Advertisement

The Journal of Membrane Biology

, Volume 30, Issue 1, pp 121–134 | Cite as

Electrochemical potential of protons in vesicles reconstituted from purified, proton-translocating adenosine triphosphatase

  • Nobuhito Sone
  • Masasuke Yoshida
  • Hajime Hirata
  • Harumasa Okamoto
  • Yasuo Kagawa
Article

Summary

Measurements were made of the difference in the electrochemical potential of protons (\(\Delta \bar \mu H^ + \)) across the membrane of vesicles reconstituted from the ATPase complex (TF0·F1) purified from a thermophilic bacterium and P-lipids. Two fluorescent dyes, anilinonaphthalene sulfonate (ANS) and 9-aminoacridine (9AA) were used as probes for measuring the membrane potential (ΔΨ) and pH difference across the membrane (Δ pH), respectively.

In the presence of Tris buffer the maximal ΔΨ and no Δ pH were produced, while in the presence of the permeant anion NO 3 the maximal Δ pH and a low ΔΨ were produced by the addition of ATP. When the ATP concentration was 0.24mm, the ΔΨ was 140–150 mV (positive inside) in Tris buffer, and the Δ pH was 2.9–3.5 units (acidic inside) in the presence of NO 3 . Addition of a saturating amount of ATP produced somewhat larger ΔΨ and Δ pH values, and the\(\Delta \bar \mu H^ + \) attained was about 310 mV.

By trapping pH indicators in the vesicles during their reconstitution it was found that the pH inside the vesicles was pH 4–5 during ATP hydrolysis.

The effects of energy transfer inhibitors, uncouplers, ionophores, and permeant anions on these vesicles were studied.

Keywords

Tris Buffer Electrochemical Potential Thermophilic Bacterium Adenosine Triphosphatase Saturate Amount 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bakker, E.P., Van Dam, K. 1974. The influence of diffusion potentials across liposomal membranes on the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate.Biochim. Biophys. Acta 339:157Google Scholar
  2. 2.
    Chance, B., Mela, L. 1967. Hydrogen ion concentration changes in mitochondrial membranes.J. Biol. Chem. 241:4588Google Scholar
  3. 3.
    Conti, F., Malerba, F. 1972. Fluorescence signals and stained lipid bilayers under applied potentials.Biophysik 8:326Google Scholar
  4. 4.
    Deamer, D.W., Prince, R., Crofts, A.R. 1972. The response of fluorescent amines to pH gradients across liposome membranes.Biochim. Biophys. Acta 274:323Google Scholar
  5. 5.
    Harold, F.M. 1970. Antimicrobial agents and membrane function.Adv. Microb. Physiol. 4:45Google Scholar
  6. 6.
    Jasaitis, A.A., Kuliene, V.V., Skulachev, V.P. 1971. Anilinonaphthalenesulfonate fluorescence changes induced by nonenzymatic generation of membrane potential in mitochondria and submitochondria particles.Biochim. Biophys. Acta 234:177Google Scholar
  7. 7.
    Kagawa, Y. 1972. Reconstitution of oxidative phosphorylation.Biochim. Biophys. Acta 265:297Google Scholar
  8. 8.
    Kagawa, Y., Kandrach, A., Racker, E. 1973. Partial resolution of the enzyme catalyzing oxidative phosphorylation, XXVI. Phospholipid specificity of the vesicles capable of energy transformation.J. Biol. Chem. 248:676Google Scholar
  9. 9.
    Kagawa, Y., Racker, E. 1971. Partial resolution of the enzymes catalyzing oxidative phosphorylation, XXV. Reconstitution of vesicles catalyzing32Pi-adenosine triphosphate exchange.J. Biol. Chem. 246:5477Google Scholar
  10. 10.
    Mitchell, P. 1966. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation.Biol. Rev. 41:445Google Scholar
  11. 11.
    Mitchell, P., Moyle, J. 1968. Proton translocation coupled to ATP hydrolysis in rat liver mitochondria.Eur. J. Biochem. 4:530Google Scholar
  12. 12.
    Mitchell, P., Moyle, J., Smith, L. 1968. Bromthymol blue as a pH indicator in mitochondrial suspensions.Eur. J. Biochem. 4:9Google Scholar
  13. 13.
    Rottenberg, H. 1975. The measurement of transmembrane electrochemical proton gradients.Bioenergetics 7:61Google Scholar
  14. 14.
    Rottenberg, H., Lee, C.P. 1975. Energy dependent hydrogen ion accumulation in submitochondrial particles.Biochemistry 14:2675Google Scholar
  15. 15.
    Schuldiner, S., Rottenberg, H., Avron, M. 1972. Determination of Δ pH in chloroplasts.Eur. J. Biochem. 25:64Google Scholar
  16. 16.
    Sone, N., Yoshida, M., Hirata, H., Kagawa, Y. 1975. Purification and properties of a dicyclohexylcarbodiimide-sensitive adenosine triphosphatase from thermophilic bacterium.J. Biol. Chem. 250:7917Google Scholar
  17. 17.
    Thayer, W. S., Hinkel, P. C. 1973. Stoichiometry of adenosine triphosphate-driven proton translocation in bovine heart submitochondrial particles.J. Biol. Chem. 248:5395Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1976

Authors and Affiliations

  • Nobuhito Sone
    • 1
  • Masasuke Yoshida
    • 1
  • Hajime Hirata
    • 1
  • Harumasa Okamoto
    • 1
  • Yasuo Kagawa
    • 1
  1. 1.Department of BiochemistryJichi Medical SchoolTochigi-KenJapan

Personalised recommendations