Solubilization and Characterization of Rat Brain ?2-Adrenergic Receptor

Abstract

.alpha.2-Adrenergic receptors labeled by [3H]-clonidine [CLD] (.alpha.2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitter-ionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). Of the original [3H]CLO binding sites in the membrane .apprx. 40% were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound .alpha.2-adrenergic receptors. Scatchard plots of [3H]CLO bindings to solubilized .alpha.2-receptors were curvilinear, indicating the existence of the 2 distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow an investigation of the regulation mechanisms of the brain .alpha.2-adrenergic receptor system at the molecular level.