Method for isolation of 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 Kd 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 Mr) in sodium dodecyl sulfate (SDS) gel electrophoresis.

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

Keywords

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

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

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