Physicochemical Characterization of Detergent‐Solubilized γ‐Aminobutyric Acid and Benzodiazepine Receptor Proteins from Bovine Brain

Abstract

[³H]Muscimol and [³H]flunitrazepam binding activities have been solubilized from bovine cortex using the ionic detergent sodium deoxycholate. The soluble receptor proteins were shown to bind [³H]muscimol with a dissociation constant, K_d, of 12 nM and a binding capacity (B_max value) of 1.56 pmol/mg protein; 7gamma;-amino[³H]- butyric acid with a K_d of 50 nM and B_max of 1.55 pmol/mg protein; and [³H]flunitrazepam with a K_d of 8 nM and a B_max of 0.8 pmol/mg protein. Gel filtration of the soluble receptor proteins showed that the γ-amino[³H]-butyric acid and [³H]flunitrazepam binding activities comigrated with a Stokes radius of 6.8 nm. The two binding activities were also found to comigrate after sedimentation in a sucrose density gradient. The hydrodynamic properties of the assumed protein-detergent complexes were determined by gel filtration and sedimentation through gradients of sucrose in H₂O or ²H₂O. Under the conditions employed, the parameters for both the putative γ-aminobutyric acid and benzodiazepine receptors were : partial specific volume, 0.73 ml g ⁻¹; sedimentation coefficient, 12.5 S; molecular weight, 355000; and frictional ratio 1.46. These observations are consistent with the conclusion that the majority of both binding activities solubilized in deoxycholate reside in a single macromolecular complex. However, Triton X-100 selectively solubilized the benzodiazepine binding activity. This suggests that the two binding activities can be at least partially separated.