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Human cell dehydroascorbate reductase. Kinetic and functional properties

R Bigley, M Riddle, D Layman

    Biochimica Et Biophysica Acta
    |May 14, 1981
    PubMed
    Summary
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    Human cells, including neutrophils and lymphocytes, possess dehydroascorbate reductase activity. This enzyme activity correlates with dehydroascorbate uptake, suggesting a key role in cellular redox balance.

    Area of Science:

    • Biochemistry
    • Cell Biology
    • Enzymology

    Background:

    • Dehydroascorbate reductase (DHAR) is crucial for the regeneration of ascorbic acid, a vital antioxidant.
    • Understanding DHAR activity in human cells is important for cellular redox homeostasis and antioxidant defense.
    • Previous studies have focused on plant and microbial DHAR, with less known about its specific role in human cell types.

    Purpose of the Study:

    • To investigate the activity and kinetic properties of dehydroascorbate reductase in human cellular extracts.
    • To determine the relationship between DHAR activity and dehydroascorbate uptake in various human cell types.
    • To elucidate the enzymatic mechanism of dehydroascorbate reduction within human cells.

    Main Methods:

    • Crude cytosol extracts from human neutrophils, lymphocytes, monocytes, and cultured fibroblasts were prepared.

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  • Dehydroascorbate reductase activity was assayed using spectrophotometric methods at pH 6.85.
  • Kinetic parameters, including Km values for dehydroascorbate and reduced glutathione, were determined.
  • Dehydroascorbate uptake by intact cells was measured and correlated with intracellular DHAR activity.

    • Main Results:

    • Dehydroascorbate reductase activity was detected in cytosol extracts of human neutrophils and lymphocytes.
    • The Michaelis constant (Km) for dehydroascorbate was 1.3 mM and for reduced glutathione was 3.8 mM in these cells.
    • Uptake rates of dehydroascorbate by intact neutrophils, monocytes, lymphocytes, and fibroblasts were directly proportional to their respective cytosol DHAR activities.
    • The findings suggest that enzymatic reduction by DHAR is the primary mechanism for dehydroascorbate processing during cellular uptake.

    Conclusions:

    • Human blood cells and fibroblasts exhibit significant dehydroascorbate reductase activity.
    • The kinetic properties of human DHAR are consistent with its role in maintaining cellular antioxidant capacity.
    • Enzymatic reduction by dehydroascorbate reductase appears to be the dominant pathway for dehydroascorbate clearance in these human cell types.