The study by Claus Braestrup and Richard F. Squires investigates the specific binding of [³H]diazepam to rat brain membranes, revealing a single, saturable binding site with an affinity constant of about 3 nM at pH 7.4. This binding constitutes more than 90% of total binding at 0°C and less than 10% at 37°C. The binding is influenced by temperature, with a sharp conformational change occurring around 18°C. Mitochondrial fractions from kidney, liver, and lung exhibit some [³H]diazepam binding, but it is not as saturable as the binding in brain membranes. Ro-4864, which is inactive in displacing [³H]diazepam from brain membranes, is highly potent in kidney mitochondria, while clonazepam, a potent inhibitor of brain binding, is weak in kidney binding. The affinity constant for [³H]diazepam binding in kidney mitochondria is about 40 nM, 15 times higher than in brain membranes. [³H]diazepam binding was also detected in intestine and skeletal muscle but was restricted to brain, where it is unevenly distributed, with higher densities in the cortex compared to the pons-medulla. Trypsin and chymotrypsin completely abolished specific [³H]diazepam binding in brain and kidney. The findings suggest that the [³H]diazepam binding site in brain may be associated with an unknown endogenous transmitter, and the binding characteristics are distinct from those of presumed neurotransmitters.The study by Claus Braestrup and Richard F. Squires investigates the specific binding of [³H]diazepam to rat brain membranes, revealing a single, saturable binding site with an affinity constant of about 3 nM at pH 7.4. This binding constitutes more than 90% of total binding at 0°C and less than 10% at 37°C. The binding is influenced by temperature, with a sharp conformational change occurring around 18°C. Mitochondrial fractions from kidney, liver, and lung exhibit some [³H]diazepam binding, but it is not as saturable as the binding in brain membranes. Ro-4864, which is inactive in displacing [³H]diazepam from brain membranes, is highly potent in kidney mitochondria, while clonazepam, a potent inhibitor of brain binding, is weak in kidney binding. The affinity constant for [³H]diazepam binding in kidney mitochondria is about 40 nM, 15 times higher than in brain membranes. [³H]diazepam binding was also detected in intestine and skeletal muscle but was restricted to brain, where it is unevenly distributed, with higher densities in the cortex compared to the pons-medulla. Trypsin and chymotrypsin completely abolished specific [³H]diazepam binding in brain and kidney. The findings suggest that the [³H]diazepam binding site in brain may be associated with an unknown endogenous transmitter, and the binding characteristics are distinct from those of presumed neurotransmitters.