Purification of a kappa-opioid receptor subtype from frog brain |
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Authors: | J Simon S Benyhe J Hepp A Khan A Borsodi M Szücs K Medzihradszky M Wollemann |
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Institution: | Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged. |
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Abstract: | A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol 3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis. |
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