Presence of endogenous inhibitors of benzodiazepine binding in developing chick optic lobe

An endogenous inhibitor of benzodiazepine receptor binding was removed from synaptic membranes of developing chick optic lobe by an exhaustive buffer washing procedure. This treatment increased [3.H]flunitrazepam binding at all stages of development, although this effect was greater at early stages (embryonic day 14-16). Scatchard analysis performed on exhaustively washed membranes at embryonic day 16 and postnatal day 15 revealed the presence of a single population of flunitrazepam binding sites with apparent dissociation constants (Kd) of 1.99 +/- 0.11 and 3.28 +/- 0.10 nM and a maximal number of binding sites (Bmax) of 1.07 +/- 0.08 and 1.22 +/- 0.11 pmol mg-1 protein, respectively. At both stages of development, the addition of inhibitory material, present in the supernatants obtained after the washing procedure, reduced the affinity of recognition sites for [3H]flunitrazepam with Kd of 4.92 +/- 0.13 and 5.62 +/- 0.12 nM, respectively while their Bmax values remained unchanged. These studies demonstrate the presence of an endogenous material in developing chick optic lobe which competitively inhibits [3H]flunitrazepam binding to its receptor site.