PURPOSE: This study was undertaken to investigate whether thioltransferase (TTase) exhibits dehydroascorbate (DHA) reductase activity in human lens epithelial cells. METHODS: TTase was investigated for DHA reductase activity in vitro by the method of glutathione reductase-coupled spectrophotometric assay. DHA reductase activities of human lens epithelial (HLE-B3) cell lysate and TTase-depleted HLE-B3 cell lysate were determined with a 6-deoxy-6-fluoro-DHA probe and 19F-nuclear magnetic resonance (NMR) spectroscopy. TTase-overexpressing and -depleted HLE-B3 cells were investigated for DHA reductase activity. RESULTS: TTase showed DHA reductase activity at a Km of 0.15 mM and Vmax of 35 nmol/min. Investigation of the DHA reductase activity in human lens epithelial (HLE-B3) cell lysate, by using a 6-deoxy-6-fluoro-DHA probe and 19F-NMR spectroscopy, revealed that cell lysate possesses significant DHA reductase activity. This activity decreased extensively when TTase was depleted from the cell lysate by immunoprecipitation. In a cell-free system with externally added DHA, nearly 70% of the recycling ability was diminished when TTase was removed from the lysate. The TTase-overexpressing cells increased DHA reductase activity twofold. HLE-B3 cells showed an ability to take up and recycle DHA, and this ability was increased approximately twofold in the TTase-transfected cells. Suppression of TTase in HLE-B3 cells by an antisense cDNA strategy resulted in a 77% decrease in DHA reductase activity. CONCLUSIONS: The data provide evidence that TTase plays a major role in ascorbic acid recycling in human lens epithelial cells.
PURPOSE: This study was undertaken to investigate whether thioltransferase (TTase) exhibits dehydroascorbate (DHA) reductase activity in human lens epithelial cells. METHODS:TTase was investigated for DHA reductase activity in vitro by the method of glutathione reductase-coupled spectrophotometric assay. DHA reductase activities of human lens epithelial (HLE-B3) cell lysate and TTase-depleted HLE-B3 cell lysate were determined with a 6-deoxy-6-fluoro-DHA probe and 19F-nuclear magnetic resonance (NMR) spectroscopy. TTase-overexpressing and -depleted HLE-B3 cells were investigated for DHA reductase activity. RESULTS:TTase showed DHA reductase activity at a Km of 0.15 mM and Vmax of 35 nmol/min. Investigation of the DHA reductase activity in human lens epithelial (HLE-B3) cell lysate, by using a 6-deoxy-6-fluoro-DHA probe and 19F-NMR spectroscopy, revealed that cell lysate possesses significant DHA reductase activity. This activity decreased extensively when TTase was depleted from the cell lysate by immunoprecipitation. In a cell-free system with externally added DHA, nearly 70% of the recycling ability was diminished when TTase was removed from the lysate. The TTase-overexpressing cells increased DHA reductase activity twofold. HLE-B3 cells showed an ability to take up and recycle DHA, and this ability was increased approximately twofold in the TTase-transfected cells. Suppression of TTase in HLE-B3 cells by an antisense cDNA strategy resulted in a 77% decrease in DHA reductase activity. CONCLUSIONS: The data provide evidence that TTase plays a major role in ascorbic acid recycling in human lens epithelial cells.