Method for isolation if kappa-opioid binding sites by dynorphin affinity chromatography

J. Simon, S. Benyhe, J. Hepp, E. Varga, K. Medzihradszky, A. Borsodi, M. Wollemann

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

A kappa-opioid receptor subtype was purified from a digitonin extract of frog brain membranes, using affinity chromatography. The affinity resin was prepared by coupling dynorphin (1-10) to AH Sepharose 4B. The purified receptor binds 4,750 pmol [3H]ethylketocyclazocine (EKC) per mg protein (5,600-fold purification over the membrane-bound receptor) with a K(d) of 9.1 nM. The addition of cholesterol-phosphatidylethanolamine (2 : 1) enhanced 3.6-fold the binding activity of the purified material, which gives a purification very close to the theoretical. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows one major band (65,000 M(r)) in sodium dodecyl sulfate (SDS) gel electrophoresis.

Original languageEnglish
Pages (from-to)549-555
Number of pages7
JournalJournal of Neuroscience Research
Volume25
Issue number4
Publication statusPublished - 1990

Fingerprint

Dynorphins
Affinity Chromatography
Opioid Analgesics
Ethylketocyclazocine
Binding Sites
Digitonin
kappa Opioid Receptor
Membranes
Sodium Dodecyl Sulfate
Anura
Electrophoresis
Proteins
Gels
Cholesterol
Ligands
Brain
aminohexyl-sepharose
phosphatidylethanolamine

Keywords

  • dynorphin (1-10)-AH Sepharose 4B
  • frog brain
  • kappa-opioid-binding sites
  • receptor purification
  • reconstitution with lipids
  • SDS gel electrophoresis

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

Method for isolation if kappa-opioid binding sites by dynorphin affinity chromatography. / Simon, J.; Benyhe, S.; Hepp, J.; Varga, E.; Medzihradszky, K.; Borsodi, A.; Wollemann, M.

In: Journal of Neuroscience Research, Vol. 25, No. 4, 1990, p. 549-555.

Research output: Contribution to journalArticle

Simon, J, Benyhe, S, Hepp, J, Varga, E, Medzihradszky, K, Borsodi, A & Wollemann, M 1990, 'Method for isolation if kappa-opioid binding sites by dynorphin affinity chromatography', Journal of Neuroscience Research, vol. 25, no. 4, pp. 549-555.
Simon, J. ; Benyhe, S. ; Hepp, J. ; Varga, E. ; Medzihradszky, K. ; Borsodi, A. ; Wollemann, M. / Method for isolation if kappa-opioid binding sites by dynorphin affinity chromatography. In: Journal of Neuroscience Research. 1990 ; Vol. 25, No. 4. pp. 549-555.
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