Purification and characterization of the human platelet alpha 2-adrenergic receptor.
Human platelet alpha 2-adrenergic receptors have been purified approximately 80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is approximately 2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of Mr 64,000. The specific binding activity of the alpha 2-adrenergic receptor after four chromatographic steps is 14.5 nmol/mg protein. This value is consistent with the expected theoretical specific activity (15.6 nmol/mg) for a protein with a molecular mass of 64,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. The purified protein can be covalently labeled with the alkylating alpha-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the Mr 64,000 protein contains the ligand binding site of the alpha 2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper alpha 2-adrenergic specificity. The alpha 2-adrenergic receptor contains an essential sulfhydryl residue. Thus, exposure of the purified receptor to the sulfhydryl-specific reagent, phenylmercuric chloride, resulted in an 80% loss of binding activity. This loss of binding activity was prevented when exposure to phenylmercuric chloride was done in the presence of alpha 2-adrenergic ligands, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified alpha 2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, alpha-chymotrypsin, and papain. In a comparison with purified beta 2-adrenergic receptors, no common partial proteolytic products were found.
Regan, JW; Nakata, H; DeMarinis, RM; Caron, MG; Lefkowitz, RJ
Volume / Issue
Start / End Page
Pubmed Central ID
Electronic International Standard Serial Number (EISSN)
International Standard Serial Number (ISSN)