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, Kd, of 12 nM and a binding capacity (Bmax value) of 1.56 pmol/mg protein; 7gamma;‐amino[3H]‐ butyric acid with a Kd of 50 nM and Bmax of 1.55 pmol/mg protein; and [3H]flunitrazepam with a Kd of 8 nM and a Bmax 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. Copyright © 1982, Wiley Blackwell. All rights reserved