PURIFICATION AND CHARACTERIZATION OF ADENYLATE CYCLASE

细胞内cAMP控制碳酸盐、脂肪、蛋白质等方面 以及动物细胞中的核酸代谢。此外，夏令营 对几种类型的神经功能起着重要的作用 包括突触传递和特定离子的活性 通道受cAMP的调节。大脑中cAMP的水平是 由腺苷环化酶直接控制，腺苷环化酶受 神经递质和钙离子。这个项目的长期目标是 项目是为了阐明大脑调节的机制 Ca~(2+)、CaM和cAMP依赖蛋白的腺苷环化酶 激活剂。我们实验室最近提纯了这种酶以 抗CaM多克隆抗体的获得及同源性研究 灵敏的腺苷环化酶牛脑前。此外，我们 最近发现了一种来自大脑的蛋白质，命名为pp90， 激活脑腺苷环化酶，我们怀疑这是一种 钙结合蛋白。该项目的具体目标包括 牛脑钙调素敏感型腺苷环化酶的纯化 用免疫亲和层析法纯化CaM 大脑中敏感的腺苷环化酶，CaM的检查， 钙调素敏感腺苷酸盐对钙离子和异丙肾上腺素的敏感性 用β肾上腺素能受体重组的环化酶，以及 阐明该酶的调节机制 CAMP依赖的蛋白激酶。此外，我们建议 黄曲霉毒素对腺苷酸环化酶的调控机制 第90页。我们还将尝试克隆编码该基因的 钙调素敏感的腺苷酸酯的催化提交序列 从牛脑中提取的环化酶。该项目应提供 关于腺苷酸盐结构的基本信息 环化酶及其调控机制 钙离子、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.