Reconstitution of beta-adrenergic receptors into phospholipid vesicles: restoration of [125I]iodohydroxybenzylpindolol binding to digitonin-solubilized receptors.
beta-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 A in diameter, and had a lipid content of 6 mumol phospholipid/mg protein. Specific binding of the beta-adrenergic ligand [3H]dihydroalprenolol ([3H]DNA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [Kd = 84 pM] and competed appropriately with (+) and (-) isomers of beta-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of beta-adrenergic receptor function in a defined lipid milieu.
D000818:Animals; D004072:Digitonin; D004134:Dimyristoylphosphatidylcholine; D004910:Erythrocyte Membrane / Q000378:metabolism; D005614:Freeze Fracturing; D007545:Isoproterenol / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008297:Male; D008854:Microscopy, Electron; D010713:Phosphatidylcholines / Q000378:metabolism*; D010869:Pindolol / Q000031:analogs & derivatives* / Q000378:metabolism; D011433:Propranolol / Q000378:metabolism; D051381:Rats; D011941:Receptors, Adrenergic / Q000378:metabolism*; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D012995:Solubility
D008081:Liposomes; D010713:Phosphatidylcholines; D011941:Receptors, Adrenergic; D011943:Receptors, Adrenergic, beta; C011878:iodohydroxybenzylpindolol; D011433:Propranolol; D010869:Pindolol; D004072:Digitonin; D007545:Isoproterenol; D004134:Dimyristoylphosphatidylcholine