Citrate Synthase Enzyme Activity Assay

Citrate synthase (CS) enzyme activity was assessed as previously described[30 (link),33 (link)]. From 40 to 70 mg samples of the gastrocnemius muscle (n=8 in each group) were homogenized in ice-cold lysis buffer (50 mM Tris-HCl, 100 mM KHPO₄, 2 mM ethylenediaminetetraacetic acid (EDTA), 0.2% wt/vol bovine serum albumin, pH was adjusted to 7.0) in frozen liquid nitrogen. Then, the homogenates were defrosted by shaking for 60 min and centrifuged at 13,000 g for 10 min at 4°C for extraction of soluble proteins. The Bradford reagent (B6916, Merck, Germany) was used for assessment of protein concentration of supernatants. Measurements of CS activity were carried out using CS reaction reagent (100 mM triethanolamine-HCl, 100 µM dithionitrobenzoic acid (DTNB), 0.5 mM Triton-X (0.25% vol/vol), 0.5 mM oxaloacetate, 0.31 mM acetyl CoA, pH 8.0) and spectrophotometer (T60 UV, PG Instruments Limited, UK) at room temperature of 21°C. 10 µL of supernatant was added to start the reaction in 1000 µL. The CS from a porcine heart was used as a standard (C3260, Merck, Germany) for assay calibration. The wavelength of 412 nm and molar extinction coefficient of 13,600 M^-1 cm^-1 were used to assess the maximum CS activity (V_max) during the first 2 min of the reaction.