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Solubilization of the Fusicoccin Receptor and a Protein Kinase from Highly Purified Plasma Membrane from Oat Roots

  • A. H. De Boer
  • T. L. Lomax
  • R. P. Sandstrom
  • R. E. Cleland
Part of the NATO ASI Series book series (NSSA, volume 133)

Abstract

Fusicoccin (FC), a diterpine glucoside Phytotoxin, that stimulates electrogenic H+ extrusion in higher plants binds specifically to a plant plasma membrane (PM) protein. A highly purified preparation of plasma membrane vesicles has been prepared from oat roots and used to study the FC-binding protein (receptor). In these right-side out vesicles the FC-binding site is protected from trypsin degradation, but disruption of the vesicle integrity with Triton X-100 or solubilization of the receptor renders it susceptible to trypsin degradation. The receptor has been successfully solubilized with octyl-glucoside (90% recovery) and can be assayed effectively by binding to glass-fiber filters coated with 1% polyethylinimine.

A Mg2+-dependent Ca2+-activated protein-kinase was solubilized from the PM vesicles through sonication. This shows that the kinase is a peripheral membrane protein. The soluble kinase has a sharp pH optimum at pH 7.

The enzyme that is thought to be activated by FC, the plasma membrane ATPase, was present in the PM vesicles with a very high specific activity (4 – 5 µmol/mg protein, min). We show evidence that the detergent activition of this enzyme is not simply due to breaking the vesicle integrity. A conformational change or removal of a regulatory subunit may cause stimulation of the H -ATPase and this may be of importance in our study of the mode of action of fusicoccin.

Keywords

Electron Spin Resonance ATPase Activity Electron Spin Resonance Measurement Peripheral Membrane Protein Plasma Membrane ATPase 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1987

Authors and Affiliations

  • A. H. De Boer
    • 1
  • T. L. Lomax
    • 1
  • R. P. Sandstrom
    • 1
  • R. E. Cleland
    • 1
  1. 1.Department of BotanyUniversity of WashingtonSeattleUSA

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