FIGURE 4.

Deglutathionylation of cTPI mediated by recombinant GRXC1 and GRXC2. BioGEE detection by streptavidin conjugated with alkaline phosphatase after native PAGE analysis of 350 ng recombinant cTPI (A,B, upper). Recombinant cTPI was S-glutathionylated in the presence of BioGEE followed by desalting as described in Section “Materials and Methods.” The enzyme was then treated for 30 min with GSH-regeneration system (GRS), 10 μM (A) GRXC1, (B) GRXC2 or both GRS + GRXC1 (A) or GRXC2 (B). A control sample without treatment (CTL) and another control deglutathionylated in 10 mM DTT (DTT) were also prepared. Aliquots of the samples were denatured and analyzed by Western blot (80 ng proteins) after SDS-PAGE using an anti-cTPI antibody to show equal protein concentrations in samples (A,B, lower). For (C,D), S-glutathionylated cTPI (0.2 μg/ml) was incubated with 0.01% BSA (CTL, black circles) and GRS (open circles) or 10 mM DTT (black squares) or 0.2 μM (C) GRXC1 (D) GRXC2 (open triangles) or both GRS + GRXs (black triangles). Aliquots of the different treatment were withdrawn at different time points to measure cTPI activity (mean ± SE, n = 3). Activity is expressed as percentage of the control (non-glutathionylated) sample.