Competitive Inhibition of ATP Synthase

Purified M. phlei c-ring in 0.6% Cymal-5 (Anatrace) was used to assay the competition of DCCD with BDQ. The concentrated sample (15 mg/ml) was diluted to 0.1 mg/ml using 20 mM cacodylate/trimethylamine/NH₃ (pH 7.5). BDQ, solubilized in dimethyl sulfoxide, was added to the indicated final concentrations, and samples were incubated for 1 hour at room temperature before adding 25 μM DCCD. Aliquots were removed at several time points (5 to 60 min), directly mixed in a 1:1 ratio with 2′,4′,-dihydroxyacetophenone matrix, and applied onto a ground steel MALDI target in duplicates. Mass spectra were acquired on a MALDI–time-of-flight (TOF)/TOF mass spectrometer (Bruker Autoflex III Smartbeam) and evaluated as previously described (32 (link)). Error bars were calculated from four individual measurements. Graphs and curves were generated using SigmaPlot.