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dc.contributor.authorKelleher, Daniel J.
dc.contributor.authorRashidbaigi, Abbas
dc.contributor.authorRuoho, Arnold E.
dc.contributor.authorJohnson, Gary L.
dc.date2022-08-11T08:08:00.000
dc.date.accessioned2022-08-23T15:38:59Z
dc.date.available2022-08-23T15:38:59Z
dc.date.issued1983-11-10
dc.date.submitted2014-10-22
dc.identifier.citationJ Biol Chem. 1983 Nov 10;258(21):12881-5.
dc.identifier.issn0021-9258 (Linking)
dc.identifier.pmid6313681
dc.identifier.urihttp://hdl.handle.net/20.500.14038/26056
dc.description.abstractbeta-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.
dc.language.isoen_US
dc.relation<a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=6313681&dopt=Abstract">Link to Article in PubMed</a>
dc.relation.urlhttp://www.jbc.org/content/258/21/12881.long
dc.subjectAdenylate Cyclase
dc.subjectAlprenolol
dc.subjectAnimals
dc.subjectChromatography, Affinity
dc.subjectDeoxycholic Acid
dc.subjectDigitonin
dc.subjectErythrocyte Membrane
dc.subjectGTP-Binding Proteins
dc.subjectHumans
dc.subjectIsoproterenol
dc.subjectKinetics
dc.subjectPhosphatidylcholines
dc.subjectReceptors, Adrenergic, beta
dc.subjectReceptors, Cell Surface
dc.subjectTurkeys
dc.subjectBiochemistry
dc.subjectBiochemistry, Biophysics, and Structural Biology
dc.subjectMolecular Biology
dc.titleRapid vesicle reconstitution of alprenolol-Sepharose-purified beta 1-adrenergic receptors. Interaction of the purified receptor with N
dc.typeJournal Article
dc.source.journaltitleThe Journal of biological chemistry
dc.source.volume258
dc.source.issue21
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/bmp_pp/196
dc.legacy.embargo2014-11-05T00:00:00-08:00
dc.identifier.contextkey6269470
html.description.abstract<p>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.</p>
dc.identifier.submissionpathbmp_pp/196
dc.contributor.departmentBiochemistry and Molecular Pharmacology
dc.contributor.departmentNeurobiology
dc.source.pages12881-5


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