Luciferin luciferase chemiluminescent method

An amount of 20 µg of total OS homogenate protein was incubated for 5 min at 37 °C in an assay solution composed by: 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 (Ap5A, adenylate kinase inhibitor), and 25µg/mL ampicillin (0.1 mL final volume). As respiratory substrates, 5 mM pyruvate plus 2.5 mM malate were added to the incubation medium. ATP synthesis was induced by the addition of 5 mM KH₂PO₄ and 0.2 mM ADP, at the same pH of the mixture. ATP formation was followed for 2 min in a luminometer (Lumi-Scint, Bioscan, Washington, D.C. USA) by the luciferin/luciferase chemiluminescent method (Roche Diagnostics Corp., Indianapolis, IN, USA). The calibration curve was obtained with ATP standard solutions (Roche Diagnostics Corp., Indianapolis, IN, USA) from 10⁻⁹ and 10⁻⁷ M in the same solution of the experiments [14 (link),31 (link)].

ATP formation from ADP and inorganic phosphate was assayed in purified OS as previously described [36 (link)]. 100 µL OS aliquots (diluted to 1.8 mg/mL) were preincubated with either 1.0 mg/mL cirsiliol or the same volume of vehicle for controls (DMSO) for 1 h in the dark at 25 °C. Then, aliquots of suspensions were incubated for 5 min at 37 °C in 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 (Ap5A, adenylate kinase inhibitor) and 25µg/mL ampicillin to a final protein concentration of 0.012 mg protein/0.1 mL, plus respiratory substrates (0.6 mM NADH and 10 mM Na succinate). ATP synthesis was induced by addition of 5 mM KH₂PO₄ and 0.1 mM ADP at the same pH of the mixture and ATP formation, followed for 1 min in a luminometer (GloMax^® 20/20, Promega Corp. Madison, Fitchburg, WI, USA) by the luciferin/luciferase chemiluminescent method (Roche Diagnostics Corp., Indianapolis, IN, USA). Calibration curve was obtained with ATP standard solutions (Roche Diagnostics Corp., Indianapolis, IN, USA) between 10⁻⁹ and 10⁻⁷ M in the same solution as the experiments.