(I-125)IODOAZIDOCOCAINE, A PHOTOAFFINITY LABEL FOR THE HALOPERIDOL-SENSITIVE SIGMA-RECEPTOR
(I-125)IODOAZIDOCOCAINE, A PHOTOAFFINITY LABEL FOR THE HALOPERIDOL-SENSITIVE SIGMA-RECEPTOR
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DOI：
10.1073/pnas.89.4.1393
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发表时间：
1992-02-15
影响因子：
11.1
通讯作者：
RUOHO, AE
中科院分区：
文献类型：
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作者：
KAHOUN, JR;RUOHO, AE
合成了一种无载体的放射性碘化可卡因光亲和标记(-)-3-(I-125)碘-4-叠氮可卡因[(I-125)IACoc]，并将其作为可卡因结合蛋白的探针。用0.5 nM(I-125)IACoc进行光亲和标记，在大鼠全脑、大鼠肝脏和人胎盘的膜上选择性衍生化26 kDa的多肽，使其具有Sigma受体的药理作用。26 kDa多肽的共价标记被1-MU-M氟哌啶醇、二(2-甲苯基)胍(DTG)、3-(3-羟基苯基)N-(1-丙基)哌啶(3-PPP)、右美沙芬和卡倍他丁抑制。观察到3-PPP[(+)-3-PPP比(-)-3-PPP]对(I-125)IACoc光标记的立体选择性保护作用。10-MU-M丙咪嗪、阿米替林、氟西汀、苯妥拉平和四苯肼也抑制了26 kDa多肽的(I-125)IACoc标记。(I-125)IACoc标记蛋白的大小与Sigma Photoolabel azido[H-3]DTG在豚鼠脑膜和肝脏膜上光标记的蛋白大小一致。(I-125)IACoc与大鼠肝微粒体结合的动力学分析显示有两个位点，K(D)值分别为19和126 pM。膜制备过程中蛋白水解物的存在或不存在并不影响光标记的sigma受体的大小，这表明26 kDa的多肽不是来自更大的蛋白质。综上所述，(I-125)IACoc是氟哌啶醇敏感的sigma受体的有效和高度特异的光亲和标记，将有助于其生物化学和分子表征。
A carrier-free radioiodinated cocaine photoaffinity label, (-)-3-(I-125)iodo-4-azidococaine [(I-125)IACoc], has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM (I-125)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. Covalent labeling of the 26-kDa polypeptide was inhibited by 1-mu-M haloperidol, di(2-tolyl)guanidine (DTG), 3-(3-hydroxyphenyl)N-(1-propyl)piperidine (3-PPP), dextromethorphan, and carbetapentane. Stereoselective protection of (I-125)IACoc photolabeling by 3-PPP [(+)-3-PPP more potent than (-)-3-PPP] was observed. (I-125)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10-mu-M imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the (I-125)IACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigma photolabel azido-[H-3]DTG. Kinetic analysis of (I-125)IACoc binding to rat liver microsomes revealed two sites with K(d) values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not after the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, (I-125)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization.