Open AccessIrreversible site‐directed labeling of the 4‐aminobutyrate binding site by tritiated meta‐ sulfonate benzene diazonium
Author(s) -
Jacques Patrice,
Perret Philippe,
Bouchet MarieJeanne,
Foucaud Bernard,
Goeldner Maurice,
Benke Dietmar
Publication year - 1999
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1999.00715.x
Subject(s) - chemistry , protein subunit , nucleophile , gabaa receptor , g alpha subunit , binding site , stereochemistry , receptor , immunoprecipitation , biochemistry , gene , catalysis
Tritiated meta‐ sulfonate benzene diazonium ([ 3 H]MSBD), a molecule structurally related to 4‐aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABA A receptor. Irreversible labeling reactions using [ 3 H]MSBD were performed on purified GABA A receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [ 3 H]MSBD was found to be specifically incorporated into proteins in the 45–60 kDa molecular mass range which were identified as α1 subunits and β2/β3 subunits by immunoprecipitation with subunit‐specific antibodies. The specific immunoprecipitation of α and β subunits confirms that binding of [ 3 H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the β subunit but reveal also the contribution of yet unidentified nucleophilic residues on the α subunit for the GABA binding site.