Figure 5.

Cysteine-dependent regulation of cICDH activity. (A) ICDH activity was measured for 800 ng of ICDH and ICDH-C363S by monitoring the change in absorbance of NADPH at 340 nm due to the isocitrate-dependent rate of NADP⁺ reduction at 25 °C in 1 mL reaction medium containing 5 mM MgCl₂, 250 μM of NADP⁺ and different (0–5 mM) concentrations of DL-isocitric acid. (B) ICDH were incubated with 1 mM GSNO and measured in the same conditions than described previously. (C) 800 ng of cICDH were incubated with 0.5 mM GSNO, 0.75 mM GSSG for 15 min at 25 °C. The samples were diluted 2-fold and then incubated with 1 mM NADPH in the presence of NTRA (3 µM) and TRXh3 or TRXh5 (4.59 µM) (D) Same experimental design as in (C), but in presence of GR (0.45 µM), GSH (0.8 mM) and GRXC1 or GRXC2 (5 µM). Error bars represent SE (n = 3). * p < 0.01, ** p < 0.001 for statistical differences compared to non-treated samples (Student’s t test).