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. 1989 Jan;89(1):250–259. doi: 10.1104/pp.89.1.250

Purification and Identification of the Fusicoccin Binding Protein from Oat Root Plasma Membrane 1

Albertus H de Boer 1,2, Bruce A Watson 1, Robert E Cleland 1
PMCID: PMC1055827  PMID: 11537448

Abstract

Fusicoccin (FC), a fungal phytotoxin, stimulates the H+-ATPase located in the plasma membrane (PM) of higher plants. The first event in the reaction chain leading to enhanced H+-efflux seems to be the binding of FC to a FC-binding protein (FCBP) in the PM. We solubilized 90% of the FCBP from oat (Avena sativa L. cv Victory) root PM in an active form with 1% octyl-glucoside. The FCBP was stabilized by the presence of protease inhibitors. The FCBP was purified by affinity chromatography using FC-linked adipic acid dihydrazide agarose (FC-AADA). Upon elution with 8 molar urea, two major protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 29,700 and 31,000 were obtained. Successive chromatography on DEAE Bio-Gel A, hexyl agarose, and FC-AADA resulted in the same two bands when the FC-AADA was eluted with sodium dodecyl sulfate. A direct correlation was made between 3H-FC-binding activity and the presence of the two protein bands. The stoichiometry of the 29,700 and 31,000 molecular weight bands was 1:2. This suggests that the FCBP occurs in the native form as a heterotrimer with an apparent molecular weight of approximately 92,000.

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Selected References

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

  1. Bidwai A. P., Takemoto J. Y. Bacterial phytotoxin, syringomycin, induces a protein kinase-mediated phosphorylation of red beet plasma membrane polypeptides. Proc Natl Acad Sci U S A. 1987 Oct;84(19):6755–6759. doi: 10.1073/pnas.84.19.6755. [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. Conti-Tronconi B. M., Raftery M. A. The nicotinic cholinergic receptor: correlation of molecular structure with functional properties. Annu Rev Biochem. 1982;51:491–530. doi: 10.1146/annurev.bi.51.070182.002423. [DOI] [PubMed] [Google Scholar]
  4. Forbush B., 3rd Characterization of right-side-out membrane vesicles rich in (Na,K)-ATPase and isolated from dog kidney outer medulla. J Biol Chem. 1982 Nov 10;257(21):12678–12684. [PubMed] [Google Scholar]
  5. Karlin A., DiPaola M., Kao P. N., Lobel P. Functional sites and transient states of the nicotinic acetylcholine receptor. Soc Gen Physiol Ser. 1987;41:43–65. [PubMed] [Google Scholar]
  6. Lamed R., Levin Y., Wilchek M. Covalent coupling of nucleotides to agarose for affinity chromatography. Biochim Biophys Acta. 1973 Apr 28;304(2):231–235. doi: 10.1016/0304-4165(73)90239-0. [DOI] [PubMed] [Google Scholar]
  7. Löbler M., Klämbt D. Auxin-binding protein from coleoptile membranes of corn (Zea mays L.). I. Purification by immunological methods and characterization. J Biol Chem. 1985 Aug 15;260(17):9848–9853. [PubMed] [Google Scholar]
  8. Marrè M. T., Romani G., Bellando M., Marrè E. Stimulation of Weak Acid Uptake and Increase in Cell Sap pH as Evidence for Fusicoccin- and K-Induced Cytosol Alkalinization. Plant Physiol. 1986 Sep;82(1):316–323. doi: 10.1104/pp.82.1.316. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Sandstrom R. P., Deboer A. H., Lomax T. L., Cleland R. E. Latency of Plasma Membrane H-ATPase in Vesicles Isolated by Aqueous Phase Partitioning : Increased substrate Accessibility or Enzyme Activation. Plant Physiol. 1987 Nov;85(3):693–698. doi: 10.1104/pp.85.3.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Shimomura S., Sotobayashi T., Futai M., Fukui T. Purification and properties of an auxin-binding protein from maize shoot membranes. J Biochem. 1986 May;99(5):1513–1524. doi: 10.1093/oxfordjournals.jbchem.a135621. [DOI] [PubMed] [Google Scholar]
  11. Sibley D. R., Benovic J. L., Caron M. G., Lefkowitz R. J. Regulation of transmembrane signaling by receptor phosphorylation. Cell. 1987 Mar 27;48(6):913–922. doi: 10.1016/0092-8674(87)90700-8. [DOI] [PubMed] [Google Scholar]
  12. Stout R. G., Cleland R. E. Partial characterization of fusicoccin binding to receptor sites on oat root membranes. Plant Physiol. 1980 Sep;66(3):353–359. doi: 10.1104/pp.66.3.353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Stryer L., Bourne H. R. G proteins: a family of signal transducers. Annu Rev Cell Biol. 1986;2:391–419. doi: 10.1146/annurev.cb.02.110186.002135. [DOI] [PubMed] [Google Scholar]

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