Fig. 4.

Oxidative stress-dependent structural changes between functional states of Prxs The structural changes of PpPrx in cells exposed to H₂O₂, MV, or gamma rays were investigated. a H₂O₂ and MV were added to the indicated final concentrations for 30 min: 1 no additions, 2 0.1 mM MV, 3 10 mM MV, 4 50 mM MV, 5 0.5 mM H₂O₂, 6 10 mM H₂O₂, and 7 50 mM H₂O₂. b Irradiation was performed at the indicated doses for 30 min: 1 no irradiation, 2 30 Gy, 3 60 Gy, 4 90 Gy, 5 120 Gy, 6 150 Gy, and 7 200 Gy. Crude extracts (3 μg) were dissolved in sample loading buffer and then resolved by native and reducing PAGE. Proteins were transferred to a nitrocellulose membrane and then analyzed by western blot using a mouse anti-PpPrx antibody. Immunoreactive proteins were detected using horseradish peroxidase-conjugated goat anti-mouse IgG. cP. putida was challenged with 20 mM H₂O₂ for 30 min and then allowed to recover for 60 min without H₂O₂. The structural changes in the PpPrx were then analyzed by native PAGE and western blotting every 15 min. P. putida cells were exposed to different conditions and then detected with an anti-PpPrx antibody: 1 normal conditions, 2 and 3 exposure to 20 mM H₂O₂ for 15 and 30 min, and 4–7 recovery without H₂O₂ for 15, 30, 45, and 60 min. d To understand the mechanism of the structural change, crude proteins were extracted from the P. putida cells under normal conditions and then exposed to various reagents; the structural changes in the PpPrx were analyzed by non-reducing PAGE and western blotting with an anti-PpPrx antibody. 1 Cell extract with 100 mM DTT; 2 cell extract without added reagents; 3 cell extract with 1 mM NADPH; 4 cell extract with 1 mM NADPH and 10 mM H₂O₂; 5 cell extract with 1 mM NADPH, 10 mM H₂O₂, and yeast Trx system; 6 cell extract with 1 mM NADPH, 10 mM H₂O₂, and yeast GSH system; 7 cell extract with 10 mM H₂O₂. The Trx and GSH systems contained Trx-thioredoxin reductase (TR) and GSH-glutathione reductase (GR). The lower panels indicate the amount of PpPrx in each sample under reducing conditions