Citrate Synthase Activity Measurement

Citrate synthase activity in the brown adipose tissue was assayed in approximately 10 mg of brown adipose tissue lysed in 20 volumes of CelLytic MT Cell Lysis containing 1% (v/v) of protease inhibitor cocktail P8340 (Sigma, Saint-Louis, MO, USA) by bead-beating. The lysate was centrifuged two times at 10,000 g during 10 min at 4°C in order to remove the lipids and the tissue debris. Tissue extract was diluted 1:10 in a 100 mM phosphate buffer (pH 7.1) containing 10 mM 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) and 30 mM acetyl-CoA. After the addition of 10 mM oxaloacetate, free coenzyme A produced from the condensation of acetyl-CoA and oxaloacetate was bound to DTNB, and resulting change in light absorbance detected spectrophotometrically at 412 nm was used to determine the activity of citrate synthase (µmol/mg/s).