P1506

2 ml tubes were preloaded with 250 μl of buffer BI (3 M HClO₂, 77 mM EDTA). 1 ml culture sample was added, vortexed and incubated (lysis, 15 min on ice). 600 μl of BII (1 M KOH, 0.5 M KCl, 0.5 M Tris) were added (neutralization). Samples vortexed and incubated (10 min, on ice), centrifuged (10 min, 0°C, 12 000 g), flash-frozen in liquid nitrogen and stored at –80°C. Extracts were thawed on ice and centrifuged (10 min, 0°C, 12 000 g). 200 μl samples were added either to 320 μl of BIII/PEP (100 mM HEPES, 50 mM MgSO₄·7H₂O, adjusted to pH 7.4 with NaOH, and 1.6 mM phosphoenolpyruvate (Sigma-Aldrich, 860077)) for ATP quantification or BIII/PEP + PK (BIII/PEP with 2 U/μl pyruvate kinase, (Sigma-Aldrich, P1506)) for ATP + ADP quantification, incubated (30 min, 37°C), and heat-inactivated (10 min, 90°C). ATP concentrations were determined using the Invitrogen ATP determination kit was used (ThermoFisher: A22066). 10 μl of each PEP or PEP + PK-treated sample was loaded in a white 96-well plate with solid bottom and kept on ice until the reaction was started. The luciferase master mix was scaled down in volume, and 90 μl of master mix was added to each well. Luminescence was recorded using a BMG Clariostar. ATP concentrations were calculated from a standard curve on the same plate.

The activity of acyl‐CoA synthetase (ACS) was measured by a continuous coupled enzymatic assay as previously described.^[47] The reaction mixture contained 100 mM Tris‐HCl buffer (pH 8.0), 10 mM ATP (10519979001, Sigma‐Aldrich), 15 mM MgCl₂, 5 mM dithiothreitol, 150 mM KCl, 1 mM potassium phosphoenolpyruvate (P7127, Sigma‐Aldrich), 0.3 mM nicotinamide adenine dinucleotide (NADH, N8129, Sigma‐Aldrich) in 100 mM triethanolamine (pH 8.2), and 500 µM sodium palmitate (P9767, Sigma‐Aldrich). 4.5 µg adenylate kinase (M3003, Sigma‐Aldrich), 3 µg pyruvate kinase (P1506, Sigma‐Aldrich), 3 µg lactate dehydrogenase (L8080, Solarbio), and 3 µg mitochondrial protein sample were added in a total reaction volume of 100 µL. The reaction system was incubated at 37 °C for 1 min, and the reaction was initiated by the addition of CoA (final concentration 600 µM) (C4282, Sigma‐Aldrich). Changes in absorbance of the reaction system at 334 nm were measured every 10 s for 15 min with a recording spectrophotometer (Thermo Fisher Scientific). The reaction rate was calculated using the slope and intercept created from a NADH standard curve.
Total cellular and mitochondrial PKA activities were measured by the nonradioactive PKA Kinase Activity Assay Kit (ab139435, Abcam) following manufacturers' instructions.