RT Journal Article
SR Electronic
T1 Immunochemical studies of the 36-kDa common beta subunit of guanine nucleotide-binding proteins: identification of a major epitope.
JF Molecular Pharmacology
JO Mol Pharmacol
FD American Society for Pharmacology and Experimental Therapeutics
SP 257
OP 264
VO 33
IS 3
A1 T Zaremba
A1 P Gierschik
A1 M Pines
A1 P Bray
A1 A Carter
A1 R Kahn
A1 C Simons
A1 R Vinitsky
A1 P Goldsmith
A1 A Spiegel
YR 1988
UL http://molpharm.aspetjournals.org/content/33/3/257.abstract
AB Twenty-four of 24 rabbits immunized with the beta subunit common to guanine nucleotide binding proteins developed antibodies reactive on immunoblots with the 15-kDa (amino-terminal) tryptic fragment of beta. 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 beta-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 beta subunit and the protein encoded by the cDNA clone suggested that the amino-terminal decapeptide of beta contains a major epitope. A synthetic decapeptide, corresponding to the amino terminus of the 36-kDa beta subunit, effectively and specifically blocked binding of antibodies in beta-immune sera (but not in beta-reactive nonimmune sera) to nitrocellulose-bound 15-kDa fragment. The 15-kDa fragment-reactive antibodies could be affinity-purified from beta-immune sera on a matrix containing bound decapeptide; affinity-purified antibodies reacted equally well with the 36- and 35-kDa forms of the beta subunit. Native transducin beta/gamma 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 beta/gamma complex. In contrast, the amino terminal decapeptide of the beta subunit is exposed on the surface of the native protein and contains a major antigenic site in both the 35- and 36-kDa forms.