M. phlei cells (10 g) were resuspended in buffer A [0.15 M NaCl, 0.1 M tricine-KOH (pH 7.5), 5 mM MgCl2, 10% (v/v) glycerol] and broken with a French press (20,000 psi, three passages). Unbroken cells were removed by centrifugation, and membranes were collected. The pellet was resuspended in buffer A and applied to a Sephadex G-50 column. The vesicle-containing fraction was collected. Vesicles were pelleted for 45 min at 60,000 rpm and resuspended in buffer A to a concentration of 5 mg/ml. All procedures were performed at 4°C.
To assay the ATP synthesis activity, 0.2 mM malonate was added to the vesicles, followed by incubation for 1 hour on ice. Reaction buffer (290 μl) [20 mM tricine-KOH (pH 7.5), 0.1 M NaCl, 5 mM KPi, 5 mM MgCl2] was mixed with 50 μg of IMVs, 50 μM ADP, 12.5 mM luciferin, 25 ng of luciferase (Roche), and inhibitors or uncouplers (Fig. 2 ). After stirring for 10 min at room temperature, the reaction was started by the addition of 10 mM succinate/KOH (pH 7.8). ATP synthesis was monitored by the increasing luciferase signal measured with a Sirius L single tube luminometer (Berthold). As an internal standard, 8 nM ATP was added. Data were evaluated using SigmaPlot (Systat).
To assay the ATP synthesis activity, 0.2 mM malonate was added to the vesicles, followed by incubation for 1 hour on ice. Reaction buffer (290 μl) [20 mM tricine-KOH (pH 7.5), 0.1 M NaCl, 5 mM KPi, 5 mM MgCl2] was mixed with 50 μg of IMVs, 50 μM ADP, 12.5 mM luciferin, 25 ng of luciferase (Roche), and inhibitors or uncouplers (