Immunoaffinity purification and characterization of gamma-aminobutyric acid (GABA)B receptor from bovine cerebral cortex.

We developed a novel method for immunoaffinity purification of gamma-aminobutyric acid (GABAB) receptor from bovine cerebral cortex using a monoclonal antibody, which specifically recognizes an 80-kDa GABA-binding protein (Nakayasu, H., Mizutani, H., Hanai, K., Kimura, H., and Kuriyama, K. (1992) Biochem. Biophys. Res. Commun. 182, 722-726). The GABA binding activity in the solubilized synaptic membrane preparation was adsorbed on the antibody-conjugated beads and was eluted as a single protein band of 80 kDa by an acidic buffer, pH 2.5. The purified preparation mimicked the GABAB receptor in its binding activity for GABA, baclofen (GABAB receptor agonist) and 2-hydroxysaclofen (a GABAB receptor antagonist). The purified preparation was reconstituted with partially purified GTP-binding protein and an adenylylcyclase preparation on a phospholipid membrane. On addition of GABA or baclofen to the reconstituted membrane, the adenylylcyclase activity was inhibited, and this inhibition was antagonized by 2-hydroxysaclofen. Moreover, this inhibition by GABA was not observed in a system reconstituted system without either the 80-kDa GABA-binding protein or the GTP-binding protein. These results are strong evidence that the 80-kDa antigenic protein is the GABAB receptor. This is the first report of the nearly complete purification of the GABAB receptor.