Physicochemical characterization of detergent-solubilized gamma-aminobutyric acid and benzodiazepine receptor proteins from bovine brain.
[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; gamma-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 gamma-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 gamma-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.
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002540:Cerebral Cortex / Q000032:analysis*; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002850:Chromatography, Gel; D003840:Deoxycholic Acid; D005445:Flunitrazepam / Q000378:metabolism; D008970:Molecular Weight; D009118:Muscimol / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D011955:Receptors, Drug / Q000302:isolation & purification / Q000378:metabolism*; D011963:Receptors, GABA-A; D012995:Solubility; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D011956:Receptors, Cell Surface; D011955:Receptors, Drug; D011963:Receptors, GABA-A; D003840:Deoxycholic Acid; D009118:Muscimol; D005680:gamma-Aminobutyric Acid; D005445:Flunitrazepam