Binding characteristics of [ 3H]opioid ligands to active opioid binding sites solubilized from rat brain membranes by glycodeoxycholate and NaCl: the recovery of binding activity by dilution

Abstract

This paper describes the binding properties of [ 3H]peptidergic opioi ligands to binding sites solubilized from rat brain membranes by the treatment with 0.125% sodium glycodeoxycholate and 1 M NaCl. The highest amount of the specific binding of [ 3H]-[ d-Ala 2-, Met 5]enkephalinamide was obtainable when 10-fold diluted solubilized preparations were incubated in the presence of 0.1 mM MnCl 2 and 100 mM NaCl at 0 °C (on ice) for 3 h. With this assay condition, the significant binding of followint [ 3H]opioid ligands, which have been thought to be selective for receptor types, was also observed: [ 3H]-[ d-A;a 2, MePhe 4, Gly-ol 5]enkephalin (μ-type), [ 3H]-[ d-Ala 2, d-Leu 5]enkephalin (δ-type) and [ 3H]dynorphin 1–9 ( K-type). The number of binding sites in solubilized preparations for each [ 3H]ligand corresponded to 40–50% recovery of original membrane-bound binding sites. The Scatchard plot of the concentration-saturation binding curve showed only one class of binding sites, with a high affinity for each [ 3H]ligand. Apparent dissociation constants between solubilized receptors and [ 3H]ligands were the same as membrane-bound ones, but the ligand specificity for each receptor-type, which was examined by binding inhibition tests with unlabeled ligands, decreased. Present results indicate that heterogeneous opioid receptors in rat brain membranes seem to be transformed into less heterogeneous forms through the treatment with glycogdeoxycholate and NaCl and the dilution process.