Fluorescence quenching studies on the characterization of energy generated at the NADH:quinone oxidoreductase and quinol oxidase segments of marine bacteria.
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.
Kim
Y J
YJ
Institute of Applied Microbiology, University of Tokyo.
Mizushima
S
S
Tokuda
H
H
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016660:NAD(P)H Dehydrogenase (Quinone); D009994:Osmolar Concentration; D010088:Oxidoreductases / Q000378:metabolism*; D011808:Quinone Reductases / Q000378:metabolism*; D012623:Seawater; D012964:Sodium / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014733:Vibrio / Q000201:enzymology*
D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D012964:Sodium; D010088:Oxidoreductases; C020094:duroquinol oxidase; D016660:NAD(P)H Dehydrogenase (Quinone); D011808:Quinone Reductases