1 mM−1 cm−1). The reaction buffer contained 10 mM potassium phosphate, pH 7.0, 0.6 mM n-dodecyl-d-maltoside, 2–4 l g−1 homogenate protein and the reaction was initiated with addition of 0.7 l g−1 reduced cytochrome c. The activity of complex IV was measured at 25 °C for 10 min. The activities of the mitochondrial respiratory chain complexes

were described as nmol min−1 mg protein−1. The homogenates (n = 5 each) were centrifuged at 800g for 10 min. and the supernatants kept at −70 °C until used for creatine kinase activity determination. The maximal period between homogenate preparation and enzyme analysis was always less than 5 days. Protein content was determined by the method described by Lowry et al. (1951) using bovine serum albumin as standard. Creatine kinase activity was measured Birinapant in brain homogenates pre-treated with 0.625 mM lauryl maltoside. The reaction mixture consisted of 60 mM Tris–HCl, pH 7.5, containing 7 mM phosphocreatine, 9 mM MgSO4 and approximately 0.4–1.2 μg protein in a final volume of 100 μL. After 15 min of preincubation at 37 °C, the reaction was started by the addition of 0.3 μmol of ADP plus 0.08 μmol of reduced glutathione. The reaction was

stopped after 10 min by the addition of 1 μmol of hydroxymercuribenzoic acid. The creatine formed was estimated according to the colorimetric method of Hughes (1962). The color was developed by the addition of 100 μL 2% α-naphthol and 100 μL 0.05% diacetyl in a final volume of 1 mL and read spectrophotometrically after 20 min at 540 nm. Results were described as nmol min−1 mg protein−1. selleck compound The prefrontal cortex, hippocampus and amygdala (n = 5

each) were homogenized (1:10, w/v) in SETH buffer (0.25 M sucrose, 1 mM EDTA, 10 mM Tris–HCl, pH 7.4). The homogenates were centrifuged at 800×g for 10 min and the supernatants were kept at −70 °C until it will be used for enzyme activity determination. Protein content was determined by the method described by Lowry et al. (1951) using bovine serum albumin as standard. Citrate synthase activity those was assayed according to the method described by Shepherd and Garland (1969). The reaction mixture contained 100 mM Tris, pH 8.0, 100 mM acetyl CoA, 100 mM 5,5-di-thiobis (2-nitrobenzoic acid), 0.1% triton X-100, and 2–4 g supernatant protein and was initiated with 100 Moxaloacetate and monitored at 412 nm for 3 min at 25 °C (the final volume of reaction mixture was 0.3 mL). The Prefrontal cortex, hippocampus and amygdala tissues (n = 5 each) were excised. The tissues were homogenized immediately in extraction buffer (mM) (1% Triton-X 100, 100 Tris, pH 7.4, containing 100 sodium pyrophosphate, 100 sodium fluoride, 10 EDTA, 10 sodium vanadate, 2 PMSF and 0.1 mg of aprotinin/ml) at 4 °C with a Polytron PTA 20S generator (Brinkmann Instruments model PT 10/35) operated at maximum speed for 30 s. The extracts were centrifuged at 11,000 rpm and 4 °C in a Beckman 70.