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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1976 Jun;73(6):1892–1896. doi: 10.1073/pnas.73.6.1892

The electrochemical gradient of protons and its relationship to active transport in Escherichia coli membrane vesicles.

S Ramos, S Schuldiner, H R Kaback
PMCID: PMC430413  PMID: 6961

Abstract

Membrane vesicles isolated from E. coli generate a trans-membrane proton gradient of 2 pH units under appropriate conditions when assayed by flow dialysis. Using the distribution of weak acids to measure the proton gradient (deltapH) and the distribution of the lipophilic cation triphenyl-methylphosphonium to measure the electrical potential across the membrane (delta psi), the vesicles are shown to generate an electrochemical proton gradient (deltamuH+) of approximately-180 mV at pH 5.5 in the presence of ascorbate and phenazine methosulfate, the major component of which is a deltapH of about -110mV. As external pH is increased, deltapH decreases, reaching 0 at pH 7.5 and above, while delta psi remains at about-75 mV and internal pH remains at pH 7.5. Moreover, the ability of various electron donors to drive transport is correlated with their ability to generate deltamuH+. In addition, deltapH and delta psi can be varied reciprocally in the presence of valinomycin and nigericin. These data and others (manuscript in preparation) provide convincing support for the role of chemiosmotic phenomena in active transport.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altendorf K., Hirata H., Harold F. M. Accumulation of lipid-soluble ions and of rubidium as indicators of the electrical potential in membrane vesicles of Escherichia coli. J Biol Chem. 1975 Feb 25;250(4):1405–1412. [PubMed] [Google Scholar]
  2. Colowick S. P., Womack F. C. Binding of diffusible molecules by macromolecules: rapid measurement by rate of dialysis. J Biol Chem. 1969 Feb 25;244(4):774–777. [PubMed] [Google Scholar]
  3. Harold F. M., Altendorf K. H., Hirata H. Probing membrane transport mechanisms with inophores. Ann N Y Acad Sci. 1974 May 10;235(0):149–160. doi: 10.1111/j.1749-6632.1974.tb43264.x. [DOI] [PubMed] [Google Scholar]
  4. Harold F. M. Conservation and transformation of energy by bacterial membranes. Bacteriol Rev. 1972 Jun;36(2):172–230. doi: 10.1128/br.36.2.172-230.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Hirata H., Altendorf K., Harold F. M. Role of an electrical potential in the coupling of metabolic energy to active transport by membrane vesicles of Escherichia coli. Proc Natl Acad Sci U S A. 1973 Jun;70(6):1804–1808. doi: 10.1073/pnas.70.6.1804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kaback H. R., Barnes E. M., Jr Mechanisms of active transport in isolated membrane vesicles. II. The mechanism of energy coupling between D-lactic dehydrogenase and beta-galactoside transport in membrane preparations from Escherichia coli. J Biol Chem. 1971 Sep 10;246(17):5523–5531. [PubMed] [Google Scholar]
  7. Kaback H. R., Milner L. S. Relationship of a membrane-bound D-(-)-lactic dehydrogenase to amino acid transport in isolated bacterial membrane preparations. Proc Natl Acad Sci U S A. 1970 Jul;66(3):1008–1015. doi: 10.1073/pnas.66.3.1008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kaback H. R., Reeves J. P., Short S. A., Lombardi F. J. Mechanisms of active transport in isolated bacterial membrane vesicles. 18. The mechanism of action of carbonylcyanide m-chlorophenylhydrazone. Arch Biochem Biophys. 1974 Jan;160(1):215–222. doi: 10.1016/s0003-9861(74)80028-7. [DOI] [PubMed] [Google Scholar]
  9. Kaback H. R. Transport across isolated bacterial cytoplasmic membranes. Biochim Biophys Acta. 1972 Aug 4;265(3):367–416. doi: 10.1016/0304-4157(72)90014-7. [DOI] [PubMed] [Google Scholar]
  10. Kaback H. R. Transport studies in bacterial membrane vesicles. Science. 1974 Dec 6;186(4167):882–892. doi: 10.1126/science.186.4167.882. [DOI] [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Lombardi F. J., Kaback H. R. Mechanisms of active transport in isolated bacterial membrane vesicles. 8. The transport of amino acids by membranes prepared from Escherichia coli. J Biol Chem. 1972 Dec 25;247(24):7844–7857. [PubMed] [Google Scholar]
  13. Lombardi F. J., Reeves J. P., Short S. A., Kaback H. R. Evaluation of the chemiosmotic interpretation of active transport in bacterial membrane vesicles. Ann N Y Acad Sci. 1974 Feb 18;227:312–327. doi: 10.1111/j.1749-6632.1974.tb14396.x. [DOI] [PubMed] [Google Scholar]
  14. MITCHELL P. Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature. 1961 Jul 8;191:144–148. doi: 10.1038/191144a0. [DOI] [PubMed] [Google Scholar]
  15. Mitchell P. Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol Rev Camb Philos Soc. 1966 Aug;41(3):445–502. doi: 10.1111/j.1469-185x.1966.tb01501.x. [DOI] [PubMed] [Google Scholar]
  16. Mitchell P. Performance and conservation of osmotic work by proton-coupled solute porter systems. J Bioenerg. 1973 Jan;4(1):63–91. doi: 10.1007/BF01516051. [DOI] [PubMed] [Google Scholar]
  17. Padan E., Zilberstein D., Rottenberg H. The proton electrochemical gradient in Escherichia coli cells. Eur J Biochem. 1976 Apr 1;63(2):533–541. doi: 10.1111/j.1432-1033.1976.tb10257.x. [DOI] [PubMed] [Google Scholar]
  18. Patel L., Schuldiner S., Kaback H. R. Reversible effects of chaotropic agents on the proton permeability of Escherichia coli membrane vesicles. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3387–3391. doi: 10.1073/pnas.72.9.3387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Reeves J. P. Transient pH changes during D-lactate oxidation by membrane vesicles. Biochem Biophys Res Commun. 1971 Nov;45(4):931–936. doi: 10.1016/0006-291x(71)90427-x. [DOI] [PubMed] [Google Scholar]
  20. Rottenberg H. The measurement of transmembrane electrochemical proton gradients. J Bioenerg. 1975 May;7(2):61–74. doi: 10.1007/BF01558427. [DOI] [PubMed] [Google Scholar]
  21. Schuldiner S., Kaback H. R. Membrane potential and active transport in membrane vesicles from Escherichia coli. Biochemistry. 1975 Dec 16;14(25):5451–5461. doi: 10.1021/bi00696a011. [DOI] [PubMed] [Google Scholar]
  22. Schuldiner S., Weil R., Kaback H. R. Energy-dependent binding of dansylgalactoside to the lac carrier protein: direct binding measurements. Proc Natl Acad Sci U S A. 1976 Jan;73(1):109–112. doi: 10.1073/pnas.73.1.109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Short S. A., Kaback H. R., Kohn L. D. Localization of D-lactate dehydrogenase in native and reconstituted Escherichia coli membrane vesicles. J Biol Chem. 1975 Jun 10;250(11):4291–4296. [PubMed] [Google Scholar]
  24. Stroobant P., Kaback H. R. Ubiquinone-mediated coupling of NADH dehydrogenase to active transport in membrane vesicles from Escherichia coli. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3970–3974. doi: 10.1073/pnas.72.10.3970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. WADDELL W. J., BUTLER T. C. Calculation of intracellular pH from the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO); application to skeletal muscle of the dog. J Clin Invest. 1959 May;38(5):720–729. doi: 10.1172/JCI103852. [DOI] [PMC free article] [PubMed] [Google Scholar]

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