MOLECULAR ASPECTS OF MULTIPLE OPIOID RECEPTORS

这份为期5年的续订申请针对的是使用 14β-(溴乙酰胺)-7，8-二氢吗啡(H_2BAM)亲和标记及 纯化Mu阿片受体。在没有二硫键还原的情况下 试剂，这种亲和配基可逆地结合到阿片类药物的结合 在大鼠脑膜上的位置。然而，在二硫键还原后 在Mu阿片结合部位，H_2BAM不可逆地与这种结合结合 通过抑制阿片类药物与Mu位点的结合而确定，但 而不是三角洲或卡帕地区。保护实验证明， 只有与u阿片结合部位结合的阿片类药物才能阻断 这个部位的烷基化作用。[~3H]H_2BAM以高比表面积合成 活动。[~3H]H_2BAM可逆性结合的亲和力和特异性 在大鼠和牛脑中不可逆地结合Mu阿片类药物结合部位 膜将通过放射受体结合分析来确定 二硫键还原剂二硫苏糖醇的存在和不存在。 将优化实验条件，以进行特定的烷基化反应 Mu阿片类药物与[~3H]H_2BAM的结合部位。乳清蛋白的分子量 用[~3H]H_2BAM特异性标记的蛋白质(S)将通过 用聚丙烯酰胺凝胶分离[~3H]H_2BAM标记膜 变性和还原条件下，然后进行荧光照相。特异性 研究将确定哪些阿片类药物可以阻止不可逆转的标记 特定蛋白质(S)。多种MU阿片类药物结合的可能性 将对这些地点进行审查。细胞系，特定的大脑区域，以及其他 将对组织进行测试，以进一步表征[~3H]H_2BAM的特异性 膜的烷基化。这些研究将产生一个关于 Mu阿片结合位点的亲和标记。蛋白质，特别是 用[~3H]H_2BAM标记，将从牛纹状体膜中纯化 在变性条件下，包括亲和层析，高 使用离子交换和分子交换的加压液相色谱 筛柱和凝胶电泳法。多肽片段来自于 纯化的[~3H]H_2BAM标记蛋白将通过酶或化学方法产生 消化。用反相高效液相色谱法对多肽和部分氨基进行纯化 多肽的酸序列将被确定。以氨基酸为基础 序列，寡核苷酸探针将被合成，并用于筛选 CDNA文库，以期最终克隆Mu阿片受体。

This 5-year renewal application is directed at using 14beta-(bromoacetamido)-7,8-dihydromorphine (H2BAM) to affinity label and purify the mu opioid receptor. In the absence of a disulfide bond reducing reagent, this affinity ligand bound reversibly to the mu opioid binding site in rat brain membranes. However, after reduction of a disulfide bond at the mu opioid binding site, H2BAM bound irreversibly to this binding site, as determined by the inhibition of opioid binding to the mu site, but not to the delta or kappa site. Protection experiments demonstrated that only opioids that bind to the mu opioid binding site were able to block the alkylation of this site. [3H]H2BAM was synthesized to a high specific activity. The affinity and specificity of [3H]H2BAM to bind reversibly and irreversibly to the mu opioid binding site in rat and bovine brain membranes will be determined by radioreceptor binding assays in the presence and absence of the disulfide bond reducing reagent dithiothreitol. Experimental conditions will be optimized for the specific alkylation of the mu opioid binding site with [3H]H2BAM. The molecular weight of protein(s) specifically labeled with [3H]H2BAM will be determined by separating [3H]H2BAM-labeled membranes on polyacrylamide gels under denaturing and reducing conditions, followed by fluorography. Specificity studies will determine which opioids can block the irreversible labeling of specific protein(s). The possibility of multiple mu opioid binding sites will be examined. Cell lines, specific brain regions, and other tissues will be tested to further characterize the specificity of [3H]H2BAM alkylation of membranes. These studies will result in a profile of the affinity labeling of the mu opioid binding site. Proteins, specifically labeled with [3H]H2BAM, will be purified from bovine striatal membranes under denaturing conditions, including affinity chromatography, high pressure liquid chromatography (HPLC), using ion exchange and molecular sieve columns, and gel electrophoresis. Peptide fragments from the purified [3H]H2BAM-labeled protein will be generated by enzyme or chemical digestion. Peptides will purified by reverse-phase HPLC, and partial amino acid sequences of the peptides will be determined. Based on the amino acid sequences, oligonucleotide probes will be synthesized, and used to screen cDNA libraries, in order to ultimately clone the mu opioid receptor.