Solubilization and Characterization of Rat Brain α2-Adrenergic Receptor |
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Authors: | Hiroaki Matsui Mikio Asakura Tohru Tsukamoto Jun Imafuku Miyuki Ino Noriko Saitoh Satoko Miyamura Kazuo Hasegawa |
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Institution: | Department of Neuropsychiatry, St. Marianna University School of Medicine, Kawasaki, Kanagawa, Japan |
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Abstract: | alpha 2-Adrenergic receptors labelled by 3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original 3H]CLO binding sites in the membranes 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 binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two 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 a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level. |
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Keywords: | α2-Adrenergic receptor Guanine nucleotide binding protein Solubilization Rat brain |
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