Rapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N
Department of Biochemistry and Molecular Pharmacology, Department of Neurobiology University of Massachusetts Medical School
Medical Subject Headings
Adenylate Cyclase; Alprenolol; Animals; Chromatography, Affinity; Deoxycholic Acid; Digitonin; Erythrocyte Membrane; GTP-Binding Proteins; Humans; Isoproterenol; Kinetics; Phosphatidylcholines; Receptors, Adrenergic, beta; Receptors, Cell Surface; Turkeys
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.
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Citation: J Biol Chem. 1983 Nov 10;258(21):12881-5.
Kelleher, Daniel J.; Rashidbaigi, Abbas; Ruoho, Arnold E.; and Johnson, Gary L., "Rapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N" (1983). Biochemistry and Molecular Pharmacology Publications and Presentations. Paper 196.