Figure 6. Synergistic action of PDIs during Prx4-driven oxidative folding.

(A) Effects of PDI on oxidative folding of RNase A catalyzed by Prx4 and ERp46. RNase A was refolded for the indicated times by Prx4 and ERp46 in the presence of an amount of PDI equimolar to that of ERp46 (2 μM) in buffer containing H₂O₂. The reaction was quenched by cysteine alkylation with AMS, and the products were subjected to non-reducing SDS-PAGE (upper panel). The representative non-reducing gel images of the three independent experiments are shown. The gel data is cropped to highlight the time course of RNase A oxidation. Time course of recovery of RNase A activity was measured by the change in Abs₂₉₅, using cCMP as a substrate (lower panel). Values represent the mean ± SD from three independent experiments. The dotted lines indicate the result of the simple addition of the recovery curves for Prx4-PDI and Prx4-ERp46 for the refolding time 0–10 min. (B) Effects of PDI on oxidative folding of RNase A catalyzed by Prx4 and P5. Procedures were as described in (A), with the exception that P5 replaced ERp46 in the reaction mixture. The representative non-reducing gel images of the three independent experiments are shown. The gel data is cropped to highlight the time course of RNase A oxidation. (C) Recovery of RNase A activity by the action of PDI. Reduced and denatured RNaseA was pre-incubated with Prx4 and either ERp46 (left) or P5 (right) in buffer containing H₂O₂ for 20 min, followed by the addition of reduced, oxidized, or Cys-less PDI (5 μM) (yellow arrow). Values are the mean ± SD of three independent experiments. (D) Effects of reduced PDIs on recovery of RNase A activity. Procedures were as described in (C), with the exception that, after the 20-min pre-incubation, the reduced forms of the indicated PDIs (5 μM) were added to the reaction solution (yellow arrow). Values are the mean ± SD of three independent experiments.