Luciferin/luciferase ATP synthesis assay

The assay was performed in clear 1.5 ml Eppendorf tubes in a final volume of 100 μl. 50 mM Hepes-NaOH pH 7.5 were used as buffer and the assay mixture contained 10 mM MgCl₂, 200 μg/ml luciferase reagent, 40 μg/ml inverted bacterial membrane vesicles, 2 mM (saturating) DTT and variable concentrations of ATPαS, ADP (typically 50–100 μM) and total phosphate (typically 10 mM). The luminescence was monitored with a Promega GloMax luminometer in steps of 30 s, allowing additions of relevant reagents to the tube when required.

The baseline was first measured (30 s), before 250 pmol ATP were added as a calibration standard (30 s). Ubiquinone Q₁, an ethanol soluble analogue of ubiquinone that can be reduced by DTT, was used to energize the membrane by bo3 oxidase proton pumping and start the ATP synthesis (3 × 30 s). ATP synthesis could be interrupted by the addition of 400 nM potassium cyanide or 10 μM CCCP (2 × 30 s).