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.
Department of Pharmacology, Kyoto Prefectural University of Medicine, Japan.
H
H
Nakayasu
M
M
Nishikawa
H
H
Mizutani
H
H
Kimura
K
K
Kuriyama
D000262:Adenylyl Cyclases / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal; D002417:Cattle; D002540:Cerebral Cortex / Q000737:chemistry*; D003488:Cyanogen Bromide; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism; D015151:Immunoblotting; D011963:Receptors, GABA-A / Q000302:isolation & purification* / Q000378:metabolism; D015398:Signal Transduction; D013570:Synaptic Membranes / Q000737:chemistry
D000911:Antibodies, Monoclonal; D011963:Receptors, GABA-A; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D003488:Cyanogen Bromide