Method for isolation of kappa-opioid binding sites by dynorphin affinity chromatography
✍ Scribed by Dr. J. Simon; S. Benyhe; J. Hepp; E. Varga; K. Medzihradszky; A. Borsodi; M. Wollemann
- Publisher
- John Wiley and Sons
- Year
- 1990
- Tongue
- English
- Weight
- 563 KB
- Volume
- 25
- Category
- Article
- ISSN
- 0360-4012
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✦ Synopsis
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, 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,) in sodium dodecyl sulfate (SDS) gel electrophoresis.