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. 1988 Apr;86(4):1315–1322. doi: 10.1104/pp.86.4.1315

Dissipation of pH Gradients in Tonoplast Vesicles and Liposomes by Mixtures of Acridine Orange and Anions

Implications for the Use of Acridine Orange as a pH Probe

Andrew J Pope 1, Roger A Leigh 1
PMCID: PMC1054671  PMID: 16666073

Abstract

Acridine orange altered the response to anions of both ATP and in-organic pyrophosphate-dependent pH gradient formation in tonoplast vesicles isolated from oat (Avena sativa L.) roots and red beet (Beta vulgaris L.) storage tissue. When used as a fluorescent pH probe in the presence of I−, ClO3−, NO3−, Br−, or SCN−, acridine orange reported lower pH gradients than either quinacrine or [14C]methylamine. Acridine orange, but not quinacrine, reduced [14C]methylamine accumulation when NO3− was present indicating that the effect was due to a real decrease in the size of the pH gradient, not a misreporting of the gradient by acridine orange. Other experiments indicated that acridine orange and NO3− increased the rate of pH gradient collapse both in tonoplast vesicles and in liposomes of phosphatidylcholine and that the effect in tonoplast vesicles was greater at 24°C than at 12°C. It is suggested that acridine orange and certain anions increase the permeability of membranes to H+, possibly because protonated acridine orange and the anions form a lipophilic ion pair within the vesicle which diffuses across the membrane thus discharging the pH gradient. The results are discussed in relation to the use of acridine orange as a pH probe. It is concluded that the recently published evidence for a NO3−/H+ symport involved in the export of NO3− from the vacuole is probably an artefact caused by acridine orange.

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Selected References

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

  1. Bennett A. B., O'neill S. D., Spanswick R. M. H-ATPase Activity from Storage Tissue of Beta vulgaris: I. Identification and Characterization of an Anion-Sensitive H-ATPase. Plant Physiol. 1984 Mar;74(3):538–544. doi: 10.1104/pp.74.3.538. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blumwald E., Poole R. J. Kinetics of Ca/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris L. Plant Physiol. 1986 Mar;80(3):727–731. doi: 10.1104/pp.80.3.727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Blumwald E., Poole R. J. Na/H Antiport in Isolated Tonoplast Vesicles from Storage Tissue of Beta vulgaris. Plant Physiol. 1985 May;78(1):163–167. doi: 10.1104/pp.78.1.163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Blumwald E., Poole R. J. Nitrate storage and retrieval in Beta vulgaris: Effects of nitrate and chloride on proton gradients in tonoplast vesicles. Proc Natl Acad Sci U S A. 1985 Jun;82(11):3683–3687. doi: 10.1073/pnas.82.11.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Briskin D. P., Thornley W. R., Wyse R. E. Membrane Transport in Isolated Vesicles from Sugarbeet Taproot : II. Evidence for a Sucrose/H-Antiport. Plant Physiol. 1985 Aug;78(4):871–875. doi: 10.1104/pp.78.4.871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Briskin D. P., Thornley W. R., Wyse R. E. Membrane transport in isolated vesicles from sugarbeet taproot : I. Isolation and characterization of energy-dependent, h-transporting vesicles. Plant Physiol. 1985 Aug;78(4):865–870. doi: 10.1104/pp.78.4.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chanson A., Fichmann J., Spear D., Taiz L. Pyrophosphate-driven proton transport by microsomal membranes of corn coleoptiles. Plant Physiol. 1985 Sep;79(1):159–164. doi: 10.1104/pp.79.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Churchill K. A., Sze H. Anion-sensitive, h-pumping ATPase in membrane vesicles from oat roots. Plant Physiol. 1983 Mar;71(3):610–617. doi: 10.1104/pp.71.3.610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Deamer D. W., Prince R. C., Crofts A. R. The response of fluorescent amines to pH gradients across liposome membranes. Biochim Biophys Acta. 1972 Aug 9;274(2):323–335. doi: 10.1016/0005-2736(72)90180-0. [DOI] [PubMed] [Google Scholar]
  11. Deane-Drummond C. E., Glass A. D. Nitrate Uptake into Barley (Hordeum vulgare) Plants : A New Approach Using ClO(3) as an Analog for NO(3). Plant Physiol. 1982 Jul;70(1):50–54. doi: 10.1104/pp.70.1.50. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dell'Antone P., Azzone G. F. Inhibition of energy-linked uptake of acridine dyes by permeant anions. FEBS Lett. 1974 Feb 1;39(1):67–72. doi: 10.1016/0014-5793(74)80018-9. [DOI] [PubMed] [Google Scholar]
  13. Dupont F. M. Variable Effects of Nitrate on ATP-Dependent Proton Transport by Barley Root Membranes. Plant Physiol. 1987 Jun;84(2):526–534. doi: 10.1104/pp.84.2.526. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Garlid K. D., Nakashima R. A. Studies on the mechanism of uncoupling by amine local anesthetics. Evidence for mitochondrial proton transport mediated by lipophilic ion pairs. J Biol Chem. 1983 Jul 10;258(13):7974–7980. [PubMed] [Google Scholar]
  15. Griffith C. J., Rea P. A., Blumwald E., Poole R. J. Mechanism of Stimulation and Inhibition of Tonoplast H-ATPase of Beta vulgaris by Chloride and Nitrate. Plant Physiol. 1986 May;81(1):120–125. doi: 10.1104/pp.81.1.120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kaestner K. H., Sze H. Potential-dependent anion transport in tonoplast vesicles from oat roots. Plant Physiol. 1987 Mar;83(3):483–489. doi: 10.1104/pp.83.3.483. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee H. C., Forte J. G. A study of H+ transport in gastric microsomal vesicles using fluorescent probes. Biochim Biophys Acta. 1978 Apr 4;508(2):339–356. doi: 10.1016/0005-2736(78)90336-x. [DOI] [PubMed] [Google Scholar]
  18. Lew R. R., Spanswick R. M. Characterization of Anion Effects on the Nitrate-Sensitive ATP-Dependent Proton Pumping Activity of Soybean (Glycine max L.) Seedling Root Microsomes. Plant Physiol. 1985 Feb;77(2):352–357. doi: 10.1104/pp.77.2.352. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Poole R. J., Briskin D. P., Krátký Z., Johnstone R. M. Density gradient localization of plasma membrane and tonoplast from storage tissue of growing and dormant red beet : characterization of proton-transport and ATPase in tonoplast vesicles. Plant Physiol. 1984 Mar;74(3):549–556. doi: 10.1104/pp.74.3.549. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Rea P. A., Poole R. J. Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles. Plant Physiol. 1985 Jan;77(1):46–52. doi: 10.1104/pp.77.1.46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schumaker K. S., Sze H. A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots. Plant Physiol. 1985 Dec;79(4):1111–1117. doi: 10.1104/pp.79.4.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Schumaker K. S., Sze H. Decrease of pH Gradients in Tonoplast Vesicles by NO(3) and Cl: Evidence for H-Coupled Anion Transport. Plant Physiol. 1987 Mar;83(3):490–496. doi: 10.1104/pp.83.3.490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang Y., Leigh R. A., Kaestner K. H., Sze H. Electrogenic h-pumping pyrophosphatase in tonoplast vesicles of oat roots. Plant Physiol. 1986 Jun;81(2):497–502. doi: 10.1104/pp.81.2.497. [DOI] [PMC free article] [PubMed] [Google Scholar]

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