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 alpha1 subunit.
Tritiated meta-sulfonate benzene diazonium ([3H]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 GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.
Jacques
P
P
Laboratoire de Chimie Bio-organique, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg, France.
Perret
P
P
Bouchet
M J
MJ
Foucaud
B
B
Goeldner
M
M
Benke
D
D
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003979:Diazonium Compounds / Q000737:chemistry / Q000378:metabolism*; D018756:GABA Antagonists / Q000378:metabolism*; D007553:Isotope Labeling; D015335:Molecular Probes; D011233:Precipitin Tests; D011963:Receptors, GABA-A / Q000276:immunology / Q000378:metabolism*; D014316:Tritium; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives*
D003979:Diazonium Compounds; D018756:GABA Antagonists; D015335:Molecular Probes; D011963:Receptors, GABA-A; D014316:Tritium; C092049:3-sulfobenzenediazonium; D005680:gamma-Aminobutyric Acid