PURIFICATION AND CHARACTERIZATION OF ADENYLYL CYCLASE

为了提供适合于阐明配体结合的材料 参与其与调节蛋白相互作用的位点和结构域， 哺乳动物腺酰环化酶纯化方法的改进 和简单化。Forskolin的新衍生物，一种萜类化合物， 与腺酰环化酶的催化亚单位直接相互作用， 评价它们在制备亲和力载体方面的效用 层析法。7-脱乙酰基-7-氨基乙基氨基甲酰衍生物 Forsklin可以通过一种简单的程序与琼脂糖偶联，从而产生一种 支持将腺酰环化酶活性提高6000倍 一步法制备粗溶牛脑膜，总收率 大约20%。提纯的材料可以用化学物质进行标记。 和源于Forskolin的光亲和配体，并被 从已发表的序列中提取的三种不同多肽的抗血清 这种酶。亲和力支持提供了极具重复性的 六个多月的净化效果。实验正在进行中，以 进一步优化支撑剂的准备程序。此外, 上述抗血清的进一步鉴定及制备 促进结构研究的其他免疫化学试剂有 被追捕。 为了补充这项工作，对蛋白质分离程序的改进 和排序是在提出的挑战的背景下开发的 通过最近分离到的与腺苷酸功能相关的蛋白质 循环酶。已经开发了几种技术来允许分析 极端疏水的蛋白质，通常在皮下摩尔水平。这一点很受欢迎 TURN允许在几种情况下完成分子克隆。 这些研究代表了定义分子的重要的第一步 许多通过腺苷作用的药物和激素的药理作用 循环酶。最终，获得的信息可能会允许合理设计 新的治疗药物类别。

In order to provide material suitable for elucidation of ligand binding sites and domains involved in its interactions with regulatory proteins, methods for purification of mammalian adenylyl cyclase have been improved and simplified. Novel derivatives of forskolin, a terpenoid compound that interacts directly with the catalytic subunit of adenylyl cyclase, were evaluated for their utility in preparation of supports for affinity chromatography. The 7-desacetyl-7-aminoethylcarbamyl derivative of forskolin could be coupled to agarose in a simple procedure that yielded a support allowing 6000 fold enrichment of adenylyl cyclase activity from crude solubilized bovine brain membranes in one step with an overall yield of approximately 20%. The purified material could be labeled with chemical and photoaffinity ligands derived from forskolin and was recognized by antisera to three different peptides derived from the published sequence of the enzyme. The affinity support has provided extremely reproducible purification results for over six months. Experiments are in progress to further optimize procedures for preparation of the support. In addition, further characterization of the antisera mentioned above and preparation of additional immunochemical reagents to facilitate structural studies are being pursued. To complement this work, refinements to procedures for protein separation and sequencing have been developed in the context of challenges presented by recently-isolated proteins that are functionally related to adenylyl cyclase. Several techniques have been developed to allow analysis of extremely hydrophobic proteins, often at subpicomolar levels. This has in turn allowed molecular cloning to be accomplished in several cases. These studies represent an essential first step in defining the molecular pharmacology of the many drugs and hormones acting through adenylyl cyclase. Ultimately, the information gained may allow rational design of new classes of therapeutic agents.