M3003

Quantification of ATP and AMP was based on the enzyme coupling method [26 (link)]. Twenty micrograms of total proteins was used. Briefly, ATP was assayed spectrophotometrically at 340 nm, following NADP⁺ reduction, at 25 °C. The reaction mixture contained the following: 1 mM NADP⁺, 10 mM MgCl₂, 5 mM glucose, and 100 mM Tris-HCl, pH 7.4, in 1 ml final volume. Samples were analyzed before and after the addition of purified hexokinase and glucose-6-phosphate dehydrogenase (4 μg; HK+G6PD, HKG6PDH-RO, Sigma-Aldrich, Italy). AMP was assayed spectrophotometrically at 340 nm, following NADH oxidation. The reaction mixture contained the following: 75 mM KCl, 5 mM MgCl₂, 0.2 mM ATP, 0.5 mM phosphoenolpyruvate, 0.2 mM NADH, 10 IU adenylate kinase (AK, M3003, Sigma-Aldrich, Italy), 25 IU pyruvate kinase plus 15 IU lactate dehydrogenase (PK+LDH, Sigma-Aldrich, Italy), and 100 mM Tris-HCl pH 8.0.

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.