PURIFICATION AND CHARACTERIZATION OF ADENYLYL CYCLASE

为配体结合的解析提供合适的材料

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.