MOLECULAR ASPECTS OF MULTIPLE OPIOID RECEPTORS

这5年的更新申请是针对使用 14 β-（溴乙酰氨基）-7，8-二氢吗啡（H2 BAM）与亲和标记物结合， 纯化μ阿片受体。 在不存在二硫键还原的情况下， 试剂，该亲和配体可逆地结合μ阿片样物质结合 大鼠脑细胞膜中的位点。 然而，在二硫键还原后， 在μ阿片样物质结合位点，H2 BAM不可逆地与这种结合 位点，如通过抑制阿片样物质与μ位点结合所确定的，但 而不是三角洲或卡帕基地 保护实验表明， 只有与μ阿片样物质结合位点结合的阿片样物质能够阻断 这个网站的链接。[3 H] H_2BAM的合成具有高特异性， 活动 [3 H] H2 BAM可逆结合的亲和力和特异性， 对大鼠和牛脑中μ阿片样物质结合位点的不可逆作用 膜将通过放射受体结合试验测定， 二硫键还原剂二硫苏糖醇的存在和不存在。 实验条件将被优化用于特定的烷基化反应。 μ阿片样物质结合位点与[3 H] H2 BAM。 的分子量 用[3 H] H2 BAM特异性标记的蛋白质将通过 在聚丙烯酰胺凝胶上分离[3 H] H2 BAM标记的膜， 变性和还原条件，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的标记 特定蛋白质（S）。 多种μ阿片类药物结合的可能性 将对网站进行检查。细胞系、特定脑区和其他 将检测组织以进一步表征[3 H] H2 BAM的特异性 膜的烷基化。 这些研究将导致一个档案的 μ阿片样物质结合位点的亲和标记。 蛋白质，特别是 用[3 H] H2 BAM标记，将从牛纹状体膜中纯化 在变性条件下，包括亲和层析， 高压液相色谱法（HPLC），使用离子交换和分子 筛柱和凝胶电泳。 肽片段， 纯化的[3 H] H2 BAM标记的蛋白质将通过酶或化学试剂产生 消化. 肽将通过反相HPLC纯化，并部分氨基化。 将测定肽的酸序列。 基于氨基酸 序列，寡核苷酸探针将被合成，并用于筛选 cDNA文库，以便最终克隆μ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.