Related Experiment Videos
Human cell dehydroascorbate reductase. Kinetic and functional properties
Insights
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.
- 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.
Abstract:
Dehydroascorbate reductase (glutathione: dehydroascorbate oxidoreductase, EC 1.8.5.1) activity was examined in crude cytosol extracts of human cells. In blood neutrophil and lymphocyte extracts, the Km at pH 6.85 for dehydroascorbate was 1.3 mM, and for reduced glutathione 3.8 mM. Rates of dehydroascorbate uptake by intact human neutrophils, monocytes, lymphocytes and cultured fibroblasts were proportional to cytosol dehydroascorbate reductase activities. Dehydroascorbate reduction during dehydroascorbate uptake by these cells may be entirely enzymatic.