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.
D001792:Blood Platelets / Q000032:analysis*; D002846:Chromatography, Affinity; D004072:Digitonin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D010643:Phenoxybenzamine / Q000378:metabolism; D010646:Phentolamine / Q000494:pharmacology; D010663:Phenylmercury Compounds / Q000494:pharmacology; D011942:Receptors, Adrenergic, alpha / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives; D012995:Solubility; D013438:Sulfhydryl Compounds / Q000032:analysis; D014316:Tritium
D010663:Phenylmercury Compounds; D011942:Receptors, Adrenergic, alpha; D013438:Sulfhydryl Compounds; C037405:heparin-sepharose; D010643:Phenoxybenzamine; D014316:Tritium; D012685:Sepharose; D004072:Digitonin; D010646:Phentolamine