European Biophysics Journal

, Volume 42, Issue 2–3, pp 147–158

Estimating the rotation rate in the vacuolar proton-ATPase in native yeast vacuolar membranes

  • Csilla Ferencz
  • Pál Petrovszki
  • Zoltán Kóta
  • Elfrieda Fodor-Ayaydin
  • Lajos Haracska
  • Attila Bóta
  • Zoltán Varga
  • András Dér
  • Derek Marsh
  • Tibor Páli
Original Paper
  • 326 Downloads

Abstract

The rate of rotation of the rotor in the yeast vacuolar proton-ATPase (V-ATPase), relative to the stator or steady parts of the enzyme, is estimated in native vacuolar membrane vesicles from Saccharomyces cerevisiae under standardised conditions. Membrane vesicles are formed spontaneously after exposing purified yeast vacuoles to osmotic shock. The fraction of total ATPase activity originating from the V-ATPase is determined by using the potent and specific inhibitor of the enzyme, concanamycin A. Inorganic phosphate liberated from ATP in the vacuolar membrane vesicle system, during ten min of ATPase activity at 20 °C, is assayed spectrophotometrically for different concanamycin A concentrations. A fit of the quadratic binding equation, assuming a single concanamycin A binding site on a monomeric V-ATPase (our data are incompatible with models assuming multiple binding sites), to the inhibitor titration curve determines the concentration of the enzyme. Combining this with the known ATP/rotation stoichiometry of the V-ATPase and the assayed concentration of inorganic phosphate liberated by the V-ATPase, leads to an average rate of ~10 Hz for full 360° rotation (and a range of 6–32 Hz, considering the ± standard deviation of the enzyme concentration), which, from the time-dependence of the activity, extrapolates to ~14 Hz (8–48 Hz) at the beginning of the reaction. These are lower-limit estimates. To our knowledge, this is the first report of the rotation rate in a V-ATPase that is not subjected to genetic or chemical modification and is not fixed to a solid support; instead it is functioning in its native membrane environment.

Keywords

ATPase Concanamycin F-ATPase Native membrane Rotary enzyme V-ATPase 

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Copyright information

© European Biophysical Societies' Association 2012

Authors and Affiliations

  • Csilla Ferencz
    • 1
  • Pál Petrovszki
    • 1
  • Zoltán Kóta
    • 1
  • Elfrieda Fodor-Ayaydin
    • 1
    • 5
  • Lajos Haracska
    • 2
  • Attila Bóta
    • 3
  • Zoltán Varga
    • 3
  • András Dér
    • 1
  • Derek Marsh
    • 4
  • Tibor Páli
    • 1
  1. 1.Institute of BiophysicsBiological Research CentreSzegedHungary
  2. 2.Institute of GeneticsBiological Research CentreSzegedHungary
  3. 3.Department of Biological Nanochemistry, Institute of Molecular PharmacologyResearch Centre for Natural SciencesBudapestHungary
  4. 4.Max Planck Institute for Biophysical ChemistryGöttingenGermany
  5. 5.Institute of BiochemistryBiological Research CentreSzegedHungary

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