Bacterial protease

The Azocasein method (Andrejko et al., 2013 (link)) was utilized to demonstrate total extracellular protease production. Here, 100 μl SN was added to 100 μl of Azocasein (Sigma-Aldrich) solution (5 mg/ml in 0.1 M Tris–HCL, pH 8.8) and incubated at 37°C for 3 h. The reaction was stopped with 10% (v/v) trichloroacetic acid (25 μl per tube) and samples were centrifuged at 14,000 × g for 15 min at RT. To each well of a 96-well plate, 50 μl of 0.5 M NaOH was added to 50 μl of Azocasein SN in triplicate. NaOH was used as a blank, bacterial protease (Sigma-Aldrich) was included as a positive control, and absorbance was measured at 405 nm. For the Azocasein assay, one protease activity unit was defined as an absorbance increase (OD_{405 nm}) of 0.02 per hour (Andrejko et al., 2013 (link)).

Brain cortical mitochondria were isolated from mice by the method of Moreira and collaborators [90 (link)], using 0.02% digitonin to free mitochondria from the synaptosomal fraction. Briefly, after animal decapitation, the hippocampus was immediately separated and homogenized at 4 °C in 10 mL of isolation medium (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/mL BSA, pH 7.4) containing 5 mg of the bacterial protease (Sigma-Aldrich, St. Louis, MO, USA). Single brain homogenates were brought to 30 mL and then centrifuged at 2500 rpm (Sorvall Evolution RC Superspeed Refrigerated Centrifuge) for 5 min. The pellet, including the fluffy synaptosomal layer, was resuspended in 10 mL of the isolation medium containing 0.02% digitonin and centrifuged at 10,000 rpm for 10 min. The brown mitochondrial pellet without the synaptosomal layer was resuspended again in 10 mL of medium and centrifuged at 10,000 rpm for 5 min. The pellet was resuspended in 10 mL of washing medium (225 mM mannitol, 75 mM sucrose, 5 mM HEPES, pH 7.4) and centrifuged at 10,000 rpm for 5 min. The final mitochondrial pellet was resuspended in the washing medium and the protein amount determined by the Biuret method calibrated with BSA [91 (link)].

Standard multi-ionic solution for plasma spectrometry is from Perkin-Elmer, Waltham, MA, USA. N-benzoyl-DL-arginine, +catechin, p-nitroanilide, β-glucosidase, bacterial protease, porcine pancreatic trypsin, bovine pancreatic α-chymotrypsin, porcine intestinal peptidase, trypsin, boron trifluoride, and vainillin were from Sigma (Sigma Chemical Co., St Louis, MO, USA). All the other chemicals were of analytical grade.