Zipser B, Ruff M R, O'Neill J B, Smith C C, Higgins W J, Pert C B
Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, MD 20892.
Brain Res. 1988 Nov 1;463(2):296-304. doi: 10.1016/0006-8993(88)90403-9.
We compared the molecular nature of the rat brain opiate receptor with that of the invertebrate leech, Haemopis marmorata, and the protozoan, Tetrahymena, in order to examine the issue of apparent receptor heterogeneity with respect to biochemical structure. A binding study with rat brain membrane verified that [125I]beta-endorphin [( 125I]beta E), a broad specificity ligand, is displaced by the antagonist (-)-naloxone, but not the inactive stereoisomer (+)-naloxone; agonists considered prototypes for mu, delta, and kappa opiate receptors all displayed stereospecific binding displacement. For SDS-PAGE analysis of the opiate receptor [125I]beta-endorphin was covalently affixed to its recognition molecule with the cross-linking reagent DSS. Primary reaction products occur at 110, 58/55, and 29 kDa. Cross-linking products of all 3 molecular weights are effectively reversed by opiate ligands, regardless of their mu, delta, or kappa specificities. Peptide mapping studies in SDS gels, using limited proteolysis, showed that the 110 kDa band can be digested into 58 and 29 kDa fragments and the 58 kDa band into a 29 kDa fragment. Additional smaller molecular weight fragments were generated from the 110, 58/55, and 29 kDa bands which shared their molecular weights. Two possible explanations for the extensive sequence homology between the three major cross-linking products are: (1) the 110 kDa species is the opiate receptor, and the 58 and 29 kDa species are proteolytic fragments; and (2) one of the lower molecular weight species is the opiate receptor, and adjacent receptors are aggregated into the 110 kDa complex through cross-linking.(ABSTRACT TRUNCATED AT 250 WORDS)
为了研究在生化结构方面明显的受体异质性问题，我们比较了大鼠脑阿片受体与无脊椎动物蚂蟥（Haemopis marmorata）及原生动物四膜虫（Tetrahymena）的分子特性。一项对大鼠脑膜的结合研究证实，具有广泛特异性的配体[125I]β-内啡肽（[125I]βE）可被拮抗剂（-）-纳洛酮取代，但不能被无活性的立体异构体（+）-纳洛酮取代；被视为μ、δ和κ阿片受体原型的激动剂均表现出立体特异性结合取代。对于阿片受体的SDS-PAGE分析，[125I]β-内啡肽通过交联剂DSS共价附着于其识别分子。主要反应产物出现在110、58/55和29 kDa处。所有这三种分子量的交联产物都能被阿片配体有效逆转，无论其μ、δ或κ特异性如何。在SDS凝胶中使用有限蛋白酶解进行的肽图谱研究表明，110 kDa条带可被消化成58和29 kDa片段，58 kDa条带可被消化成29 kDa片段。从110、58/55和29 kDa条带产生了额外的分子量相同的较小分子量片段。对于三种主要交联产物之间广泛的序列同源性，有两种可能的解释：（1）110 kDa的物种是阿片受体，58和29 kDa的物种是蛋白水解片段；（2）较低分子量的物种之一是阿片受体，相邻受体通过交联聚集形成110 kDa复合物。（摘要截于250字）
