Fujioka T, Inoue F, Kuriyama M, Miura Y, Higashijima T
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Hyogo, Japan.
Chem Pharm Bull (Tokyo). 1990 Jan;38(1):168-71. doi: 10.1248/cpb.38.168.
Digitonin-solubilized opioid receptors from rat brain were purified with an affinity resin, AH-Sepharose coupled with [D-Ala2, D-Leu5]enkephalin (DADLE). Radioreceptor binding assay showed that the purified materials had specific opioid-binding activity of 310 pmol/mg protein on DADLE binding. Analyses by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that the materials were rich in two polypeptides; the major component had a molecular weight of 62000-64000. To establish the materials responsible for binding opiates, the purified materials were cross-linked with 125I-labeled beta-endorphin using bis[2-(succinimidooxycarbonyloxy)-ethyl]sulfone as a cross-linker. The molecular weight of 62000-64000, the major band of the purified materials on SDS-PAGE, agreed closely with that determined by the cross-linking experiment. The results suggest that the purified materials contained opioid-binding materials (opioid receptors).
用亲和树脂AH-Sepharose偶联[D-丙氨酸2，D-亮氨酸5]脑啡肽（DADLE）纯化大鼠脑内洋地黄皂苷增溶的阿片受体。放射受体结合试验表明，纯化后的物质对DADLE结合具有310 pmol/mg蛋白的特异性阿片结合活性。在十二烷基硫酸钠存在下进行聚丙烯酰胺凝胶电泳（SDS-PAGE）分析表明，该物质富含两种多肽；主要成分的分子量为62000 - 64000。为确定负责结合阿片类药物的物质，使用双[2-（琥珀酰亚胺氧基羰基氧基）-乙基]砜作为交联剂，将纯化后的物质与125I标记的β-内啡肽交联。SDS-PAGE上纯化物质的主要条带分子量为62000 - 64000，与交联实验测定的结果非常吻合。结果表明，纯化后的物质含有阿片结合物质（阿片受体）。
