Thioltranferase mediated ascorbate recycling in human lens epithelial cells.
OBJECTIVE
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.
Fernando
M Rohan
MR
Redox Biology Center and Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0905, USA.
Satake
Makoto
M
Monnier
Vincent M
VM
Lou
Marjorie F
MF
D019138:Antibodies, Blocking / Q000494:pharmacology; D001205:Ascorbic Acid / Q000378:metabolism*; D002460:Cell Line; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology*; D005456:Fluorescent Dyes; D054477:Glutaredoxins; D005978:Glutathione / Q000494:pharmacology; D006801:Humans; D007908:Lens, Crystalline / Q000166:cytology / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000502:physiology*; D011490:Protein Disulfide Reductase (Glutathione); D013997:Time Factors
D019138:Antibodies, Blocking; D005456:Fluorescent Dyes; D054477:Glutaredoxins; D010088:Oxidoreductases; D011490:Protein Disulfide Reductase (Glutathione); C020666:glutathione dehydrogenase (ascorbate); D005978:Glutathione; D001205:Ascorbic Acid