Figure 4.

The Trx system reverses H₂O₂-inactivated PTP1B. A, reduced PTP1B (600 nm) was preincubated for 30 min either alone or with 2 μm Trx1, 0.5 μm TrxR1 (specific activity, 9.75 units/mg), and 200 μm NADPH, with or without Prx2 (10 μm). H₂O₂ (100 μm) was then added, and samples were taken at 5, 15, and 30 min for measurement of PTP activity (n = 3; mean ± S.E.; *, p < 0.05). B, H₂O₂ consumption by PTP1B and components of the Trx/Prx2 system. Combinations of Trx1 (2 μm), TrxR1 (0.5 μm, 18 units/mg), Prx2 (10 μm), and NADPH (200 μm) with and without reduced PTP1B (600 nm) were treated with 100 μm H₂O₂ at 22 °C, and concentrations of H₂O₂ at the indicated time points were determined by ferrous oxidation of xylenol orange assay (n = 3; mean ± S.E.; *, p < 0.05). C, reactivation of H₂O₂-inactivated PTP1B (cleaved form). Reduced PTP1B was treated with 1 mm H₂O₂ for 5 min and then with catalase to remove residual H₂O₂ and reactivated with 10 mm DTT or TrxR1 (2.5 μm) (specific activity, 18 units/mg) and NADPH (1 mm) with or without Trx1 (10 μm). After 45 min at 22 °C, samples were analyzed for PTP activity (n = 3; mean ± S.E.; *, p < 0.05). D, reactivation of H₂O₂-inactivated PTP1B catalytic domain. Reduced PTP1B was treated with 1 mm H₂O₂ for 5 min and then with catalase and reactivated with 10 mm DTT or TrxR1 (2.5 μm) (specific activity, 22 units/mg), NADPH (300 μm), and Trx1 (10 μm). After 5, 10, and 40 min at 22 °C, samples were analyzed for PTP activity (n = 3; mean ± S.E.; *, p < 0.05).