During the past year, characteristics of an affinity support for purification of adenylyl cyclase, including ligand density and blocking protocol, have been optimized, allowing simple and reproducible synthesis of the support. Continued experiments with these supports have established that they have a useful lifetime of well over 18 months of continuous use. The addition of a simple selective solubilization procedure early in the purification has yielded adenylyl cyclase of extremely high purity and specific activity using a straightforward two day procedure. A number of antisera to peptides corresponding to different regions of the adenylyl cyclase molecule have been characterized. In conjunction with the photoaffinity labeling technology and procedures for separation and immunodetection of peptides obtained from larger molecules by enzymatic or chemical fragmentation that have been developed in the laboratory, these reagents will be useful in identifying binding sites within the enzyme. In addition, specific antibodies of this type will allow assessment of the distribution of this form of adenylyl cyclase in different tissues and brain regions. Comparison of these results with those obtained from parallel photoaffinity labeling studies may yield information regarding other members of the adenylyl cyclase family.
