Part of the succinate monoanion folds into a ring, where the negative charge is delocalized between the two carboxylic groups, and in this form it becomes membrane permeable (2). Consequently, it diffuses through the membrane from the inside to the outside, following its concentration gradient until the developing electric potential becomes equal to the concentration difference of the succinate monoanion on both sides (3). The membrane potential thus formed is essential for the synthesis of ATP by the chloroplast ATP synthase (4). Note that the membrane potential rapidly collapses by proton movements from the inside to the outside. The membrane permeability for protons is increased by both   and pH.
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Figure 3 -dependent uptake of several dicarboxylic acids or [14C]SCN- into liposomes. The buffer kept at pH 8.5 (A), pH 5.0 (B) or pH 4.0 (C) contained 2 mM of 14C-labelled NaSCN ( ), 2 mM [14C]maleinate ( ), 2 mM [14C]malonate ( ), 2 mM [14C]succinate ( ) or 2 mM [14C]fumarate ( ). A K+/valinomycin diffusion potential of 60 mV was induced by the addition of KCl ( ) and the uptake of the radioactivity was determined. For details, see Materials and methods.
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 | Figure 4 Effect of various phospholipid:valinomycin ratios on the electric potential elicited by succinate diffusion. The uptake of [14C]thiocyanate into liposomes was measured after applying the 'acid bath procedure'. Following incubation in succinate buffer, pH 5.0, the suspension was diluted 1:1 into Tricine buffer, pH 8.5, containing 34  M [14C]NaSCN with no valinomycin () or lipid:valinomycin ratios of 920:1 (), 230:1 () or 92:1 (). For details, see Materials and methods.
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Figure 5 Relative contribution of the electric potential () and the H+ or Na+ concentration gradient (pH; pNa+) to the rate of ATP formation by three different ATP synthases. (A) Reconstituted chloroplast ATP synthase. K+/valinomycin diffusion potentials were applied in the absence ( ) or presence () of pH = 206 mV. (B) Reconstituted E.coli ATP synthase. K+/valinomycin diffusion potentials were applied in the absence ( ) or presence () of pH = 206 mV. (C) Reconstituted P.modestum ATP synthase. K+/valinomycin diffusion potentials were applied in the absence of pNa+ (5 mM NaCl present on either side of the membrane) ( ) or in the presence of pNa+ = 77 mV (outside 5 mM NaCl, inside 100 mM NaCl) (). ATP was determined as described in Materials and methods. Please note that ATP synthesis at = 0 was measured in the presence of valinomycin and 1 mM KCl on either side of the membrane in order to avoid interference with the generation of an electric potential through the movement of ions during ATP synthesis.
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 | Figure 6 ATP synthesis and electric potential () generated by the 'acid bath procedure' in thylakoid membranes. (A) ATP synthesis by isolated thylakoid membranes. Thylakoid membranes were subjected to an acid–base transition as described in Materials and methods using 20 mM succinate () or 20 mM fumarate (), pH 5.0, as the acidic, and 100 mM Tris–HCl, pH 8.5, as the basic buffer. In control experiments, thylakoids equilibrated in the acid stage with succinate () or fumarate ( ) were subjected to the basic stage buffer containing, additionally, 2 mM sodium tetraphenylboron. (B) Electric potentials generated across thylakoid membranes by the 'acid bath procedure'. During the acid stage, thylakoids were loaded with 20 mM succinate (, ) or 20 mM fumarate (, ), pH 5.0. The suspensions subsequently were diluted 1:1 into 100 mM Tris–HCl buffer, pH 8.5, containing 23 M [14C]NaSCN (0.5 Ci) without (, ) or with 2 mM sodium tetraphenylboron (, ). The membrane potential was calculated from the uptake of [14C]thiocyanate as described in Materials and methods.
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