In catecholamine-secreting cells, the catecholamine is stored in intracellular vesicles. These storage vesicles contain ascorbic acid (vitamin C) which is used by dopamine B-hydroxylase to convert dopamine to norepinephrine. The mechanism of ascorbate accumulation and the way in which ascorbate is regenerated from its oxidized form, dehydroascorbate, are unknown. This project will elucidate these processes in bovine chromaffin granules, the storage vesicles of the adrenal medulla. I propose that ascorbate within the granules is reduced by ascorbate in the cytoplasm and that the electron transfer from extragranular to intragranular ascorbate is driven by the transmembrane pH gradient and electrical potential. I propose further that cytochrome b-561 (a membrane protein also known as chromomembrin B) mediates the transfer of electrons from external to internal ascorbate. Finally, I propose that ascorbate is accumulated because dehydroascorbate permeates the chromaffin-granule membrane but is reduced to ascorbate more completely internally than externally. This project, designed to test these hypotheses, will consist of three parts: First, we will develop assays for measuring electron transfer across the chromaffin-granule membrane and we will study the effects of inhibitors and such factors as temperature, membrane potential and pH gradient. Second, we will show that cytochrome b-561 mediates this process by demonstrating electron transfer across phospholipid vesicle membranes into which the purified cytochrome has been reconstituted. Finally, we will see whether the equilibrium transmembrane gradient in ascorbate concentration correlates with the steady-state pH gradient and membrane potential as predicted by theory. This project will define, for the first time, a cellular mechanism for accumulating and regenerating ascorbic acid. It will contribute to the field of bioenergetics by demonstrating a novel chemiosmotic coupling between the proton gradient and electron flow. Finally, it will clarify the role of ascorbic acid in catecholamine metabolism and in the functioning of the nervous system.