ALPHA-RECEPTOR PURIFICATION ROLE IN NEUROTRANSMISSION

我们的实验室已经将哺乳动物α-1肾上腺素能 大鼠肝细胞膜受体。 这种蛋白质的特征是 斯托克半径为49 A，沉降速度为7.1 S， 59，000道尔顿的亚基。 此外，高比活度， 已经合成了α-1受体的高亲和性探针， 表征了 我们现在建议广泛研究 受体的特性，包括其 亚基结构通过双向电泳和后肽 交联，通过等电聚焦测定其p1， 通过凝集素亲和色谱法评估糖基化， 构建纯化的受体蛋白的肽图谱。 使用 放射性标记的受体探针，未标记的流体动力学性质 受体将得到确认，可能的种间和组织间 研究分子异质性。 为了补充这些研究， 针对纯化受体的多克隆（引发）和单克隆抗体 和特异性分离的肽片段。 这些 抗体将有助于受体的进一步纯化， 免疫吸附色谱法，用于识别结合位点区域 负责亚型选择性，并在识别 受体蛋白的技术独立于其结合活性。 到 深入了解受体蛋白的表达和调节， 并结合其氨基酸组成，我们进一步提出分离cDNA α 1受体mRNA的克隆。 这个目标现在应该是 这是可行的，因为我们有能力实现受体的高级别纯化 由于蛋白质微测序的最新发展， 分离稀有mRNA。 最后，初步研究针对 α-2肾上腺素能受体的纯化和表征 血小板膜，将继续使用开发的亲和 色谱技术和新合成的受体特异性探针 潜在地能够与受体结合位点共价相互作用。

Our laboratory has purified to homogeneity the mammalian alpha-1 adrenergic receptor from rat hepatic membranes. This proten is characterized by a Stoke's radius of 49A, a sedimentation velocity of 7.1S and a principle subunit of 59,000 daltons. Additionally, high-specific activity, high-affinity probes for the alpha-1 receptor have been synthesized and characterized. We propose now to extensively investigate the molecular properties of the receptor, including the detailed determination of its subunit structure by two-dimensional electrophoresis and after peptide cross-linking, the determination of its p1 by isoelectric focusing, the assessment of glycosylation by lectin affinity chromatography, and the construction of peptide maps of the purified receptor-protein. Using the radiolabeled receptor probes, the hydrodynamic properties of the unlabeled receptor will be confirmed, and possible interspecies and intertissue molecular heterogeneity investigated. To complement these studies, polyclonal (elicited) and monoclonal antibodies to the purified receptor and to specific isolated peptide fragments, will be raised. These antibodies will aid in the further pufification of the receptor by immunoadsorbent chromatography, in identifying binding site regions responsible for subtype selectivity, and in the identification of the receptor-protein by a technique independent of its binding activity. To gain insight into the expression and regulation of the receptor-protein, and into its amino acid composition, we further propose to isolate a cDNA cloneencoding for the alpha-1 receptor mRNA. This goal should now be feasible with our ability to effect high-grade purification of the receptor and because of recent developments in protein microsequencing, and in the isolation of rare mRNAs. Finally, preliminary studies directed towards the purification and characterization of the alpha-2 adrenergic receptor from platelet membranes, will be continued using developed affinity chromatography techniques and newly synthesized receptor-specific probes potentially capable of covalent interaction with the receptor-binding site.