EVIDENCE FOR TRANSMEMBRANE ELECTRON-TRANSFER COUPLED TO PROTON SECRETION IN PLASMA-MEMBRANE VESICLES LOADED BY ELECTROPORATION

We demonstrate an in vitro trans plasma membrane electron transport from an encapsulated nucleotide to an external electron acceptor. Right-side-out vesicles prepared from soybean hypocotyls by aqueous two-phase partitioning were loaded with NADH by electroporation. Absence of calcium ions and an electric field strength of over 2 kV/cm was necessary for opening of the vesicles and importation of NADH. The presence of calcium ions was necessary for vesicle sealing. External NADH was removed with oxalacetate and malate dehydrogenase. If an impermeable electron acceptor was added to the exterior of the sealed vesicles, the oxidation of encapsulated NADH was increased, indicating a transmembrane electron transfer from an internal e(-) donor to the external e(-) acceptor analogous to that observed with intact cells. The ratio of NADH oxidized to hexacyanoferrat III reduced was 0.67. This indicates that NADH oxidation with oxygen as acceptor still occurred. Oxidation of the internal NADH was not affected by actinomycin D. Inhibition was also observed by the auxin 2,4-D, but by the inactive analog 2,3-D too. Oxidation of internal NADH both in absence and presence of the external acceptor, HCF III, showed a rapid decline in activity, which is relieved by the detergent Triton X-100, or the protonophore FCCP. Maximum NADH oxidation both with and without HCF III required an internal acidic pH gradient across the vesicle membrane. The results are consistent with a proton-gradient driving transplasma membrane NADH dehydrogenase which can transfer electrons to oxygen or an external impermeable oxidant.