PURIFICATION AND CHARACTERIZATION OF ADENYLATE CYCLASE

细胞内cAMP控制着碳水化合物、脂质、蛋白质等方面

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.