Purification and characterization of the human platelet α2-adrenergic receptor

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

Research output: Contribution to journalArticle

60 Citations (Scopus)

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 - 1986
Externally publishedYes

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Platelets
Adrenergic Receptors
Purification
Blood Platelets
Ligands
Adrenergic Agents
Proteins
Proteolysis
Phenoxybenzamine
Sulfhydryl Reagents
Competitive Binding
Papain
Dithiothreitol
Chymotrypsin
Molecular mass
Electrophoresis
Sodium Dodecyl Sulfate
Labeling
Staphylococcus aureus
Polyacrylamide Gel Electrophoresis

ASJC Scopus subject areas

  • Biochemistry

Cite this

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.

Purification and characterization of the human platelet α2-adrenergic receptor. / Regan, John W; Nakata, H.; DeMarinis, R. M.; Caron, M. G.; Lefkowitz, R. J.

In: Journal of Biological Chemistry, Vol. 261, No. 8, 1986, p. 3894-3900.

Research output: Contribution to journalArticle

Regan, JW, Nakata, H, DeMarinis, RM, Caron, MG & Lefkowitz, RJ 1986, 'Purification and characterization of the human platelet α2-adrenergic receptor', Journal of Biological Chemistry, vol. 261, no. 8, pp. 3894-3900.
Regan, John W ; Nakata, H. ; DeMarinis, R. M. ; Caron, M. G. ; Lefkowitz, R. J. / Purification and characterization of the human platelet α2-adrenergic receptor. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 8. pp. 3894-3900.
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