Figure 2.

Reduced Prx2 protects PTP1B from inactivation by H₂O₂. A, analysis of the PTP1B His-tagged cleaved variant and PTP1B catalytic domain on SDS-PAGE stained with Coomassie Blue. The purity was ∼72% and ∼ 99%, respectively, as determined by densitometry using ImageJ. Side-by-side comparison of activity of the reduced PTP1B cleaved variant and PTP1B catalytic domain showed a mean substrate turnover of 269 and 355 min⁻¹, respectively (n = 3). B, H₂O₂-dependent inactivation of recombinant PTP1B. Reduced PTP1B (600 nm) was exposed to 100 μm or 1 mm H₂O₂ for the designated times and subsequently analyzed for PTP activity using a p-nitrophenyl phosphate (pNPP) substrate (n = 3; mean ± S.E.; *, p < 0.05). PTP1B activities are expressed as percentages of untreated controls. C, activity of PTP1B following treatment with H₂O₂ and reduced or oxidized Prx2. Fully reduced PTP1B (600 nm) was incubated for 30 min with 20 μm reduced Prx2 and 20 μm H₂O₂ or reduced or oxidized Prx alone and then assayed for PTP activity (n = 3; mean ± S.E.; *, p < 0.05; H₂O₂-treated compared with controls). D, Prx2 oxidation state after treatment with reduced PTP1B. Equimolar reduced PTP1B (150 nm) and oxidized Prx2 were incubated for 30 min and analyzed by non-reducing SDS-PAGE. A silver-stained gel (representative of three independent experiments) shows the positions of individual proteins as well as reduced Prx2 for comparison. The bottom and top oxidized Prx2 bands correspond to dimers containing one or two disulfides, respectively. E, activity of PTP1B following treatment with H₂O₂ or oxidized Prx1. Fully reduced PTP1B (600 nm) was incubated for 30 min with 10 μm H₂O₂ or 10 μm oxidized Prx alone and then assayed for PTP activity (n = 3; mean ± S.E.; *, p < 0.05; H₂O₂-treated compared with controls).