Rate of transmembrane electron transfer in chromaffin-vesicle ghosts

Abstract

The chromaffin vesicle of the adrenal medulla contains a transmembrane electron carrier that may provide reducing equivalents for dopamine beta-hydroxylase in vivo. This electron-transfer system can be assayed by trapping ascorbic acid inside resealed membrane vesicles (ghosts), adding an external electron acceptor such as ferricytochrome c or ferricyanide, and following the reduction of these acceptors spectrophotometrically. Cytochrome c reduction is more rapid at high pH and is proportional to the amount of chromaffin-vesicle ghosts, at least at low ghost concentrations. At pH 7.0, ghosts loaded with 100 mM ascorbic acid reduce 60 microM cytochrome c at a rate of 0.035 +/- 0.010 mu equiv min-1 (mg of protein)-1 and 200 microM ferricyanide at a rate of 2.3 +/- 0.3 mu equiv min-1 (mg of protein)-1. The rate of cytochrome c reduction is accelerated to 0.105 +/- 0.021 mu equiv min-1 (mg of protein)-1 when cytochrome c is pretreated with equimolar ferrocyanide. Pretreatment of cytochrome c with ferricyanide also causes a rapid rate of reduction, but only after an initial delay. The ferrocyanide-stimulated rate of cytochrome c reduction is further accelerated by the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), probably because FCCP dissipates the membrane potential generated by electron transfer. These rates of electron transfer are sufficient to account for electron transfer to dopamine beta-hydroxylase in vivo and are consistent with the mediation of electron transfer by cytochrome b-561.

Ascorbate depletion as a consequence of product recycling during dopamine .beta.-monooxygenase catalyzed selenoxidation

“…From a pharmacological point of view, the rapid oxygenation of PAESe by DBM with its associated ascorbate oxidation cycle could be of great interest. It is thought that the primary process for the regeneration of reduced ascorbate in adrenergic neurotransmitter vesicles is the semidehydroascorbate reductase/cyt B-561 system (Diliberto & Allen, 1981;Njus et al, 1982;Harnadek et al, 1985), which operates on the semidehydroascorbate produced from ascorbate during normal enzymatic turnover by DBM. Since we have demonstrated both qualitatively and quantitatively that no cyt c trappable semidehydroascorbate is produced during the nonenzymatic depletion of fully reduced ascorbate via PAESeO recycling, it is tempting to suggest that, if the electron equivalents for DBM arise solely from the recycling of vesicular semidehydroascorbate, the ASCH2 regenerating system would be incapable of preventing the PAESe-dependent depletion of vesicular ascorbate.…”

Determination of Transport Parameters of Permeant Substrates of the Vesicular Amine Transporter

“…3), it reaches a steady state established by opposing transport and permeation processes. In these experiments, the pH gradient, monitored using acridine orange (14), was not affected by substrate addition over the range of substrate concentrations used here (data not shown). Consequently, we may analyze the transport/permeation steady state assuming a constant pH gradient.…”