PURIFICATION AND CHARACTERIZATION OF ADENYLATE CYCLASE

腺苷酸环化酶的纯化和表征

• 批准号: 3400844
• 负责人: DANIEL R STORM
• 金额: $ 12.24万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-04-01 至 1995-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3400844
• 关键词: adenosine triphosphate; adenylate cyclase; affinity chromatography; antibody; beta adrenergic receptor; brain; calcium; calmodulin; complementary DNA; cyclic AMP; enzyme inhibitors; enzyme mechanism; enzyme reconstitution; enzyme structure; fluorescence spectrometry; genetic manipulation; isoproterenol; laboratory mouse; laboratory rabbit; membrane proteins; molecular cloning; protein kinase; protein kinase A; protein kinase C

Intracelluar cAMP controls aspects of carbolydrate, lipid, protein and nucleic acid metabolism in animal cells. Futhermore, cAMP plays an important role for several types of neuronal function including synaptic transmission, and the activity of specific ion channels is regulated by cAMP. The levels of cAMP in brain are directly controlled by adenylate cyclase which is regulated by neurotransmitters and Ca2+. The long-term objectives of this project are to elucidate the mechanisms for regulation of brain adenylate cyclase by Ca2+, CaM, and cAMP dependent protein kinase. Our laboratory has recently purified this enzyme to homogeneity and obtained polyclonal antibodies against the CaM sensitive adenylate cyclase front bovine brain. Furthermore, we have recently discovered a protein from brain, designated pp90, that activates brain adenylate cyclase which we suspect is a calcium binding protein. Specific aims of this project include the purification of CaM sensitive adenylate cyclase from bovine brain using immunoaffinity chromatography, purification of the CaM sensitive adenylate cyclase from brain, examination of the CaM, Ca2+, and isoproterenol sensitivity of the CaM sensitive adenylate cyclase reconstituted with beta adrenergic receptors, and elucidation of the mechanism for regulation of the enzyme by cAMP dependent protein kinase. Furthermore, we propose to elucidate the mechanism for regulation of adenylate cyclase by pp90. We will also attempt to clone cDNA's encoding the sequence of the catalytic submit of the CaM sensitive adenylate cyclase from bovine brain. This project should provide fundemental information concerning the structure of adenylate cyclases and the mechanisms for regulation of the enzyme by Ca2+, protein kinases, and catecholamines.

细胞内 cAMP 控制碳水化合物、脂质、蛋白质的各个方面 和动物细胞中的核酸代谢。 此外，环磷酸腺苷 对多种类型的神经元功能起着重要作用 包括突触传递和特定离子的活性 通道受 cAMP 调节。脑内 cAMP 水平为 直接受腺苷酸环化酶控制，而腺苷酸环化酶受以下因素调节 神经递质和Ca2+。 本次活动的长期目标 该项目旨在阐明大脑调节机制 Ca2+、CaM 和 cAMP 依赖性蛋白的腺苷酸环化酶 激酶。 我们的实验室最近纯化了这种酶 均质性并获得抗 CaM 的多克隆抗体 敏感的腺苷酸环化酶前部牛脑。 此外，我们 最近发现了一种来自大脑的蛋白质，命名为 pp90， 激活大脑腺苷酸环化酶，我们怀疑它是 钙结合蛋白。 该项目的具体目标包括 牛脑中 CaM 敏感腺苷酸环化酶的纯化 使用免疫亲和层析，纯化 CaM 来自大脑的敏感腺苷酸环化酶，CaM 检查， CaM 敏感腺苷酸的 Ca2+ 和异丙肾上腺素敏感性 用β肾上腺素能受体重建环化酶，以及 阐明酶的调节机制 cAMP 依赖性蛋白激酶。 此外，我们建议 阐明腺苷酸环化酶的调节机制 第 90 页。 我们还将尝试克隆编码 CaM 敏感腺苷酸的催化提交序列 来自牛脑的环化酶。 该项目应提供 有关腺苷酸结构的基本信息 环化酶及其调节机制 Ca2+、蛋白激酶和儿茶酚胺。