Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors.

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Abstract
A rat brain polypeptide termed diazepam-binding inhibitor (DBI) and thought to be chemically and functionally related to the endogenous effector of the benzodiazepine recognition site was purified to homogeneity. This peptide gives a single band of protein on NaDodSO4 [sodium dodecyl sulfate] and acidic urea gel electrophoresis. A single UV-absorbing peak was obtained by HPLC [inhibitory constant] using 3 different columns and solvent systems. DBI has a molecular mass of .apprxeq. 11,000 daltons. Carboxyl-terminus analysis shows that tyrosine is the only residue while the amino-terminus was blocked. Cyanogen bromide treatment of DBI yields 3 polypeptide fragments, and the sequences of 2 of them were determined for a total of 45 amino acids. DBI is a competitive inhibitor for a total of 45 amino acids. DBI is a competitive inhibitor for a total of 45 amino acids. DBI is a competitive inhibitor for the binding of [3H]diazepam, [3H]flunitrazepam, .beta.-[3H]carboline propyl esters and 3H-labeled Ro 15-1788 [ethyl 8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazoci,5a] Cl,4]-benzodiazepine-3-carboxylate]. The Ki [inhibitory constant] for [3H]-diazepam and .beta.-[3H]carboline binding were 4 and 1 .mu.m, respectively. Doses of DBI that inhibited [3H]diazepam binding by > 50% fail to change [3H]etorphine, [3H]GABA [3H]-quinuclidinyl benzilate, [3H]dihydroalprenolol, [3H]adenosine and [3H]imipramine binding tested at their respective Kd values. DBI injected intraventricularly at doses of 5-10 nmol completely reversed the anticonflict action of diazepam on unpunished drinking and, similar to the anxiety-inducing .beta.-carboline derivative FG7142 (.beta.-carboline-3-carboxylic acid methyl ester), facilitated the shock-induced suppression of drinking by lowering the threshold for this response.