Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1985 Aug;78(4):865–870. doi: 10.1104/pp.78.4.865

Membrane Transport in Isolated Vesicles from Sugarbeet Taproot 1

I. Isolation and Characterization of Energy-Dependent, H+-Transporting Vesicles

Donald P Briskin 1,2, W Robert Thornley 1,2, Roger E Wyse 1,2
PMCID: PMC1064839  PMID: 16664342

Abstract

Sealed membrane vesicles were isolated from homogenates of sugarbeet (Beta vulgaris L.) taproot by a combination of differential centrifugation, extraction with KI, and dextran gradient centrifugation. Relative to the KI-extracted microsomes, the content of plasma membranes, mitochondrial membranes, and Golgi membranes was much reduced in the final vesicle fraction. A component of ATPase activity that was inhibited by nitrate co-enriched with the capacity of the vesicles to form a steady state pH gradient during the purification procedure. This suggests that the nitrate-sensitive ATPase may be involved in driving H+-transport, and this is consistent with the observation that H+-transport, in the final vesicle fraction was inhibited by nitrate. Proton transport in the sugarbeet vesicles was substrate specific for ATP, insensitive to sodium vanadate and oligomycin but was inhibited by diethylstilbestrol and N,N′-dicyclohexylcarbodiimide. The formation of a pH gradient in the vesicles was enhanced by halide ions in the sequence I− > Br− > Cl− while F− was inhibitory. These stimulatory effects occur from both a direct stimulation of the ATPase by anions and a reduction in the vesicle membrane potential. In the presence of Cl−, alkali cations reduce the pH gradient relative to that observed with bis-tris-propane, possibly by H+/alkali cation exchange. Based upon the properties of the H+-transporting vesicles, it is proposed that they are most likely derived from the tonoplast so that this vesicle preparation would represent a convenient system for studying the mechanism of transport at this membrane boundary.

Full text

PDF
866

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. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Briskin D. P., Poole R. J. Characterization of a k-stimulated adenosine triphosphatase associated with the plasma membrane of red beet. Plant Physiol. 1983 Feb;71(2):350–355. doi: 10.1104/pp.71.2.350. [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. 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]
  6. Diamond J. M., Wright E. M. Biological membranes: the physical basis of ion and nonelectrolyte selectivity. Annu Rev Physiol. 1969;31:581–646. doi: 10.1146/annurev.ph.31.030169.003053. [DOI] [PubMed] [Google Scholar]
  7. Franzusoff A. J., Cirillo V. P. Glucose transport activity in isolated plasma membrane vesicles from Saccharomyces cerevisiae. J Biol Chem. 1983 Mar 25;258(6):3608–3614. [PubMed] [Google Scholar]
  8. Gallagher S. R., Leonard R. T. Effect of vanadate, molybdate, and azide on membrane-associated ATPase and soluble phosphatase activities of corn roots. Plant Physiol. 1982 Nov;70(5):1335–1340. doi: 10.1104/pp.70.5.1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Gunn R. B. Co- and counter-transport mechanisms in cell membranes. Annu Rev Physiol. 1980;42:249–259. doi: 10.1146/annurev.ph.42.030180.001341. [DOI] [PubMed] [Google Scholar]
  10. Kaback H. R. The lac carrier protein in Escherichia coli. J Membr Biol. 1983;76(2):95–112. doi: 10.1007/BF02000610. [DOI] [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. Mitchell P. Vectorial chemistry and the molecular mechanics of chemiosmotic coupling: power transmission by proticity. Biochem Soc Trans. 1976;4(3):399–430. doi: 10.1042/bst0040399. [DOI] [PubMed] [Google Scholar]
  13. O'neill S. D., Bennett A. B., Spanswick R. M. Characterization of a NO(3)-Sensitive H-ATPase from Corn Roots. Plant Physiol. 1983 Jul;72(3):837–846. doi: 10.1104/pp.72.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Smith J. C., Hallidy L., Topp M. R. The behavior of the fluorescence lifetime and polarization of oxonol potential-sensitive extrinsic probes in solution and in beef heart submitochondrial particles. J Membr Biol. 1981;60(3):173–185. doi: 10.1007/BF01992556. [DOI] [PubMed] [Google Scholar]
  17. Sze H. Nigericin-stimulated ATPase activity in microsomal vesicles of tobacco callus. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5904–5908. doi: 10.1073/pnas.77.10.5904. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Turner R. J. Quantitative studies of cotransport systems: models and vesicles. J Membr Biol. 1983;76(1):1–15. doi: 10.1007/BF01871450. [DOI] [PubMed] [Google Scholar]
  19. Waggoner A. S. Dye indicators of membrane potential. Annu Rev Biophys Bioeng. 1979;8:47–68. doi: 10.1146/annurev.bb.08.060179.000403. [DOI] [PubMed] [Google Scholar]

Articles from Plant Physiology are provided here courtesy of Oxford University Press

RESOURCES