Summary
An electrochemical potential difference for H+ was established across the plasma membrane of the anaerobeStreptococcus lactis by addition of sulfuric acid to cells suspended in potassium phosphate at pH 8 along with valinomycin or permeant anions. Subsequent acidification of the cell was measured by the distribution of salicyclic acid. A comparison between cells treated or untreated with the inhibitor N,N′-dicyclohexylcarbodiimide was used to reveal that portion of net proton entry attributable to a direct coupling between H+ inflow and synthesis of ATP catalyzed by the reversible proton-translocating ATPase of this microorganism. When the imposed electrochemical proton gradient was below 180–190 mV, proton entry was at the rate expected of passive flux, for both control cells and cells treated with the ATPase inhibitor. However, at higher driving force acidification of control cells was markedly accelerated, coincident with ATP synthesis, while acidification of cells treated with the inhibitor continued at the rate characteristic of passive inflow. This observed threshold (180–190 mV) was identified as the reversal potential for this H+ “pump”. Parallel measurements showed that the free energy of hydrolysis for ATP in these washed cells was 8.4 kcal/mole (370 mV). The comparison between the reversal (threshold) potential and the free energy of hydrolysis for ATP indicates a stoichiometry of 2 H+/ATP for the coupling of proton movements to ATP formation in bacteria.
Similar content being viewed by others
References
Ames, B.N. 1966. Assay of inorganic phosphate, total phosphate and phosphatases.Methods Enzymol. 8:115–118
Blok, M.C., Gier, J. de, Deenen, L.L.M. van 1974. Kinetics of the valinomycin-induced potassium ion leak from liposomes with potassium thiocyanate enclosed.Biochim. Biophys. Acta 367:210–224
Boyer, P.D. 1975. A model for conformational coupling of membrane potential and proton translocation to ATP synthesis and to active transport.FEBS Lett. 58:1–6
Downie, J.A., Gibson, F., Cox, G.B. 1979. Membrane adenosine triphosphatases of prokaryotic cells.Annu. Rev. Biochem. 48:103–131
Drift, C., van der, Janssen, D.B., Wezenbeck, P.M.G.F. van 1978. Hydrolysis and synthesis of ATP by membrane-bound ATPase from a motileStreptococcus.Arch. Microbiol. 119:31–36
Fillingame, R.H. 1981. Biochemistry and genetics of bacterial H+-translocating ATPase.Curr. Top. Bioenerg. 11:35–106
Finkelstein, A. 1976. Water and nonelectrolyte permeability of lipid bilayer membranes.J. Gen. Physiol. 68:127–135
Futai, M., Kanazawa, H. 1980. Role of subunits in proton-translocating ATPase (F0−F1).Curr. Top. Bioenerg. 10:181–215
Gomez-Fernandez, J.C., Harris, D.A. 1978. A thermodynamic analysis of the interaction between the mitochondrial coupling adenosine triphosphatase and its naturally occurring inhibitor protein.Biochem. J. 176:967–975
Guidotti, G. 1979. Coupling of ion transport to enzyme activity.In: The Neurosciences 4th Study Program. F.O. Shmitt and F.G. Worden, editors. pp. 831–840. M.I.T. Press, Cambridge
Guynn, R.W., Veech, R.L. 1973. The equilibrium constants of the adenosine triphosphate hydrolysis and the adenosine triphosphate-citrate lyase reactions.J. Biol. Chem. 248:6966–6972
Harold, F.M. 1977. Membranes and energy transduction in bacteria.Curr. Top. Bioenerg. 6:83–149
Harold, F.M., Papineau, D. 1972. Cation transport and electrogenesis byStreptococcus faecalis. I. The membrane potential.J. Membrane Biol. 8:27–44
Harold, F.M., Pavlasova, E., Baarda, J.R. 1970. A transmembrane pH gradient inStreptococcus faecalis: Origin and dissipation by proton conductors and N,N′-dicyclohexylcarbodiimide.Biochim. Biophys. Acta 196:235–244
Harris, D.A., Crofts, A.R. 1978. The initial stages of photophosphorylation. Studies using excitation by saturating, short flashes of light.Biochim. Biophys. Acta 502:87–102
Kagawa, Y. 1978. Reconstitution of the energy transformer, gate and channel: Subunit reassembly, crystalline ATPase and ATP synthesis.Biochim. Biophys. Acta 505:45–93
Kashket, E.R. 1981. Proton motive force in growingStreptococcus lactis andStaphylococcus aureus cells under aerobic and anaerobic conditions.J. Bacteriol. 146:369–376
Kashket, E.R., Wilson, T.H. 1972. Role of metabolic energy in the transport of β-galactosides byStreptococcus lactis.J. Bacteriol. 109:784–789
Kielland, J. 1937. Individual activity coefficients of ions in aqueous solutions.J. Am. Chem. Soc. 59:1675–1678
