Measuring ATP Synthase Activity

The F_oF₁ ATP-synthase (ATP Synthase) activity was evaluated incubating 2 × 10⁵ cells at 25 °C for 10 min in a medium containing: 50 mM Tris-HCl (pH 7.4), 50 mM KCl, 1 mM EGTA, 2 mM MgCl₂, 0.6 mM ouabain, 0.25 mM di(adenosine)-5-Penta-phosphate (an adenylate kinase inhibitor), and 25 μg/mL ampicillin (0.1 mL final volume); then, 10 mM pyruvate plus 5 mM malate or 20 mM succinate were employed to stimulate complexes I, III, and IV or complexes II, III, and IV pathways, respectively [17 (link)]. As for OCR evaluation, 3 µM BPTES, 4 µM Etomoxir, and 2 µM UK5099 were used for the cellular energy substrate affinity evaluation. In this case, cells were suspended in the growth medium diluted 1:1 with the solution described above. In each case, ATP synthesis was induced by adding 0.1 mM ADP. The reaction was monitored every 30 s for 2 min with a luminometer (GloMax^® 20/20 Luminometer, Promega Italia, Milano, Italy), using the luciferin/luciferase chemiluminescent method (luciferin/luciferase ATP bioluminescence assay kit CLS II, Roche, Basel, Switzerland). ATP standard solutions in a concentration range between 10⁻⁸ and 10⁻⁵ M were used for calibration. Data were expressed as nmol ATP/min/10⁶ cells [24 (link)].