Figure 6

Thioredoxins reduce glutathionylated proteins in vitro. (A) Thioredoxin and glutaredoxin activities for purified proteins were determined using standard enzyme assays. Oxidoreductase activity was measured by the ability to reduce the mixed disulphide formed between GSH and HED. The reaction rate for Grx1 was followed by the oxidation of NADPH by glutathione reductase. Thioredoxins were tested in this assay by substituting Grx1 and Glr1, with Trx1 or Trx2 and Trr1, respectively. Protein disulphide reductase activity was measured using insulin as a substrate. The reduction of disulphide bonds in insulin was followed by the oxidation of NADPH by thioredoxin reductase (Trx1 and Trx2) or glutathione reductase (Grx1). Reaction rates are given as μmoles of NADPH oxidized per minute (×10^-3) and represent the means of at least three independent determinations. (B) MALDI-TOF analysis of glutathionylated creatine kinase. The ion at 2871.2 m/z corresponds to the peptide containing the active site cysteine residue (control). Incubation with a 10-fold molar excess of GSSG (+GSSG) resulted in a new peak with a mass shift of 305 Da (3176.4 m/z) consistent with the addition of a glutathione molecule. Thioredoxins (Trx1 or Trx2) can reduce glutathionylated creatine kinase and the modified peptide was shifted to the unmodified form. In contrast, glutaredoxins (Grx1 or Grx2) did not display deglutathionylase activity and the glutathionylated peptide was still detected (3176.4 m/z)