Eppler C M, Hulmes J D, Wang J B, Johnson B, Corbett M, Luthin D R, Uhl G R, Linden J
Agricultural Research Division, American Cyanamid Co., Princeton, New Jersey 08543.
J Biol Chem. 1993 Dec 15;268(35):26447-51.
A rat brain opioid receptor protein was isolated by binding [epsilon-biotinyl-Lys32] beta-endorphin to membranes, solubilizing the receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine and purifying on immobilized streptavidin and wheat germ agglutinin. The purified glycoprotein had a molecular mass of 60-70 kDa. Recovery of this protein was blocked by the nonselective opioid antagonist naloxone and the highly mu-selective agonist [D-Ala2,N-methyl-Phe4,Glyol5]-enkephalin but not by the highly delta-selective agonist [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin when these compounds were added as competitors at the binding step. The 60-70-kDa receptor protein co-purified through the streptavidin column with 40-kDa protein recognized by anti-Gi alpha antibodies. GTP and Na+ influenced dissociation of the solubilized R.125I-L complex and elution of the receptor and G protein from streptavidin in fashions consistent with the pharmacology of mu-opioid receptors. A 23-amino acid residue sequence from the purified receptor differs at 4 positions from a similar sequence in the murine delta-opioid receptor and is encoded within a novel rat brain cDNA isolated by polymerase chain reaction with oligonucleotide primers related to the murine delta-opioid receptor gene.
通过将[ε-生物素基-Lys32]β-内啡肽与膜结合，用脱氧胆酸盐-溶血磷脂酰胆碱溶解受体-配体（R.L）复合物，并在固定化链霉亲和素和麦胚凝集素上进行纯化，分离出大鼠脑阿片受体蛋白。纯化的糖蛋白分子量为60-70 kDa。当在结合步骤中加入这些化合物作为竞争剂时，该蛋白的回收被非选择性阿片拮抗剂纳洛酮和高度μ选择性激动剂[D-Ala2，N-甲基-Phe4，Glyol5]-脑啡肽阻断，但未被高度δ选择性激动剂[D-Pen2，4'-Cl-Phe4，D-Pen5]脑啡肽阻断。60-70 kDa的受体蛋白与抗Giα抗体识别的40 kDa蛋白一起通过链霉亲和素柱共纯化。GTP和Na+以与μ阿片受体药理学一致的方式影响溶解的R.125I-L复合物的解离以及受体和G蛋白从链霉亲和素上的洗脱。纯化受体的23个氨基酸残基序列与小鼠δ阿片受体中的类似序列在4个位置不同，并且由与小鼠δ阿片受体基因相关的寡核苷酸引物通过聚合酶链反应分离出的新的大鼠脑cDNA编码。
