In the past year we have purified several of the components of the opiate receptor - adenylate cyclase system. Adenylate cyclase has been purified from rat brain and the GTP-binding regulatory proteins Gi and Gs were purified from rabbit liver. These purified proteins were reconstituted into liposomes from detergent solutions by dialysis in the presence of phospholipids. In the reconstituted vesicles adenylate cyclase activity is stimulated by Gs and the stimulated activity is inhibited by Gi or by the beta-gamma subunit complex of bovine transducin. Thus a completely defined system has been developed with which to study opiate receptor function. We have also used specific antibodies which recognize the alpha subunits of Gi or of Go to quantitate these proteins in brain and other tissues and cells. These studies showed that brain membranes contain 1-2% of their total protein as Go, and allowed study of the ontogeny of the proteins in neo-natal rat brain. The antibodies also led to the discovery of a novel G protein which is the predominant pertussis-toxin substrate of C6 glioma cells. Studies aimed at clarifying the function of the several G proteins have been carried out with bradykinin and opiate receptor coupled processes in NG108-15 hybrid cells.
