RECONSTITUTION AND ISOLATION OF THE OPIATE RECEPTOR

拟议研究的目的是了解， 分子水平上的阿片受体功能在哺乳动物神经 系统 目前存在用于溶解和分离的方法 来自神经和细胞膜的活性阿片结合多肽。 然而，确定的这些特异性阿片剂结合总量 制备物是最初存在于膜中的一部分。 此外，阿片类药物介导的生物反应性，如抑制 NG 108 -15杂交细胞膜中腺苷酸环化酶活性的测定 神经瘤细胞系，尚未从洗涤剂中重建 萃取物 这项建议的具体目标是通过以下方式创造条件： 特异性阿片结合和剂量依赖性阿片抑制 腺苷酸环化酶活性的定量重建， NG 108 -15细胞膜的溶解制剂。 固有损耗 在溶解和分级过程中发生的阿片剂结合将 与受体多肽的物理损失相区别， 将特异性结合的放射性标记的阿片剂交联到细胞膜上， 然后在多肽分离后定量放射性标记， SDS-聚丙烯酰胺凝胶。 增溶策略将侧重于使用 非离子型去污剂分离δ-阿片受体 分化的细胞，在那里它与腺苷酸环化酶紧密偶联，和 从快速生长的细胞中分离出来，而不是在那里。 可溶性受体将是 重构为单层脂质囊泡或重构为 混合（去污剂-脂质-蛋白质）胶束。 在后一种情况下，个人 将脂质物质（或蛋白质如Ni）加入到洗涤剂-受体中 混合胶束以恢复结合活性。 所得 脂质-蛋白质化学计量将通过色谱法测定 混合胶束。 建立所需的明确的化学计量， 特异性阿片剂结合需要同质受体多肽。 因此，混合胶束形式的功能性受体将是 通过各种色谱技术进行分离，包括亲和层析 与固定的内啡肽和抗受体抗体柱结合。 阿片类药物特异性结合的差异， 脂质-蛋白质化学计量将提供关于特异性的信息， 阿片类药物的作用。 功能性阿片结合的分离 多肽将使我们能够获得详细的化学信息， 实际绑定站点域。 新药的开发 从了解这些要求，预计将有一个 对医学产生了巨大的影响

The aim of the proposed research is to gain an understanding, on the molecular level, of opiate receptor function in the mammalian nervous system. Methodology currently exists for the solubilization and isolation of active opiate binding polypeptides from both nerve and cell membranes. However, the total specific opiate binding determined for these preparations is a fraction of that originally present in membranes. Moreover, opiate-mediated biological responsiveness, such as the inhibition of adenylate cyclase activity in membranes of the NG108-15 hybrid neurotumor cell line, has not yet been reconstituted from detergent extracts. The specific aim of this proposal is to develop conditions by which both specific opiate binding and the dose dependent opiate inhibition of adenylate cyclase activity are reconstituted quantitatively from solubilized preparations of NG108-15 cell membranes. Losses of intrinsic opiate binding which occur during solubilization and fractionation will be discriminated from the physical loss of receptor polypeptide by covalently crosslinking specifically bound, radiolabeled opiate to cell membranes and then quantitating the radiolabel following the resolution of polypeptide on SDS-polyacrylamide gels. Solubilization strategies will focus on the use of non-ionic detergents to isolate Delta-opiate receptor from differentiated cells, where it is tightly coupled to adenylate cyclase, and from rapidly growing cells, where it is not. Solubilized receptor will be reconstituted into unilammelar lipid vesicles or into mixed(detergent-lipid-protein)micelles. In the latter case, individual lipid species (or proteins such as Ni) will be added to detergent-receptor mixed micelles so as to regain binding activity. The resulting lipid-protein stoichiometry will be evaluated by chromatographically sizing the mixed micelles. Establishing the unambiguous stoichiometry needed for specific opiate binding will require homogeneous receptor polypeptide. Therefore, the functional receptor, in mixed micellar form, will be fractionated by various chromatographic techniques, including affinity binding to columns of immobilized endorphin and anti-receptor antibody. Differences in specific opiate binding resulting from changes in lipid-protein stoichiometry will provide information about the specificity of opiate drug action. Isolation of the functional opiate binding polypeptide(s) will allow us to obtain detailed chemical information for the actual binding site domain. The development of new drugs that results from an understanding of these requirements is expected to have a tremondous impact in medicine.