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Ascorbic acid regeneration in chromaffin granules. In situ kinetics
K R Dhariwal1, M Shirvan, M Levine
1Laboratory of Cell Biology and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892.
The Journal of Biological Chemistry
|March 25, 1991
Summary
Ascorbic acid (vitamin C) is regenerated inside secretory vesicles by external ascorbic acid. This specific process, crucial for vesicle function, occurs efficiently at physiological concentrations.
Area of Science:
- Biochemistry
- Cell Biology
- Neuroscience
Background:
- Ascorbic acid (vitamin C) plays vital roles in cellular functions, including neurotransmitter synthesis and antioxidant defense.
- Chromaffin cells are crucial for catecholamine release, and maintaining intravesicular ascorbic acid levels is essential for their function.
Purpose of the Study:
- To investigate the kinetics, specificity, and mechanism of ascorbic acid regeneration within chromaffin secretory vesicles.
- To elucidate the factors influencing the intragranular regeneration of ascorbic acid by extragranular sources.
Main Methods:
- Utilized intact chromaffin secretory vesicles for kinetic and mechanistic studies.
- Employed Lineweaver-Burk and Eadie-Hofstee analyses to determine kinetic parameters (Km).
- Tested specificity using various reducing agents and structural analogs (isoascorbic acid, D-glucose).
Main Results:
- The apparent Km for internal ascorbic acid regeneration was approximately 280-290 microM.
- Regeneration was specifically mediated by extragranular ascorbic acid or isoascorbic acid, not other reducing agents.
- The process was independent of membrane potential and ATPase activity but potentially linked to the pH gradient across the vesicle membrane.
- Specificity was suggested by the lack of inhibition by D-glucose.
Conclusions:
- Intragranular ascorbic acid regeneration is a specific, efficient process in chromaffin vesicles.
- The mechanism appears coupled to the pH difference across the membrane, not membrane potential or ATPase activity.
- Given the low Km relative to cytosolic concentration, regeneration likely occurs at Vmax in situ, ensuring adequate intravesicular vitamin C levels.