ALPHA-RECEPTOR PURIFICATION ROLE IN NEUROTRANSMISSION

我们实验室已将哺乳动物的α-1肾上腺素能 大鼠肝脏膜上的受体。这种蛋白的特征是 斯托克半径为49A，沉积速度为7.1s，原理 59,000道尔顿的亚单位。此外，高比活度， 已经合成了针对α-1受体的高亲和力探针，并 特色化的。我们现在建议广泛研究分子 受体的性质，包括其详细的测定 双向电泳法测定亚基结构及后肽 交联剂，等电聚焦测定其p1， 凝集素亲和层析对糖基化的评价 纯化的受体蛋白肽图的构建。使用 放射性标记受体探针，未标记的流体力学性质 受体将得到确认，可能是物种间和组织间 研究了分子的异质性。为了补充这些研究， 针对纯化受体的多克隆(诱导)抗体和单抗 并针对特定的分离多肽片段，将被提出。这些 抗体将通过以下方式进一步促进受体的假化 免疫吸附层析用于鉴定结合部位区域 负责亚型选择性，并在识别 受体-蛋白质通过一种独立于其结合活性的技术。至 深入了解受体蛋白的表达和调控， 在其氨基酸组成中，我们进一步建议分离出一个 编码α-1受体基因的克隆。这个目标现在应该是 以我们的能力实现受体的高级纯化是可行的 由于蛋白质微测序的最新发展，以及在 稀有核糖核酸的分离。最后，针对以下方面的初步研究 沙门氏菌α-2肾上腺素能受体的纯化与鉴定 血小板膜，将继续使用开发的亲和力 层析技术和新合成的受体特异性探针 潜在地能够与受体结合部位发生共价相互作用。

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.