Endogenous Modulator of Benzodiazepine Binding in Rat Cortex

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Abstract
Benzodiazepine binding sites, solubilized with 1% digitonin, were used to study specific [3H]flunitrazepam ([3H]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 [3H]FNP binding. Kinetic analysis showed inhibition was noncompetitive. The inhibitory activity was sensitive to trypsin. Extracts of repeatedly frozen, thawed and washed membrane preparations still possessed inhibitory activity. Digitonin apparently solubilizes a membrane protein that inhibits benzodiazepine binding. PEG seems to remove this substance from the binding sites.