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Bioenergetics pp 233-240 | Cite as

Reconstitution of H+ATPase into Planar Phospholipid Bilayers and Its Kinetic Analysis

  • Hajime Hirata
  • Eiro Muneyuki

Summary

The proton-translocating ATPase of the thermophilic bacterium PS3 (TFoF1) was incorporated into planar phospholipid bilayers, and its electrogenicity and kinetic characteristics were examined. A short-circuit current of up to 1 nA/cm2 was generated upon the addition of ATP. The generation of the electric current was progressively suppressed by inhibitors of TF1. From the reversal potential, the electrogenicity of TFoF1 was indicated to be some 180 mV. The relationship between the electric current induced by ATP and the concentration of ATP revealed that the magnitude of the electric current followed simple Michaelis-Menten type kinetics and the Km was found to be 0.14 mM under the conditions studied. Furthermore, there was no apparent dependence of the Km on externally applied membrane potential. These results suggest that the voltage dependence resides in some steps that defines the apparent Vmax rather than Km in the reaction cycle.

Keywords

Electric Current Voltage Dependence Steady State Current Electric Capacitance Proton Translocation 
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.

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References

  1. 1.
    Boyer, P.D., Cross, R.L., and Momsen, W. (1973) Proc. Natl. Acad. Sci. 70, 2873–2839CrossRefGoogle Scholar
  2. 2.
    Grubmeyer, C., and Penefsky, H.S. (1981) J. Biol. Chem. 256, 3718–3727PubMedGoogle Scholar
  3. 3.
    Takabe, T., and Hammes, G.G. (1981) Biochemistry 20, 6859–6864PubMedCrossRefGoogle Scholar
  4. 4.
    Wong, S.Y., Matsuno-Yagi, A., and Hatefi, Y. (1984) Biochemistry 23, 5004–5009PubMedCrossRefGoogle Scholar
  5. 5.
    Kagawa, Y., and Sone, N. (1979) Methods Enzymol. 55, 364–372PubMedCrossRefGoogle Scholar
  6. 6.
    Hirata, H., Ohno, K., Sone, N., Kagawa, Y., and Hamamoto, T. (1986) J. Biol. Chem. 261, 9839–9843PubMedGoogle Scholar
  7. 7.
    Muneyuki, E., Kagawa, Y., and Hirata, H. (1989) J. Biol. Chem. 264, 6092–6096PubMedGoogle Scholar
  8. 8.
    Montai, M., and Mueller, P. (1972) Proc. Natl. Acad. Sci. USA 69, 3561–3566CrossRefGoogle Scholar
  9. 9.
    Cross, R.L., Grubmeyer, C., and Penefsky, H.S. (1982) J. Biol. Chem. 257, 12101–12105PubMedGoogle Scholar
  10. 10.
    Gresser, M.J., Myers, J.A., and Boyer, P.D. (1982) J. Biol. Chem. 257, 12030–12038PubMedGoogle Scholar
  11. 11.
    Rogner, M., and Gräber, P. (1986) Eur. J. Biochem. 159, 255–261PubMedCrossRefGoogle Scholar
  12. 12.
    Muneyuki, E., and Hirata, H. (1988) FEBS Lett. 234, 455–458PubMedCrossRefGoogle Scholar
  13. 13.
    Hatefi, Y., Yagi, T., Phelps, D.C., Wong, S-Y., Vik, S.B., and Galante, Y.M. (1982) Proc. Natl. Acad. Sci. USA 79, 1756–1760PubMedCrossRefGoogle Scholar

Copyright information

© Plenum Press, New York 1990

Authors and Affiliations

  • Hajime Hirata
    • 1
  • Eiro Muneyuki
    • 1
  1. 1.Department of BiochemistryJichi Medical SchoolTochigiJapan

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