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.
Otero de Bengtsson
M S
MS
Instituto de Quimica y Fisicoquimica Biológicas (UBA-CONICET), Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires, Argentina.
Lacorazza
H D
HD
Biscoglio de Jiménez Bonino
M J
MJ
Medina
J H
JH
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology*; D004229:Dithiothreitol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D005445:Flunitrazepam / Q000378:metabolism*; D007461:Iodoacetates / Q000494:pharmacology*; D019807:Iodoacetic Acid; D007700:Kinetics; D011963:Receptors, GABA-A / Q000187:drug effects / Q000378:metabolism*
D004220:Disulfides; D007461:Iodoacetates; D011963:Receptors, GABA-A; D005445:Flunitrazepam; D004228:Dithionitrobenzoic Acid; D004229:Dithiothreitol; D019807:Iodoacetic Acid