Involvement of a Disulfide Bond in the Binding of Flunitrazepam to the Benzodiazepine Receptor from Bovine Cerebral Cortex

: 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 mM 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 mM 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.