FIGURE 2.

Specific reduction of the Cys²⁰⁸–Cys²⁴¹ disulfide of Ero1α by PDI and ERp46. A, redox states of Eo1α mutants during Ero1α-catalyzed PDI oxidation. All experiments were initiated by mixing Ero1α-Cysless or Ero1α-CyslessSS (4 μm each) with reduced PDIs (10 μm each) in air-saturated buffer at 30 °C. At 0.25 min into the reaction, the mixture was quenched with TCA, washed with acetone, and modified by AMS. The redox states of Ero1α mutants were assessed by non-reducing SDS-PAGE, followed by immunoblotting with Ero1α antibody (left panel). The right panel indicates the quantification and statistical analysis of the red fraction of Ero1α-Cysless shown in the left panel (n = 3, means ± S.D.). Note that Ero1α-CyslessSS migrates more slowly on a non-reducing gel than does Ero1α-Cysless, because of the absence of the Cys²⁰⁸–Cys²⁴¹ long range disulfide. n.s., not significant; *, p < 0.05; **, p < 0.01. B, time course of the redox state changes of Ero1α mutants during incubation of Ero1α-Cysless or Ero1α-CyslessSS (4 μm each) with reduced PDIs (10 μm each). All experiments were performed as described for Fig. 1C. The redox states of Ero1α mutants were detected as described in A. C, quantification and statistical analysis of the red fraction of Ero1α-Cysless shown in B (n = 3, means ± S.D.). D, redox states of Ero1α-Cysless during reaction with the reduced form of PDI mutants in which a CXXC sequence in either the a or a′ domain is replaced with AXXA. All experiments were performed as described for A (left panel). The right panel indicates the quantification of red Ero1α-Cysless species shown in the left panel (n = 3, means ± S.D.). **, p < 0.01; ***, p < 0.001. E, time course of the redox state changes of Eo1α-Cysless during incubation with the reduced form of PDI active-site mutants. All experiments were performed as described in Fig. 1C. F, Quantification and statistical analysis of the red fraction of Ero1α-Cysless shown in E (n = 3, means ± S.D.). WB, Western blotting.