Mammalian α1-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit
(1986) In Journal of Biological Chemistry 261(17). p.7710-7716- Abstract
α1-Adrenergic receptors from the cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl)pentanoyl]- 1-piperazinyl]-quinazoline), an α1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent M(r) = 80,000 that co-migrates with the peptide labeled by the specific... (More)
α1-Adrenergic receptors from the cultured smooth muscle cell line (DDT1 MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl)pentanoyl]- 1-piperazinyl]-quinazoline), an α1 receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent M(r) = 80,000 that co-migrates with the peptide labeled by the specific α1-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5[(4-azido-3-[125I]iodophenyl )pentanoyl]-1-piperazinly] quinazoline. The specific activity (~13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of M(r) = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate α1-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure α1-adrenergic receptor and the pure human platelet α2-adrenergic receptor (Regan, J.W., Nakata H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.
(Less)
- author
- Lomasney, J. W. ; Leeb-Lundberg, L. M.F. LU ; Cotecchia, S. ; Regan, J. W. ; DeBernardis, J. F. ; Caron, M. G. and Lefkowitz, R. J.
- publishing date
- 1986-12-01
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Journal of Biological Chemistry
- volume
- 261
- issue
- 17
- pages
- 7710 - 7716
- publisher
- American Society for Biochemistry and Molecular Biology
- external identifiers
-
- pmid:3011796
- scopus:0023037070
- ISSN
- 0021-9258
- language
- English
- LU publication?
- no
- id
- 26513727-816c-465e-b8e9-83fc17c33ac2
- date added to LUP
- 2019-06-04 14:39:18
- date last changed
- 2024-01-01 09:17:30
@article{26513727-816c-465e-b8e9-83fc17c33ac2, abstract = {{<p>α<sub>1</sub>-Adrenergic receptors from the cultured smooth muscle cell line (DDT<sub>1</sub> MF-2) have been solubilized with digitonin and purified to apparent homogeneity by sequential chromatography on a biospecific affinity support (Sepharose-A55453 (4-amino-6,7-dimethoxy-2-[4-[5-(4-amino-3-phenyl)pentanoyl]- 1-piperazinyl]-quinazoline), an α<sub>1</sub> receptor-selective antagonist), a wheat germ agglutinin-agarose gel, and a high performance steric exclusion liquid chromatography column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveals a peptide with an apparent M(r) = 80,000 that co-migrates with the peptide labeled by the specific α<sub>1</sub>-adrenergic receptor photoaffinity probe 4-amino-6,7-dimethoxy-2-[4-[5[(4-azido-3-[<sup>125</sup>I]iodophenyl )pentanoyl]-1-piperazinly] quinazoline. The specific activity (~13,600 pmol of ligand binding/mg of protein) of purified receptor preparations is consistent with that expected for a pure peptide of M(r) = 80,000 containing a single ligand binding site. Overall yields approximate 14% of initial crude particulate binding. The purified receptor preparations bind agonist and antagonist ligands with appropriate α<sub>1</sub>-adrenergic specificity, stereoselectivity, and affinity. Peptide maps of the pure α<sub>1</sub>-adrenergic receptor and the pure human platelet α<sub>2</sub>-adrenergic receptor (Regan, J.W., Nakata H., DeMarinis, R.M., Caron, M.G., and Lefkowitz, R.J. (1986) J. Biol. Chem. 261, 3894-3900) using several different proteases suggest that these two receptors show little if any structural homology.</p>}}, author = {{Lomasney, J. W. and Leeb-Lundberg, L. M.F. and Cotecchia, S. and Regan, J. W. and DeBernardis, J. F. and Caron, M. G. and Lefkowitz, R. J.}}, issn = {{0021-9258}}, language = {{eng}}, month = {{12}}, number = {{17}}, pages = {{7710--7716}}, publisher = {{American Society for Biochemistry and Molecular Biology}}, series = {{Journal of Biological Chemistry}}, title = {{Mammalian α<sub>1</sub>-adrenergic receptor. Purification and characterization of the native receptor ligand binding subunit}}, volume = {{261}}, year = {{1986}}, }