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. 1988 Apr;86(4):1257–1263. doi: 10.1104/pp.86.4.1257

Quantitative and Rapid Estimation of H+ Fluxes in Membrane Vesicles 1

Software for Analysis of Fluorescence Quenching and Relaxation

Ian R Jennings 1,2, Philip A Rea 1,2,2, Roger A Leigh 1,2, Dale Sanders 1,2
PMCID: PMC1054661  PMID: 16666064

Abstract

Proton transport is often visualized in membrane vesicles by use of fluorescent monoamines which accumulate in acidic intravesicular compartments and undergo concentration-dependent fluorescence quenching. Software for an IBM microcomputer is described which permits logging and editing of changes in fluorescence monitored by a Perkin-Elmer LS-5 luminescence spectrometer. An accurate estimate of the instantaneous rate of fluorescence quenching or recovery is then facilitated by least squares fitting of fluorescence data to a nonlinear function. The software is tested with tonoplast vesicles from Beta vulgaris. Quenching of acridine orange fluorescence by ATP-driven (primary) transport and relaxation of quenching by Na+/H+ antiport can both be fitted with single exponential functions. Initial rates of ATP- and Na+ -dependent fluorescence changes are derived and can be used for Km determinations. The method constitutes a simple and efficient alternative to manual analysis of analog fluorescence traces and results in a reliable quantitative measurement of the relative rate of proton transport in membrane vesicle preparations.

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Selected References

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

  1. Bentrup F. W., Gogarten-Boekels M., Hoffmann B., Gogarten J. P., Baumann C. ATP-dependent acidification and tonoplast hyperpolarization in isolated vacuoles from green suspension cells of Chenopodium rubrum L. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2431–2433. doi: 10.1073/pnas.83.8.2431. [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. 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]
  5. 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]
  6. 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]
  7. Gabathuler R., Cleland R. E. Auxin regulation of a proton translocating ATPase in pea root plasma membrane vesicles. Plant Physiol. 1985 Dec;79(4):1080–1085. doi: 10.1104/pp.79.4.1080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Junge W., Hong Y. Q., Qian L. P., Viale A. Cooperative transient trapping of photosystem II protons by the integral membrane portion (CF0) of chloroplast ATP-synthase after mild extraction of the four-subunit catalytic part (CF1). Proc Natl Acad Sci U S A. 1984 May;81(10):3078–3082. doi: 10.1073/pnas.81.10.3078. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Maloney P. C. Membrane H+ conductance of Streptococcus lactis. J Bacteriol. 1979 Oct;140(1):197–205. doi: 10.1128/jb.140.1.197-205.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mettler I. J., Mandala S., Taiz L. Characterization of in vitro proton pumping by microsomal vesicles isolated from corn coleoptiles. Plant Physiol. 1982 Dec;70(6):1738–1742. doi: 10.1104/pp.70.6.1738. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Perlin D. S., San Francisco M. J., Slayman C. W., Rosen B. P. H+/ATP stoichiometry of proton pumps from Neurospora crassa and Escherichia coli. Arch Biochem Biophys. 1986 Jul;248(1):53–61. doi: 10.1016/0003-9861(86)90400-5. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Rottenberg H. The measurement of membrane potential and deltapH in cells, organelles, and vesicles. Methods Enzymol. 1979;55:547–569. doi: 10.1016/0076-6879(79)55066-6. [DOI] [PubMed] [Google Scholar]
  15. 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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