Endogenous effector of the benzodiazepine binding site: purification and characterization

Abstract

A protein has been isolated from the small intestine and bile duct which inhibits the binding of [3H]diazepam to specific benzodiazepine binding sites on synaptosomal membranes. When ion-exchange chromatography and gel filtration chromatography are used, this protein has been purified to apparent homogeneity. "Nepenthin" has been chosen as a name for this protein, which has an approximate molecular weight of 16 000, as determined by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography. Purified nepenthin is a competitive inhibitor of [3H]diazepam binding with a Ki = 4.6 X 10(-8) M. It does not inhibit the binding of specific ligands to the enkephalin, beta-adrenergic, gamma-aminobutyrate, or dopamine binding sites in the CNS. Neither gamma-aminobutyric acid nor glycine alters the inhibition of [3H]diazepam binding by this protein. Nepenthin can be extensively treated with proteases (trypsin, chymotrypsin, and Pronase), and inhibition of diazepam binding remains stable, indicating that a lower molecular weight fragment retains activity. Antibodies raised against this purified effector have been used in in situ double antibody labeling studies with rat brain slices. These studies indicate that cells containing an immunologically similar material are present in the deep cortical region of the forebrain.