Progress in the past year has centered about the characterization and use of highly purified preparations of opiate receptors and the regulatory protein(s) which couple the receptors to adenylate cyclase. We have raised antibodies which specifically recognize opiate receptors in solution as well as in the solid state. These antibodies, after affinity purification should be useful for cloning, histochemical, and metabolic studies of the receptors. The two components of the inhibitory regulatory protein preparation which we and others have purified from bovine brain are currently referred to as Ni and No. We have prepared and characterized antibodies that specifically recognize the Alpha subunits of each (the Beta and Gamma subunits are apparently identical). The two proteins are difficult to separate from one another. We are therefore exploring the suitability of tissues other than brain as potential exclusive sources of only one or the other of these proteins. Liver, for example, contains no detectable No, but does have useful amounts of Ni. Bradykinin receptors in some cells stimulate phosphatidylinositol turnover in a process mediated by Ni or No. We have found that such receptors also stimulate GTPase and inhibit adenylate cyclase activities in NG108-15 neuroblastoma x glioma hybrid cell membranes. Our data suggest that bradykinin and opiate receptors may be coupled to different N proteins. We have demonstrated the reconstitutive coupling of opiate receptors, Ni (or No) and adenylate cyclase in liposomes prepared from solutions of these proteins. Availability of a reconstitution assay should allow characterization of the activities of each of the purified components of the membrane signal transduction apparatus as well as identification of any as yet unknown components.
