Isolation, characterization, and purification to homogeneity of an endogenous polypeptide with agonistic action on benzodiazepine receptors.
A 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 and acidic urea gel electrophoresis. A single UV-absorbing peak was obtained by HPLC using three different columns and solvent systems. DBI has a molecular mass of approximately equal to 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 three polypeptide fragments, and the sequences of two of them have been determined 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. The Ki for [3H]-diazepam and beta-[3H]carboline binding were 4 and 1 microM, respectively. Doses of DBI that inhibited [3H]diazepam binding by greater than 50% fail to change [3H]etorphine, gamma-amino[3H]butyric acid, [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 FG 7142 (beta-carboline-3-carboxylic acid methyl ester), facilitated the shock-induced suppression of drinking by lowering the threshold for this response.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001569:Benzodiazepines / Q000378:metabolism*; D001921:Brain / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003975:Diazepam / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000502:physiology*; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D000596:Amino Acids; D009419:Nerve Tissue Proteins; D011956:Receptors, Cell Surface; D011963:Receptors, GABA-A; D001569:Benzodiazepines; D005680:gamma-Aminobutyric Acid; D003975:Diazepam