Figure 3.

In vitro phosphotransferase assays of PKG Iα under reducing and oxidizing conditions. A–D, cGMP-dependent activation of PKG Iα WT (black circles) and PKG Iα C42S (red squares) (A), Δ53 PKG Iα (purple squares) (B), PKG Iα C117S (green triangles) (C), and PKG Iα C195S (blue triangles) (D) under reducing (1 mm TCEP; solid lines) and oxidizing (500 μm H₂O₂, 30 min; dashed lines) conditions. Data are represented as the mean ± S.D. (error bars). E, nonreducing, denaturing SDS-PAGE of PKG Iα constructs treated with increasing concentrations of H₂O₂ (0–2 mm). The dashed line indicates the point at which the concentration of hydrogen peroxide overcomes the concentration of TCEP in the buffer. H₂O₂ values corrected for the presence of TCEP are shown in red. The panel for WT is the same as shown in Fig. 2D to show comparisons of the WT enzyme with mutants.