Thioltranferase mediated ascorbate 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-6fluoro-DHA probe and F-19-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 K-m of 0.15 mM and V-max of 35 nmol/min. Investigation of the DHA reductase activity in human lens epithelial (HLE-B3) cell lysate, by using a 6-deoxy-6fluoro-DHA probe and F-19-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.