Simon J, Benyhe S, Hepp J, Khan A, Borsodi A, Szücs M, Medzihradszky K, Wollemann M
Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Szeged.
Neuropeptides. 1987 Jul;10(1):19-28. doi: 10.1016/0143-4179(87)90085-0.
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.
利用亲和色谱法从经洋地黄皂苷增溶处理的蛙脑膜制剂中纯化出一种κ-阿片受体亚型。亲和树脂是通过将D-丙氨酸2-亮氨酸5-脑啡肽偶联到琼脂糖-6B基质上制备而成。用50 μmol纳洛酮洗脱受体后，通过琼脂糖-6B凝胶渗透色谱法将κ-亚型与μ-亚型和δ-亚型分离。纯化后的受体每毫克蛋白质结合3900 pmol [3H]-乙基酮环唑新（相对于膜结合受体有4300倍的纯化倍数），解离常数为8.3 nM。纯化后的受体蛋白对κ-选择性配体具有高亲和力。纯化后的组分在十二烷基硫酸钠凝胶电泳中显示出两条带（分子量分别为65000和58000）。
