MOLECULAR ASPECTS OF THE MULTIPLE OPIOID RECEPTORS

这项提议是针对使用针对 阿片受体阐明其分子和生理基础 多个阿片受体。一种能产生单抗OR-689.2.4 抑制阿片类药物与神经膜的结合并促使 从增溶制剂中获得了受体。一种程序 已被开发用于从该IgM中获得Fab片段。The Fab 将研究片段是否能够抑制阿片类药物与 神经膜、细胞系中不同类型的阿片受体， 外周组织和增溶受体制剂。的影响 Na+和GTP对抗体抑制阿片类药物结合能力的影响 被研究以确定抗体是否改变了阿片类药物的抑制 Na+和GTP引起的结合，或者如果Na+和GTP改变抗体的结合 抑制阿片类药物结合的能力。这些研究可能会进一步阐明 Na+和GTP抑制阿片激动剂结合但不抑制的机制 拮抗剂绑定。以确定抗体是否可以改变 阿片类药物介导的生理反应，Fab片段将被测试 确定它们是否能够调节阿片类药物介导的抑制 NG108-15细胞株和神经细胞中腺苷环化酶活性的变化 膜。抗体还将被测试其修饰能力。 采用甩尾法和扭体法进行镇痛实验。这些研究可能 确定抗体是作为激动剂还是作为拮抗剂。这个 来自OR-689.2.4的Fab片段，共价连接到琼脂糖凝胶上形成 亲和柱将用于纯化阿片受体。 从抗体亲和柱中特别洗脱的物质将是 测定其分子特征和结合阿片类药物的能力 配基。最后，还会产生更多的单抗， 使用从抗体亲和层析出的材料和 部分纯化的受体制剂作为抗原。抗体将会是 提供给合作者，用于从分子生物学到 到整个动物的化验。

This proposal is directed at using monoclonal antibodies directed against the opioid receptor to elucidate the molecular and physiological basis of the multiple opioid receptors. A monoclonal antibody, OR-689.2.4, capable of inhibiting opioid binding to neural membranes and precipitating the receptor from a solubilized preparation, has been obtained. A procedure has been developed for obtaining Fab fragments from this IgM. The Fab fragments will be studied for their ability to inhibit opioid binding to the different types of opioid receptors in neural membranes, cell lines, peripheral tissue, and solubilized receptor preparations. The effect of Na+ and GTP on the ability of the antibody to inhibit opioid binding will be studied to determine if the antibody modifies the inhibition of opioid binding caused by Na+ and GTP, or if Na+ and GTP alter the antibody's ability to inhibit opioid binding. These studies may further elucidate the mechanism by which Na+ and GTP inhibit opioid agonist binding but not antagonist binding. To determine if the antibody can alter an opioid-mediated physiological response, the Fab fragments will be tested to ascertain if they are capable of modulating the opioid-mediated inhibition of adenylate cyclase activity in the NG108-15 cell line and neural membranes. The antibody will also be tested for its ability to modify analgesia, using the tail flick and writhing assays. These studies may determine whether the antibody is acting as an agonist or antagonist. The Fab fragments from OR-689.2.4, covalently conjugated to Sepharose to form an affinity column, will be used to purify the opioid receptor. Specifically eluted material from the antibody affinity column will be assayed for its molecular characteristics and its ability to bind opioid ligands. Finally, additional monoclonal antibodies will be generated, using the material eluted from the antibody affinity column and the partially purified receptor preparation as antigen. The antibody will be provided to collaborators to use in studies ranging from molecular biology to whole animal assays.