Generation of membrane potential (inside-positive) and DELTA-pH (inside-acidic) at two kinds of NADH:quinone oxidoreductase segments, the Na+-motive segment and another segment, of Vibrio alginolyticus was examined by monitoring the quenching of fluorescence of oxonol V and that of quinacrine, respectively, with inside-out membrane vesicles. Transient generation of membrane potential at the segment occurred when ubiquinone-1 was added in the presence of KCN and NADH. The membrane potential was resistant to a proton conductor, carbonylcyanide m-chlorophenylhydrazone, indicating that the membrane potential was generated specifically at the Na+-motive segment. On the other hand, neither membrane potential nor DELTA-pH was generated at another segment. The Na+-motive segment did not generate DELTA-pH, indicating that only Na+ is extruded at this segment. Furthermore, generation of membrane potential and DELTA-pH at the NADH:quinone oxidoreductase segment of V. anguillarum was examined by using the fluorescence quenching technique. This segment of the bacterium was also found to generate DELTA-psi by the extrusion of Na+ but not H+. These results revealed that the fluorescence quenching technique is useful for the rapid identification and characterization of the respiratory segment involved in Na+ translocation.
