RECONSTITUTION AND ISOLATION OF THE OPIATE RECEPTOR

拟议研究的目的是了解 哺乳动物神经中阿片受体功能的分子水平 系统。目前存在增溶和分离的方法学 从神经膜和细胞膜中提取的活性阿片结合多肽。 然而，为这些药物确定的总的特定阿片结合量 制剂是最初存在于膜中的一小部分。 此外，阿片剂介导的生物反应性，如抑制 NG108-15杂交菌细胞膜中腺苷环化酶活力的变化 神经肿瘤细胞系，尚未从洗涤剂中重组 萃取物。这项提议的具体目标是通过以下方式创造条件 特异性阿片剂结合和剂量依赖性阿片类药物抑制 的腺苷酸环化酶活性从 NG108-15细胞膜的增溶制剂。本征损失 在溶解和分级过程中发生的阿片剂结合将是 用共价法鉴别受体多肽的物理损失 将特定结合的、放射性标记的阿片剂交联到细胞膜和 然后对多肽拆分后的放射性标记进行定量 十二烷基硫酸钠--聚丙烯酰胺凝胶。增溶策略将侧重于使用 非离子洗涤剂用于分离阿片受体的研究 分化的细胞，在那里它与腺苷环化酶紧密相连，并且 从快速生长的细胞中分离出来，而不是这样。溶解的受体将是 重组为单蜜素脂泡或重组为 混合(洗涤剂-脂肪-蛋白质)胶束。在后一种情况下，个人 脂质物种(或蛋白质，如镍)将被添加到洗涤剂受体中 混合胶束以恢复结合活性。由此产生的 脂肪-蛋白质化学计量学将通过层析分级进行评估。 混合胶束。建立所需的明确的化学计量比 阿片类药物的特异性结合需要同质受体多肽。 因此，以混合胶束形式存在的功能性受体将是 通过各种层析技术进行分级，包括亲和力 结合固定化内啡肽和抗受体抗体柱。 阿片类药物结合的变化导致的特异性结合的差异 脂肪-蛋白质化学计量学将提供有关特异性的信息 鸦片类药物的作用。功能性阿片类药物结合的分离 多肽(S)将使我们获得详细的化学信息 实际的结合部位域。新药的开发产生了 根据对这些要求的理解，预计会有一个 在医学上的可怕影响。

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.