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Thioltranferase mediated ascorbate recycling in human lens epithelial cells
M Rohan Fernando1, Makoto Satake, Vincent M Monnier
1Redox Biology Center and Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0905, USA.
Investigative Ophthalmology & Visual Science
|December 24, 2003
Summary
Thioltransferase (TTase) demonstrates dehydroascorbate (DHA) reductase activity in human lens epithelial cells, playing a key role in ascorbic acid recycling. This finding highlights TTase
Area of Science:
- Biochemistry
- Cell Biology
- Ophthalmology
Background:
- Ascorbic acid is crucial for maintaining lens health.
- Dehydroascorbate (DHA) is the oxidized form of ascorbic acid.
- Efficient recycling of DHA is essential for cellular antioxidant defense.
Purpose of the Study:
- To investigate the dehydroascorbate (DHA) reductase activity of thioltransferase (TTase).
- To determine the role of TTase in ascorbic acid recycling within human lens epithelial cells.
Main Methods:
- In vitro glutathione reductase-coupled spectrophotometric assay to measure TTase DHA reductase activity.
- 19F-NMR spectroscopy using a 6-deoxy-6-fluoro-DHA probe to assess activity in cell lysates.
- Analysis of TTase-overexpressing and TTase-depleted human lens epithelial cells (HLE-B3).
Main Results:
- TTase exhibited significant DHA reductase activity (Km = 0.15 mM, Vmax = 35 nmol/min).
- Depletion of TTase from HLE-B3 cell lysate markedly reduced DHA reductase activity (by ~70%).
- Overexpression of TTase in HLE-B3 cells doubled DHA reductase activity and DHA uptake/recycling capacity.
Conclusions:
- Thioltransferase (TTase) possesses significant dehydroascorbate (DHA) reductase activity.
- TTase plays a critical role in the ascorbic acid recycling pathway in human lens epithelial cells.
- These findings suggest TTase is vital for maintaining antioxidant balance in the lens.