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MR Fernando, MF Lou; Thioltransferase Can Catalyze Dehydroascorbate Reduction in Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2364.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Ascorbate is an important antioxidant in the lens. We have undertaken this study to investigate the enzymes responsible for the dehydroascorbate (DHA) reduction in human lens epithelial cells. Methods: In vitro DHA reductase activity of 4 candidate enzymes, including recombinant human lens thioltransferase (RHLT), recombinant human lens thioredoxin-1 (RHLTRx-1), bovine liver protein disulfide isomerase (PDI from Sigma) and rat liver albumin (A from Sigma) was assayed at 250C in a mixture containing Na phosphate buffer (100 mM, pH 7.5), EDTA (1 mM), NADPH (0.3 mM), GSH (1 mM) and glutathione reductase (2U). Purified pig liver thioredoxin reductase (1 µM,TR) was included for RHLTRx-1 assay. The reaction was started by adding fresh DHA (1mM) in the mixture and followed the decline of absorbance at 340 nm for 5 min. One unit was defined as the catalyzed oxidation of 1 µM NADPH/min. In vivo study was carried out by using the lysate of confluent (0.05 millions) cultured human lens epithelial cells (HLE-B3). The lysis buffer contained 50 mM Tris buffer, pH 7.5 and 0.5% Igepal. In some studies, TTase was removed from cell lysate by immunoprecipitating with anti-TTase antibody. Results: RHLT showed significant DHA reductase activity with 0.15 mM Km for DHA and 35 nmole/min Vmax. PDI and A displayed very low DHA reductase activity, at 17.5 mM and 43 mM Km for DHA and 8 nmole/min and 0.85 nmole/min Vmax, respectively. RHLTRx-1 together with TR showed no DHA reductase activity in vitro. HLE-B3 cell lysate yielded 4 mU of DHA reductase activity, which was progressively diminished when TTase was gradually removed from the cells with increasing concentrations of anti-TTase antibody. The antibody at 10 µg could completely inhibit the DHA reductase activity of 5 x 10 4 HLE-B3 cells. Conclusion: Our data provide evidence that TTase possesses catalytic activity to reduce dehydroascorbate to ascorbate in the human lens epithelial cells. This additional catalytic function, together with the dethiolase function demonstrated previously, confirms its important role of redox regulation in the cells.
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