Use of specific antibodies to quantitate the guanine

We immunized rabbits with purified guanine nucleotide-binding proteins (G proteins) from bovine brain and obtained an antiserum, RV3, that reacts specifically with the a subunit (39 kDa) of a G protein of unknown function, termed G0, as well as with the f subunit (35 kDa) common to all G proteins. RV3 showed no crossreactivity with the a subunits of the stimulatory (Gj) or inhibitory (G;) G proteins associated with adenylate cyclase, nor with that of the rod outer segment G protein, transducin. Immunoblots with crude and affimity- purified antiserum showed that RV3 specifically recognizes the Go a subunit and the f6 subunit in crude brain membranes. Using RV3, we found approximately equal amounts of G. in brain membranes from frog, chicken, rat, cow, and man. Quantitative immunoblotting gave G. a subunit/.8 subunit ratios 1 in cerebral cortex, raising the possibility that free G. a subunit (unassociated with /3 subunit) may exist in brain. The concentration of G. a subunit in cerebral cortex is about 5 times that of Gi a subunit. The results show that G. Is an im- munochemically distinct, highly conserved protein distributed throughout the brain, with particularly high concentrations in forebrain.