Use of specific antibodies to quantitate the guanine nucleotide-binding protein G(o) in brain

We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the α subunit (39 kDa) of a G protein of unknown function, termed G(o), as well as with the β subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the α subunits of the stimulatory (G(s) or inhibitory (G(i)) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affinity-purified antiserum showed that RV3 specifically recognizes the G(o) α subunit and the β subunit in crude brain membranes. Using RV3, we found approximately equal amounts of G(o) in brain membranes from frog, chicken, rat, cow, and man. Quantitative immunoblotting gave G(o) α subunit/β subunit ratios ~ 1 in cerebral cortex, raising the possibility that free G(o) α subunit (unassociated with β subunit) may exist in brain. The concentration of G(o) α subunit in cerebral cortex is about 5 times that of G(i) α subunit. The results show that G(o) is an immunochemically distinct, highly conserved protein distributed throughout the brain, with particularly high concentrations in forebrain.