Secretory vesicles of the adrenal medulla, and of the neurons of the peripheral catecholaminergic system, contain a mixed-function oxidase, dopamine-Beta-hydroxylase, and a b-type cytochrome. The former converts dopamine to norepinephrine, using molecular oxygen and a source of electrons. Ascorbate has been thought to be the physiological electron donor but we have previously found the vesicle membrane impermeable to ascorbate. The function of the cytochrome is unknown but it might serve to convey electrons from a cytoplasmic donor to dopamine Beta-hydroxylase. Njus et al. have recently shown that electrons can be transferred across the membranes of resealed secretory vesicles. To understand the function of this membrane better, we propose to locate possible sites of electron transfer on both sides of the membrane, using a method already demonstrated by others to be useful in studies of other cytochromes. Our approach will be to study the labeling patterns and reaction kinetics of chromous ion with electron-accepting proteins of the secretory vesicle membrane. The conversion of chromous ion to chromic ion due to electron transfer has been shown with cytochrome c to produce irreversible labeling of the cytochrome chromic ion. This approach is expected to identify all proteins capable of carrying electrons. The results should discriminate among those that are on the external face of the vesicle membrane, those on the internal face and those that functionally span the membrane. Determination of reaction stoichiometry (and in the case of cytochrome b, kinetic parameters) is expected to identify any proteins that transfer electrons as functional dimers or higher order complexes.
