Mitochondrial and Nuclear Isoform of Thioltransferase (Grx2) Has Peroxidase Activity in Lens Epithelial Cells

Abstract

Abstract: : Purpose: We have undertaken this study to investigate the physiological functions of mitochondrial and nuclear isoform of thioltransferase (Grx2) in mouse lens epithelial cells. Methods: cDNA for mouse Grx2 was cloned into pET21d(+) vector and recombinant mouse Grx2 was expressed in E. coli and purified using His–bind column. Grx2 over–expressing mouse lens epithelial cells were generated using inducible mammalian gene expression system of p–Tet on technique. Transfected cells were incubated with 5 µM doxycycline for 48 hours to induce Grx2 over–expression. Mitochondria and nucleus were isolated from normal and transfected cells and lysed. Glutathione dependent peroxidase activity of recombinant mouse Grx2 or cell lysates was assayed at 37^oC in a mixture containing potassium phosphate buffer (100 mM, pH 7.0), EDTA (2 mM), NaN₃ (2mM), NADPH (0.2 mM), glutathione (0.1mM) and glutathione reductase (GR, 2U). The reaction was started by the addition of 0.25 mM H₂O₂, cumene hydroperoxide or tert–butyl hydroperoxide in the mixture and the decline of absorbance at 340 nm was monitored for 5 min. For the measurement of thioredoxin–dependent peroxidase activity of recombinant mouse Grx2 or cell lysates, a reaction mixture containing 50 mM HEPES–NaOH (pH 7.3), 20 µM thioredoxin, 1 µM thioredoxin reductase and 0.5 mM NADPH was used. The reaction was started by the addition of 0.5 mM H₂O₂ and the decline of absorbance at 340 nm was followed for 5 min. Results: Mouse recombinant Grx2 could reduce H₂O₂, cumene hydroperoxide and tert–butyl hydroperoxide using GSH, GR and NADPH as electron donor system. Grx2 also exhibited significant peroxidase activity in the presence of thioredoxin/thioredoxin reductase/NADPH system. The nuclear peroxidase activity increased 2–fold, while the mitochondrial peroxidase activity increased 6–fold in the Grx2 over–expressed cells over the normal cells. Conclusions:These results demonstrate that Grx2 plays an important role as an antioxidant enzyme in the mitochondrial and nuclear fractions of the lens epithelial cells.

Keywords: oxidation/oxidative or free radical damage • cataract • antioxidants