PURIFICATION AND CHARACTERIZATION OF ADENYLYL CYCLASE

腺苷酸环化酶的纯化和表征

• 批准号: 3811071
• 负责人: M MOOS
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3811071
• 关键词: adenylate cyclase; affinity chromatography; affinity labeling; antibody; brain; carbamates; cell membrane; chemical binding; chemical synthesis; chromatography; cow; enzyme structure; forskolin; hydropathy; molecular cloning; protein purification

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.

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， 哺乳动物腺苷酸环化酶的纯化方法已经得到改进 并简化。毛喉素的新衍生物，毛喉素是一种萜类化合物， 直接与腺苷酸环化酶的催化亚基相互作用， 评价它们在制备亲和性载体中的效用 层析式I化合物的7-脱乙酰基-7-氨基乙基氨基甲酰基衍生物， 毛喉素可以在一个简单的程序中偶联到琼脂糖上， 允许腺苷酸环化酶活性从 一步溶解牛脑膜粗品，总收率 大约20%。纯化的材料可以用化学标记 和来自毛喉素的光亲和配体， 针对三种不同肽的抗血清，所述三种不同肽衍生自以下公开序列： 酶亲和支持物提供了极其可重复的 六个月以上的净化效果。实验正在进行中， 进一步优化载体制备程序。此外，本发明还提供了一种方法， 上述抗血清的进一步表征和 有助于结构研究的其它免疫化学试剂是 被人追杀 为了补充这项工作，改进了蛋白质分离程序 和排序的背景下提出的挑战 最近分离的与腺苷酸相关的蛋白质 环化酶已经开发了几种技术来允许分析 非常疏水的蛋白质，通常在亚皮摩尔水平。这在 在几种情况下，转基因技术使分子克隆得以完成。 这些研究代表了定义分子生物学的重要的第一步。 许多药物和激素通过腺苷酸起作用的药理学 环化酶最终，所获得的信息可以允许合理设计 新型治疗剂。