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.
|Original language||English (US)|
|Number of pages||7|
|Journal||Journal of Biological Chemistry|
|State||Published - 1986|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology