Skip to main content
PMC home page
Plant Physiology logoLink to Plant Physiology
. 1990 Feb;92(2):340–345. doi: 10.1104/pp.92.2.340

Solubilization and Reconstitution of the Oat Root Vacuolar H+/Ca2+ Exchanger 1

Karen S Schumaker 1,2, Heven Sze 1
PMCID: PMC1062295  PMID: 16667279

Abstract

Calcium is sequestered into vacuoles of oat (Avena sativa L.) root cells via a H+/Ca2+ antiporter, and vesicles derived from the vacuolar membrane (tonoplast) catalyze an uptake of calcium which is dependent on protons (pH gradient [ΔpH] dependent). The first step toward purification and identification of the H+/Ca2+ antiporter is to solubilize and reconstitute the transport activity in liposomes. The vacuolar H+/Ca2+ antiporter was solubilized with octylglucoside in the presence of soybean phospholipids and glycerol. After centrifugation, the soluble proteins were reconstituted into liposomes by detergent dilution. A ΔpH (acid inside) was generated in the proteoliposomes with an NH4Cl gradient (NH4+in » NH4+out) as determined by methylamine uptake. Fundamental properties of ΔpH dependent calcium uptake such as the Km for calcium (∼15 micromolar) and the sensitivity to inhibitors such as N,N′-dicyclohexylcarbodiimide, ruthenium red, and lanthanum, were similar to those found in membrane vesicles, indicating that the H+/Ca2+ antiporter has been reconstituted in active form.

Full text

PDF
343

Images in this article

344Figure 6
on p.344

Selected References

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

  1. Ambudkar S. V., Lynn A. R., Maloney P. C., Rosen B. P. Reconstitution of ATP-dependent calcium transport from streptococci. J Biol Chem. 1986 Nov 25;261(33):15596–15600. [PubMed] [Google Scholar]
  2. Ambudkar S. V., Maloney P. C. Bacterial anion exchange. Use of osmolytes during solubilization and reconstitution of phosphate-linked antiport from Streptococcus lactis. J Biol Chem. 1986 Aug 5;261(22):10079–10086. [PubMed] [Google Scholar]
  3. Barzilai A., Spanier R., Rahamimoff H. Isolation, purification, and reconstitution of the Na+ gradient-dependent Ca2+ transporter (Na+-Ca2+ exchanger) from brain synaptic plasma membranes. Proc Natl Acad Sci U S A. 1984 Oct;81(20):6521–6525. doi: 10.1073/pnas.81.20.6521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Guern J., Mathieu Y., Kurkdjian A., Manigault P., Manigault J., Gillet B., Beloeil J. C., Lallemand J. Y. Regulation of Vacuolar pH of Plant Cells: II. A P NMR Study of the Modifications of Vacuolar pH in Isolated Vacuoles Induced by Proton Pumping and Cation/H Exchanges. Plant Physiol. 1989 Jan;89(1):27–36. doi: 10.1104/pp.89.1.27. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Hale C. C., Slaughter R. S., Ahrens D. C., Reeves J. P. Identification and partial purification of the cardiac sodium-calcium exchange protein. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6569–6573. doi: 10.1073/pnas.81.21.6569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Hjelmeland L. M., Chrambach A. Solubilization of functional membrane proteins. Methods Enzymol. 1984;104:305–318. doi: 10.1016/s0076-6879(84)04097-0. [DOI] [PubMed] [Google Scholar]
  8. Kaplan R. S., Pedersen P. L. Determination of microgram quantities of protein in the presence of milligram levels of lipid with amido black 10B. Anal Biochem. 1985 Oct;150(1):97–104. doi: 10.1016/0003-2697(85)90445-2. [DOI] [PubMed] [Google Scholar]
  9. Lai S. P., Randall S. K., Sze H. Peripheral and integral subunits of the tonoplast H+-ATPase from oat roots. J Biol Chem. 1988 Nov 15;263(32):16731–16737. [PubMed] [Google Scholar]
  10. Miyamoto H., Racker E. Solubilization and partial purification of the Ca2+/Na+ antiporter from the plasma membrane of bovine heart. J Biol Chem. 1980 Apr 10;255(7):2656–2658. [PubMed] [Google Scholar]
  11. Nakamura T., Hsu C., Rosen B. P. Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters. J Biol Chem. 1986 Jan 15;261(2):678–683. [PubMed] [Google Scholar]
  12. 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]
  13. Schumaker K. S., Sze H. Calcium transport into the vacuole of oat roots. Characterization of H+/Ca2+ exchange activity. J Biol Chem. 1986 Sep 15;261(26):12172–12178. [PubMed] [Google Scholar]
  14. Wakabayashi S., Goshima K. Partial purification of Na+-Ca2+ antiporter from plasma membrane of chick heart. Biochim Biophys Acta. 1982 Dec 8;693(1):125–133. doi: 10.1016/0005-2736(82)90478-3. [DOI] [PubMed] [Google Scholar]

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

RESOURCES