PURIFICATION AND CHARACTERIZATION OF ADENYLATE CYCLASE

细胞内cAMP控制多环芳烃、脂质、蛋白质 以及动物细胞中的核酸代谢。 辅酶A，cAMP 在几种神经元功能中起着重要作用 包括突触传递和特定离子的活性 通道由cAMP调节。大脑中的cAMP水平为 由腺苷酸环化酶直接控制，腺苷酸环化酶由 神经递质和Ca2+。 这一长期目标 该项目旨在阐明大脑的调节机制， 腺苷酸环化酶，钙调素和cAMP依赖蛋白 激酶。 我们的实验室最近提纯了这种酶， 同源性和获得的多克隆抗体对钙调素 敏感腺苷酸环化酶。 而且我们 最近发现了一种来自大脑的蛋白质，命名为pp90， 激活脑腺苷酸环化酶，我们怀疑这是一种 钙结合蛋白 该项目的具体目标包括： 牛脑钙调素敏感性腺苷酸环化酶的纯化 使用免疫亲和层析，纯化CaM 敏感的腺苷酸环化酶，钙调素的检查， 钙调素敏感性腺苷酸的钙敏感性和异丙肾上腺素敏感性 用β肾上腺素能受体重建环化酶，和 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， pp90. 我们还将尝试克隆编码 CaM敏感腺苷酸催化底物的序列 牛脑环化酶。 该项目将提供 关于腺苷酸结构的基本信息 环化酶和调节酶的机制， Ca2+、蛋白激酶和儿茶酚胺。

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.