Determining Acly Activity in Liver Tissue

Acly activity was determined as described in ref. ^{59 (link)}. In brief, liver tissue was lysed in ice-cold 220 mM mannitol, 70 mM sucrose, 5 mM potassium HEPES buffer, pH 7.5 containing 1 mM dithiothreitol. Lysates were centrifuged at 600 × g for 10 min to precipitate the nuclei and debris. Then, the supernatant was centrifuged at 5500 × g for 10 min to precipitate the mitochondrial fraction, and the supernatant was then centrifuged at 20,000 × g for 20 min to generate a cytosolic fraction used to measure enzyme activity. Enzymatic activity was measured in 5 mM citrate, 0.3 mM coenzyme A, 3 mM ATP, 0.15 mM NADH, 10 mM MgCl₂, 10 mM dithiothreitol, and 6 units/ml of malate dehydrogenase in 100 mM Tris chloride buffer, pH 8.5 at 37 °C. The NADH disappearance was monitored at 340 nm for 1 min for background, after which 5 mM citrate was added to determine Acly activity.