Purification of an endogenous benzodiazepine-like substance from the mammalian brain.

An anti-benzodiazepine monoclonal antibody has been used to demonstrate the existence of benzodiazepine-like molecules in the brain and for the purification of these molecules. Immunocytochemical experiments show that these molecules are neuronal and not glial and that they are ubiquitously distributed throughout the brain. Immunoblots indicate the presence of benzodiazepine-like epitopes in several brain peptides. An endogenous substance that binds to the central-type benzodiazepine receptor with agonist properties has been purified to homogeneity from the bovine brain. The purification consisted on immunoaffinity chromatography on immobilized monoclonal anti-benzodiazepine antibody followed by gel filtration on Sephadex G-25 and two reverse phase HPLCs. The purified substance has a small molecular weight and its activity is protease resistant. The endogenous substance blocks the binding of agonists, inverse agonists and antagonists to the central-type benzodiazepine receptor but it does not inhibit the binding of Ro5-4864 to the "peripheral-type" benzodiazepine receptor. The neurotransmitter gamma-amino-butyric acid increases the affinity of the benzodiazepine receptor for the purified substance. Thus this benzodiazepine-like substance is different from the endogenous benzodiazepine receptor ligands reported by others.