Enzyme reactions were confirmed by monitoring the formation of products as well as the disappearance of reactants via HPLC by incubating the activity bands with the appropriate reaction mixtures. GDH expression was determined utilizing the method described in Mailloux et al. (2009a, b). Briefly, the protein samples were solubilized in 62.5 mM Tris-HCl (pH 6.8), 2% SDS, and 2%β-mercaptoethanol at 100 °C for 5 min. Following solubilization, the protein samples were then loaded into a 10% isocratic gel and electrophoresed using a discontinuous buffer system. 5-FU mw Following electrophoresis, the proteins were transferred electrophoretically to

a Hybond™- polyvinylidene difluoride membrane for immunoblotting. Nonspecific binding sites were blocked by treating the membrane with 5% nonfat skim milk dissolved in TTBS [20 mM Tris-HCl, 0.8% NaCl, and 1% Tween-20 (pH 7.6)] for 1 h. Polyclonal antibodies for GDH were obtained from Abcam. The secondary TGF-beta inhibitor antibodies (Li-Cor, Lincoln, NE) consisted of infrared 700 nm tagged goat anti-rabbit. Visualization of the immunoblot was documented using an Odyssey infrared imaging system (Li-Cor). The H2O2-mediated

regulation of KGDH, GDH, and ICDH was studied as follows: 10 mg of protein equivalent of H2O2-treated cells were transferred into the control (without H2O2) medium and a 10 mg protein equivalent of control cells were incubated in a 100/500 μM H2O2-containing medium. Following a 4–8-h incubation period, the cells were isolated and fractionated as described previously to determine enzymatic activities and/or expression. For a proper comparison, control cells (24 h) and H2O2-treated cells (28 h) in a similar growth phase were utilized to inoculate the different media, respectively. Two milligrams of protein equivalent of CFE from control and stressed cells were placed in a reaction SPTBN5 mixture consisting

of 5 mM histidine and 5 mM citrate, in the presence or absence of 5 mM fluorocitrate, an inhibitor of aconitase, in a phosphate buffer (Nasser et al., 2006). After 30 min, the reaction was halted by placing the mixture at 100 °C for 10 min. The reaction mixture was then subjected to HPLC analysis to monitor the production of KG. Data were expressed as means±SDs. Statistical correlations of data were checked for significance using the Student’s t-test (P≤0.05). All experiments were performed at least twice and in triplicate. While both citrate and histidine were utilized readily by the microorganism, it appeared that in the stationary phase of growth, nearly the entire amino acid was consumed (Fig. 1). The biomass yield was relatively similar in these two situations, with the H2O2-stressed bacteria attaining the stationary phase of growth at a slightly later time. Metabolomic analyses of the CFEs revealed that the H2O2-stressed cells contained significantly more KG and succinate (Fig. 2).