Immunochemical studies of the 36-kDa common β subunit of guanine nucleotide-binding proteins: Identification of a major epitope

Twenty-four of 24 rabbits immunized with the β subunit common to guanine nucleotide binding proteins developed antibodies reactive on immunoblots with the 15-kDa (amino-terminal) tryptic fragment of β. Only 2 of 24 developed antibodies reactive with the 26-kDa (carboxy-terminal) tryptic fragment. The 15-kDa fragment-reactive antibodies were also detected in several nonimmune sera. Antibodies reactive with the 15-kDa fragment could be affinity-purified from all β-immune sera by adsorption to a fusion protein encoded by a cDNA clone identified by expression vector screening. The 15-kDa fragment antibodies in nonimmune sera did not bind to the fusion protein. Limited amino acid sequence homology between the 36-kDa β subunit and the protein encoded by the cDNA clone suggested that the amino-terminal decapeptide of β contains a major epitope. A synthetic decapeptide, corresponding to the amino terminus of the 36-kDa β subunit, effectively and specifically blocked binding of antibodies in β-immune sera (but not in β-reactive nonimmune sera) to nitrocellulose-bound 15-kDa fragment. The 15-kDa fragment-reactive antibodies could be affinity-purified from β-immune sera on a matrix containing bound decapeptide; affinity-purified antibodies reacted equally well with the 36- and 35-kDa forms of the β subunit. Native transducin β/γ complexes readily blocked binding of 15-kDa fragment-reactive antibodies in immune but not nonimmune sera from binding to the nitrocellulose-bound fragment. The results show that nonimmune sera may contain antibodies directed against an epitope of the 15-kDa fragment that is buried in the native β/γ complex. In contrast, the amino terminal decapeptide of the β subunit is exposed on the surface of the native protein and contains a major antigenic site in both the 35- and 36-kDa forms.