Tetrodotoxin receptors in membrane fragments Purification from Electrophorus electricus electroplax and binding properties

Hans H. Grünhagen, Gerhard Dahl, Peter Reiter

Research output: Contribution to journalArticle

8 Scopus citations

Abstract

A tetrodotoxin receptor-rich preparation of membrane fragments from the electric organ of Electrophorus electricus is described. The specific binding of neurotoxins and freeze-fracture electron microscopy are used as tools to identify and to characterize membrane fractions. Freeze-fracture electron micrographs of the electric organ demonstrate a high density of membrane particles in the extrasynaptic regions. Density gradient fractions show a broad distribution of [3H]tetrodotoxin, [3H]saxitoxin and 125I-labelled bungarotoxin binding in the range of 1.04-1.15 g/ml sucrose densities, with specific neurotoxin binding up to approx. 5 pmol/mg protein. Carrier-free column electrophoresis of density gradient fractions yields a subfraction with tetrodotoxin and α-neurotoxin binding up to 30 pmol/mg protein. The major part of the membrane fragments forms vesicles, which are separated by lectin chromatography into an outside-out and inside-out population. The latter represents at least 50% of the material of a density gradient fraction. For the association of tetrodotoxin, a bimolecular kinetic constant kf ≥ 3 · 105 M-1 · s-1 is determined. The dissociation constant is kb = 2.5 · 10-2 s-1. These data are in agreement with a thermodynamic dissociation constant of KD = 20 nM as determined earlier for E. electricus membrane fragments by equilibrium methods (Grünhagen, H.H., Rack, M., Stämpfli, R., Fasold, H. and Reiter, P. (1981) Arch. Biochem. Biophys. 206, in the press).

Original languageEnglish (US)
Pages (from-to)267-285
Number of pages19
JournalBBA - Biomembranes
Volume642
Issue number2
DOIs
StatePublished - Apr 6 1981
Externally publishedYes

Keywords

  • (Electrophorus electricus)
  • Carrier-free electrophoresis
  • Lectin chromatography
  • Tetrodotoxin binding
  • Tetrodotoxin receptor

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Cell Biology
  • Medicine(all)

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