Endogenous Modulator of Benzodiazepine Binding in Rat Cortex

Benzodiazepine binding sites, solubilized with 1% digitonin, were used to study specific [ 3 H]flunitrazepam ([ 3 H]FNP) binding. Specific binding increased nonlinearly with increasing amounts of digitonin extract in the assay. Specific binding was increased, and the relationship to amount of extract became linear. in the presence of 2% polyethylene glycol 6000 (PEG). Heat treatment destroyed binding activity of the extract, but not ability to inhibit [ 3 H]FNP binding. Kinetic analysis showed inhibition to be noncompetitive. The inhibitory activity was sensitive to trypsin. Extracts of repeatedly frozen, thawed, and washed membrane preparations still possessed inhibitory activity. It is suggested that digitonin solubilizes a membrane protein that inhibits benzodiazepine binding. PEG apparently removes this substance from the binding sites.