Quantifying ATP and AMP Levels

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.