Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1986 Jan;80(1):161–166. doi: 10.1104/pp.80.1.161

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L. 1,2

Shizuo Yoshida 1, Takefumi Kawata 1, Matsuo Uemura 1, Teruo Niki 1
PMCID: PMC1075075  PMID: 16664574

Abstract

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO3−-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg2+, a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.

Full text

PDF
166

Images in this article

164Fig. 6
on p.164
165Fig. 7
on p.165

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. Buckhout T. J. Characterization of Ca Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots. Plant Physiol. 1984 Dec;76(4):962–967. doi: 10.1104/pp.76.4.962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chanson A., McNaughton E., Taiz L. Evidence for a KCl-Stimulated, Mg-ATPase on the Golgi of Corn Coleoptiles. Plant Physiol. 1984 Oct;76(2):498–507. doi: 10.1104/pp.76.2.498. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Churchill K. A., Holaway B., Sze H. Separation of two types of electrogenic h-pumping ATPases from oat roots. Plant Physiol. 1983 Dec;73(4):921–928. doi: 10.1104/pp.73.4.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Dupont F. M., Bennett A. B., Spanswick R. M. Localization of a proton-translocating ATPase on sucrose gradients. Plant Physiol. 1982 Oct;70(4):1115–1119. doi: 10.1104/pp.70.4.1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Dupont F. M., Giorgi D. L., Spanswick R. M. Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots. Plant Physiol. 1982 Dec;70(6):1694–1699. doi: 10.1104/pp.70.6.1694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Marty F., Branton D. Analytical characterization of beetroot vacuole membrane. J Cell Biol. 1980 Oct;87(1):72–83. doi: 10.1083/jcb.87.1.72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. McCarty D. R., Keegstra K., Selman B. R. Characterization and localization of the ATPase associated with pea chloroplast envelope membranes. Plant Physiol. 1984 Nov;76(3):584–588. doi: 10.1104/pp.76.3.584. [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. 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]
  13. Uemura M., Yoshida S. Involvement of Plasma Membrane Alterations in Cold Acclimation of Winter Rye Seedlings (Secale cereale L. cv Puma). Plant Physiol. 1984 Jul;75(3):818–826. doi: 10.1104/pp.75.3.818. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Wood J. G., Sarinana F. O. The staining of sciatic nerve glycoproteins on polyacrylamide gels with concanavalin A-peroxidase. Anal Biochem. 1975 Nov;69(1):320–322. doi: 10.1016/0003-2697(75)90597-7. [DOI] [PubMed] [Google Scholar]
  15. Yoshida S., Kawata T., Uemura M., Niki T. Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L. Plant Physiol. 1986 Jan;80(1):152–160. doi: 10.1104/pp.80.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Yoshida S., Uemura M., Niki T., Sakai A., Gusta L. V. Partition of membrane particles in aqueous two-polymer phase system and its practical use for purification of plasma membranes from plants. Plant Physiol. 1983 May;72(1):105–114. doi: 10.1104/pp.72.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES