Irreversible site‐directed labeling of the 4‐aminobutyrate binding site by tritiated meta‐ sulfonate benzene diazonium Contribution of a nucleophilic amino acid residue of the α1 subunit

• Published in: European journal of biochemistry Vol. 265; no. 1; pp. 189 - 194
• Main Authors: Jacques, Patrice, Perret, Philippe, Bouchet, Marie‐Jeanne, Foucaud, Bernard, Goeldner, Maurice, Benke, Dietmar
• Format: Journal Article
• Language: English
• Published: 01.10.1999
• DOI: 10.1046/j.1432-1327.1999.00715.x
• ISSN: 0014-2956

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.