Kavanaugh M P, Tester B C, Scherz M W, Keana J F, Weber E
Vollum Institute for Advanced Biomedical Research, Oregon Health Sciences University, Portland 97201.
Proc Natl Acad Sci U S A. 1988 Apr;85(8):2844-8. doi: 10.1073/pnas.85.8.2844.
The sigma-type opiate receptor is a distinct binding site in the brain that may mediate some of the psychotomimetic effects caused by benzomorphan opiates and phencyclidine in humans. We have developed a synthetic, highly selective ligand for this receptor, 1,3-di-o-tolylguanidine (DTG). To identify the binding protein(s) of the sigma receptor, we have now synthesized a radiolabeled azide derivative of DTG, 1-(4-azido-2-methyl[6-3H]phenyl)-3-(2-methyl[4,6-3H]phenyl)-guanidine ([3H]N3DTG). In guinea pig brain membrane binding assays conducted in the dark, [3H]N3DTG bound reversibly, selectively, and with high affinity (Kd = 10 nM) to sigma receptors. The drug specificity profile of reversible [3H]-N3DTG binding was identical to that of [3H]DTG and 3H-labeled (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine binding indicating that [3H]N3DTG is a selective sigma receptor ligand. Guinea pig brain membranes were photoaffinity-labeled with [3H]N3DTG. NaDodSO4/PAGE of detergent-solubilized membrane extract identified a single 29-kDa radioactive band. Sepharose Cl-6B gel chromatography of photolabeled brain membranes solubilized with the nondenaturing detergent sodium cholate showed a radioactive complex with a Stoke's radius of 4.6 nm (Mr, 150,000) that may represent the intact sigma receptor complex. NaDodSO4/PAGE of this complex showed that the radiolabeled material was a 29-kDa polypeptide that may be the binding subunit of the sigma receptor. The specific sigma receptor photoaffinity ligand described here should be a useful tool for purifying and characterizing the sigma receptor.
σ型阿片受体是大脑中一个独特的结合位点，它可能介导了苯并吗啡烷类阿片和苯环利定在人体中引起的一些拟精神病效应。我们已经开发出一种针对该受体的合成高选择性配体，即1,3-二邻甲苯基胍（DTG）。为了鉴定σ受体的结合蛋白，我们现已合成了DTG的放射性叠氮衍生物，1-(4-叠氮基-2-甲基[6-³H]苯基)-3-(2-甲基[4,6-³H]苯基)-胍（[³H]N3DTG）。在黑暗中进行的豚鼠脑膜结合试验中，[³H]N3DTG以可逆、选择性且高亲和力（Kd = 10 nM）的方式与σ受体结合。可逆性[³H]-N3DTG结合的药物特异性谱与[³H]DTG和³H标记的(+)-3-(3-羟基苯基)-N-(1-丙基)哌啶结合的谱相同，表明[³H]N3DTG是一种选择性σ受体配体。用[³H]N3DTG对豚鼠脑膜进行光亲和标记。去污剂增溶的膜提取物的十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（NaDodSO4/PAGE）鉴定出一条单一的29 kDa放射性条带。用非变性去污剂胆酸钠增溶的光标记脑膜的琼脂糖Cl-6B凝胶色谱显示出一种斯托克斯半径为4.6 nm（相对分子质量，150,000）的放射性复合物，它可能代表完整的σ受体复合物。该复合物的NaDodSO4/PAGE显示，放射性标记物质是一种29 kDa的多肽，它可能是σ受体的结合亚基。本文所述的特异性σ受体光亲和配体应该是纯化和表征σ受体的有用工具。
