Purification and characterization of the human platelet α2-adrenergic receptor

J. W. Regan, H. Nakata, R. M. DeMarinis, M. G. Caron, R. J. Lefkowitz

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

Human platelet α2-adrenergic receptors have been purified ~80,000-fold to apparent homogeneity by a five-step chromatographic procedure. The overall yield starting from the membranes is ~2%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of radioiodinated protein from purified receptor preparations shows a single major band of M(r) 64,000. The specific binding activity of the α2-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-bind site/receptor molecule. The purified protein can be covalently labeled with the alkylating α-adrenergic ligand, [3H]phenoxybenzamine. This labeling is specific, and it shows that the M(r) 64,000 protein contains the ligand binding site of the α2-adrenergic receptor. In addition, the competitive binding of ligands to the purified receptor protein shows the proper α2-adrenergic specificity. The α2-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 α2-adrenergic ligand, and it was reversed by subsequent exposure to dithiothreitol. Partial proteolysis of purified α2-adrenergic receptors was obtained with Staphylococcus aureus V-8 protease, α-chymotrypsin, and papain. In a comparison with purified β2-adrenergic receptors, no common partial proteolytic products were found.

Original languageEnglish (US)
Pages (from-to)3894-3900
Number of pages7
JournalJournal of Biological Chemistry
Volume261
Issue number8
StatePublished - Dec 1 1986
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Regan, J. W., Nakata, H., DeMarinis, R. M., Caron, M. G., & Lefkowitz, R. J. (1986). Purification and characterization of the human platelet α2-adrenergic receptor. Journal of Biological Chemistry, 261(8), 3894-3900.