Involvement of a Disulfide Bond in the Binding of Flunitrazepam to the Benzodiazepine Receptor from Bovine Cerebral Cortex |
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Authors: | Marcela S Otero de Bengtsson H Daniel Lacorazza Mirtha J Biscoglio de Jiménez Bonino Jorge H Medina |
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Institution: | Instituto de Quimica y Fisicoquimica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires;Instituto de Biologia Celular, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina |
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Abstract: | Abstract: The effects of chemical modification of a disulfide bond(s) (-SS-) or sulfhydryl group(s) (-SH) on the 3H]-flunitrazepam (3H]FNZ) binding to membrane-bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD-R) from bovine cerebral cortex were examined. Reduction of -SS- with dithiothreitol (DTT) brought about a reversible, time- and dose-dependent inhibition of 3H]FNZ binding to the membrane-bound BZD-R. Alkylation of the membranes with the -SH-modifying reagent iodoacetamide (IAA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) produced a slight inhibition of 3H]FNZ binding in a dose-dependent manner. Scatchard analysis of saturation curves of 3H]FNZ binding in the presence and absence of 5 m M DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane-bound and the immunoprecipitated BZD-R led to 3H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of -SS- does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of 3H]FNZ binding by FNZ (an agonist) or by Ro 15–1788 (an antagonist) suggests the presence of -SS- at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with 3H]FNZ revealed a clear-cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 m M DTT/IAA-treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD-R. |
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Keywords: | Benzodiazepine receptor Bovine cerebral cortex Disulfide bond [3H]Flunitrazepam |
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