FIGURE 4.

TOPK increases the peroxidase activity of Prx1 and suppresses H₂O₂ accumulation. A, in vitro peroxidase activity was determined as described under “Materials and Methods” using active TOPK and FPLC purified His-Prx1-Wt (Wt) or His-Prx1-S32A (S32A) fusion proteins. Peroxidase activities of nonphosphorylated Wt and S32A were used as controls. To convert the spectrophotometric data (A₅₇₀) to peroxidase activity (mU/ml), a peroxidase standard curve was prepared according to the manufacturer's instructions. B, peroxidase activity was determined as for (A), but using RPMI7951 cells (2 × 10⁴) transfected with control siRNA or TOPK siRNA. Samples were harvested at different times following UVB (4 kJ/m²) treatment. Data are shown as means ± S.D. of three samples from two independent experiments. The asterisk (*) indicates a significantly increased (p < 0.001) activity induced by UVB in control siRNA cells compared with untreated or UVB-treated TOPK siRNA cells. C, oxidation of Prx1 to Prx1-SO₃ was assessed by Western blotting using anti-Prx1-SO₃ (recognizes oxidized Cys-52 in Prx1) after UVB (4 kJ/m²) in RPMI7951 cells transfected with control siRNA or TOPK siRNA. Total TOPK, Prx1, or β-actin protein levels were used as internal controls to confirm TOPK deficiency and equal protein loading. D, effect of TOPK on H₂O₂ accumulation was measured as for (A) in mock, Prx1 Wt or Prx1 mutant (S32A) stably transfected cells. To convert the spectrophotometric data (A₅₇₀) to reflect the levels of H₂O₂ (μm), an H₂O₂ standard curve was prepared according to the manufacturer's instructions.