Kozlov, I.A., Skulachev, V.P. 1977. H+-adenosine triphosphatase and membrane energy coupling.Biochim. Biophys. Acta 463:29–89
Lehninger, A.L., Reynafarje, B., Alexandre, A., Villalobo, A. 1980. Respiration-coupled H+ ejection by mitochondria.Ann. N. Y. Acad. Sci. 341:585–592
Maloney, P.C. 1977. Obligatory coupling between proton entry and the synthesis of adenosine 5′-triphosphate inStreptococcus lactis.J. Bacteriol. 132:564–575
Maloney, P.C. 1978. Coupling between H+ entry and ATP formation inEscherichia coli.Biochem. Biophys. Res. Commun. 83:1496–1501
Maloney, P.C. 1979. Membrane H+ conductance ofStreptococcus lactis.J. Bacteriol. 140:197–205
Maloney, P.C. 1982. Coupling between H+ entry and ATP synthesis in bacteria.Curr. Top. Membr. Transp. 16:175–193
Maloney, P.C., Kashket, E.R., Wilson, T.H. 1974. A protonmotive force drives ATP synthesis in bacteria.Proc. Natl. Acad. Sci. USA 71:3896–3900
Maloney, P.C., Kashket, E.R., Wilson, T.H. 1975. Methods for studying transport in bacteria.In: Methods in Membrane Biology. E. Korn, editor. Vol. 5, pp. 1–50. Plenum Press, New York
Maloney, P.C., Schattschneider, S. 1980. Voltage sensitivity of the proton-translocating adenosine 5′-triphosphatase inStreptococcus lactis.FEBS Lett. 110:337–340
Maloney, P.C., Wilson, T.H. 1975. ATP synthesis driven by a protonmotive force inStreptococcus lactis.J. Membrane Biol. 25:285–310
Martin, R.G., Berberich, M.A., Ames, B.N., Davis, W.W., Goldberger, R.F., Yourno, J.D. 1971. Enzymes and intermediates of histidine biosynthesis inSalmonella typhimurium.Methods Enzymol. 17B:3–39
Mason, P.W., Carbone, D.P., Cushman, R.A., Waggoner, A.S. 1981. The importance of inorganic phosphate in regulation of energy metabolism ofStreptococcus lactis.J. Biol. Chem. 256:1861–1866
Mitchell, P. 1969. Chemiosmotic coupling and energy transduction.Theor. Exp. Biophys. 2:159–216
Mitchell, P. 1974. A chemiosmotic molecular mechanism for proton-translocating adenosine triphosphatases.FEBS Lett. 43:189–194
Mitchell, P. 1979. Compartmentation and communication in living systems. Ligand conduction: A general catalytic principle in chemical, osmotic and chemiosmotic reaction systems.Eur. J. Biochem. 95:1–20
Mitchell, P., Moyle, J. 1967. Acid-base titration across the membrane system of rat-liver mitochondria. Catalysis by uncouplers.Biochem. J. 104:588–600
Morowitz, H.J. 1978. Proton semiconductors and energy transduction in biological systems.Am. J. Physiol. 235:R99-R114
Nichols, J.W., Deamer, D.W. 1980. Net proton-hydroxyl permeability of large unilamellar liposomes measured by an acid-base titration technique.Proc. Natl. Acad. Sci. USA 77:2038–2042
Nichols, J.W., Hill, M.W., Bangham, A.D., Deamer, D.W. 1980. Measurement of net proton-hydroxyl permeability of large unilamellar liposomes with the fluorescent pH probe, 9-aminoacridine.Biochim. Biophys. Acta 596:393–403
Pedersen, P.L., Schwerzmann, K., Cintron, N. 1981. Regulation of the synthesis and hydrolysis of ATP in biological systems: Role of peptide inhibitors of H+-ATPases.Curr. Top. Bioenerg. 11:149–199
Petty, K.M., Jackson, J.B. 1979. Two protons transferred per ATP synthesised after flash activation of chromatophores from photosynthetic bacteria.FEBS Lett. 97:367–372
Rosen, B.P., Kashket, E.R. 1978. Energetics of active transport.In: Bacterial Transport. B.P. Rosen, editor. pp. 559–620. Marcel Dekker, New York
Scholes, P., Mitchell, P. 1970. Acid-base titration across the plasma membrane ofMicrococcus denitrificans: Factors affecting the effective proton conductance and the respiratory rate.J. Bioenerg. 1:61–72
Smith, J.B., Sternweis, P.C. 1977. Purification of membrane attachment and inhibitory subunits of the proton translocating adenosine triphosphatase fromEscherichia coli.Biochemistry 16:306–311
Sternweis, P.C., Smith, J.B. 1980. Characterization of the inhibitory (ε) subunit of the proton-translocating adenosine triphosphatase fromEscherichia coli.Biochemistry 19:526–531
Wilson, D.M., Alderete, J.F., Maloney, P.C., Wilson, T.H. 1976. Protonmotive force as the source of energy for adenosine 5′-triphosphate synthesis inEscherichia coli.J. Bacteriol. 126:327–337
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Maloney, P.C., Hansen, F.C. Stoichiometry of proton movements coupled to ATP synthesis driven by a pH gradient inStreptococcus lactis . J. Membrain Biol. 66, 63–75 (1982). https://doi.org/10.1007/BF01868482
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF01868482