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M.R. Fernando, V.N. Gladyshev, M.F. Lou; The Presence of Mitochondrial Thioltransferase (Grx2) and its Protective and Regenerative Roles of Ascorbic Acid in Human Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):325.
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Purpose: A new mammalian mitochondrial Glutaredoxin (Grx2) has been identified and characterized. We have undertaken this study to investigate the presence of this Grx2 and its functions in human lens epithelial cells. Methods: Human and mouse Grx2 were expressed in Escherichia coli, and were isolated by His-bind column chromatography. Rabbit anti-sera was raised against purified recombinant mouse Grx2. These antibodies cross reacted with human Grx2. Human lens epithelial cells (HLE-B3) were homogenized, fractionated and cytosolic and mitochondrial fractions were used for Western blotting. Dehydroascorbate (DHA) reductase activity of the recombinant human Grx2 (hGrx2) and the recombinant mouse Grx2 (mGrx2) was assayed at 25o C in a reaction mixture containing Na phosphate buffer (100 mM, pH 7.5), EDTA (1 mM), NADPH (0.2 mM), GSH (0.5mM) and glutathione reductase (GR, 2U). The reaction was started by adding fresh DHA (1 mM) in the mixture and followed the decline of absorbance at 340 nm for 5 min. Ascorbate (100 µM) oxidation by ascorbate oxidase (0.05 U/ml) and prevention of this oxidation in the presence of hGrx2, GSH, GR and NADPH was studied using a mixture containing Na phosphate buffer (100 mM, pH 7.5), EDTA (1 mM), NADPH (0.2 mM) GSH (0.5mM) and GR (2U). Several controls experments were carried out. Ascorbate concentration was determined at 0 time and then 15 min intervals up to 60 min using absorbance at 265 nm. Results: Western blot analysis of HLE-B3 cells revealed that Grx2 was present only in the mitochondrial fraction. Both hGrx2 and mGrx2 showed significant DHA reductase activity with Km of 0.1 mM and 0.12 mM for DHA, respectively. Oxidation of ascorbate by ascorbate oxidase was 98% completed within 60 min. GSH (0.5 mM) was ineffective in preventing such oxidation, but a 50% protection was observed in the presence of GR (2U) and NADPH (0.2 mM). Addition of hGrx2 to this reaction mixture completely prevented ascorbate from being oxidized by ascorbate oxidase. Conclusions: The ability of Grx2 in protecting ascorbate from oxidation may be important in maintaining the reducing environment of mitochondria and thereby the integrity of the organelle.
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