Ascorbic acid is required for the synthesis of both catecholamines and peptide hormones. In catecholamine synthesis, dopamine is hydroxylated to norepinephrine by dopamine Beta-hydroxylase. Many peptide hormones are amidated on the carboxy terminus by a peptide amidating monooxygenase. Both of these ascorbate-requiring enzymes are found within the secretory vesicles that store the hormones, so these vesicles must have a mechanism for regenerating internal ascorbic acid. These enzymes probably use ascorbic acid as a one-electron donor and produce semidehydroascorbate. The hypothesis to be tested is that semidehydroascorbate is reduced back to ascorbate by cytochrome b-561, a protein in the vesicle membranes, and that cytochrome b-561 in turn draws electrons from a cytosolic electron donor. Thus, cytochrome b-561 functions as a transmembrane electron carrier. Adrenal medullary chromaffin vesicles contain dopamine Beta-hydroxylase and neurohypophyseal secretory vesicles contain peptide amidating monooxygenase. Both vesicles also contain ascorbic acid and membrane-bound cytochrome b-561. It has already been shown that both membranes have the capacity to transfer electrons outward from internal ascorbate to an external electron acceptor such as cytochrome c or ferricyanide. The objectives of this project are 1) to demonstrate that electrons can be transferred into resealed chromaffin-vesicle ghosts by monitoring reduction of the trapped redox dye 2,6-dibromophenolindo-3'-phenol sulfonate (DBIP-SO3), 2) to show that external electron donors that reduce internal DBIP-SO3 will also reduce internal semidehydroascorbate (assayed by electron para-magnetic resonance) thereby maintaining internal ascorbate levels (assayed by high performance liquid chromatography), 3) to survey other organelles (platelet dense granules) for cytochrome b-561-mediated electron transfer, and 4) to show that ascorbate-requiring enzymes (peptide amidating monooxygenase, prolyl hydroxylase, lysyl hydroxylase) use ascorbate as a one-electron donor and yield semidehydroascorbate as a product.
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