Document Detail

Thioltranferase mediated ascorbate recycling in human lens epithelial cells.
MedLine Citation:
PMID:  14691178     Owner:  NLM     Status:  MEDLINE    
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.
M Rohan Fernando; Makoto Satake; Vincent M Monnier; Marjorie F Lou
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Investigative ophthalmology & visual science     Volume:  45     ISSN:  0146-0404     ISO Abbreviation:  Invest. Ophthalmol. Vis. Sci.     Publication Date:  2004 Jan 
Date Detail:
Created Date:  2003-12-23     Completed Date:  2004-01-20     Revised Date:  2007-11-15    
Medline Journal Info:
Nlm Unique ID:  7703701     Medline TA:  Invest Ophthalmol Vis Sci     Country:  United States    
Other Details:
Languages:  eng     Pagination:  230-7     Citation Subset:  IM    
Redox Biology Center and Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska 68583-0905, USA.
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MeSH Terms
Antibodies, Blocking / pharmacology
Ascorbic Acid / metabolism*
Cell Line
Epithelial Cells / drug effects,  enzymology*
Fluorescent Dyes
Glutathione / pharmacology
Lens, Crystalline / cytology,  enzymology*
Magnetic Resonance Spectroscopy
Oxidoreductases / antagonists & inhibitors,  metabolism*,  physiology*
Protein Disulfide Reductase (Glutathione)*
Time Factors
Grant Support
Reg. No./Substance:
0/Antibodies, Blocking; 0/Fluorescent Dyes; 0/Glutaredoxins; 50-81-7/Ascorbic Acid; 70-18-8/Glutathione; EC 1.-/Oxidoreductases; EC Disulfide Reductase (Glutathione); EC dehydrogenase (ascorbate)

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