Rapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N.
beta-Adrenergic receptors from turkey erythrocyte membranes have been purified 1000-4000-fold using alprenolol-Sepharose affinity chromatography. Addition of deoxycholate solubilized egg phosphatidylcholine to the beta-adrenergic receptor, that is 5-10% pure and in 0.1% digitonin, followed by Sephadex G-50 gel filtration in buffers containing 30 mM MgCl2 results in 65-70% of the receptor being incorporated into phospholipid vesicles. The beta-adrenergic receptor as detected by photoaffinity labeling using [125I]azidobenzylpindolol in membranes and after alprenolol-Sepharose chromatography is a Mr = 40,000 peptide. Addition of deoxycholate extracts of human erythrocyte membranes, which contain the guanine nucleotide stimulatory regulatory protein of adenylate cyclase (Ns) but not beta-adrenergic receptor, were used to reconstitute a guanine nucleotide-mediated change in agonist affinity for the receptor. These results demonstrate that the alprenolol-Sepharose affinity purified beta-adrenergic receptor is functional in both ligand binding and coupling to Ns. The procedure is rapid, efficient and should be generally applicable to beta-adrenergic receptor and Ns from several different membrane systems.
D000262:Adenylyl Cyclases / Q000378:metabolism; D000526:Alprenolol / Q000378:metabolism*; D000818:Animals; D002846:Chromatography, Affinity; D003840:Deoxycholic Acid; D004072:Digitonin; D004910:Erythrocyte Membrane / Q000378:metabolism*; D019204:GTP-Binding Proteins; D006801:Humans; D007545:Isoproterenol / Q000378:metabolism; D007700:Kinetics; D010713:Phosphatidylcholines; D011943:Receptors, Adrenergic, beta / Q000302:isolation & purification / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D014422:Turkeys
D010713:Phosphatidylcholines; D011943:Receptors, Adrenergic, beta; D011956:Receptors, Cell Surface; D003840:Deoxycholic Acid; D000526:Alprenolol; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D004072:Digitonin; D007545:Isoproterenol