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. 1982 Dec;79(23):7223–7227. doi: 10.1073/pnas.79.23.7223

Affinity chromatography of human platelet α2-adrenergic receptors

John W Regan *, Nicholas Barden *, Robert J Lefkowitz *, Marc G Caron *, Robert M DeMarinis †, Arnold J Krog †, Kenneth G Holden †, William D Matthews ‡, J Paul Hieble ‡
PMCID: PMC347311  PMID: 6130523

Abstract

Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving α2-adrenergic receptors. In order to facilitate purification of these inhibitory receptors we have prepared a highly effective biospecific affinity adsorbent. The immobilized ligand SKF 101253 is a 3-benzazepine with α2-adrenergic antagonist activity. SKF 101253 is coupled to Sepharose CL-4B by using a bifunctional reagent (1,4-butanediol diglycidyl ether) which also provides a hydrophilic spacer moiety between the ligand and the gel matrix. Membranes from human platelets, containing α2-adrenergic receptors, can be specifically labeled with [3H]yohimbine and can be solubilized with digitonin without loss of their α2-adrenergic binding characteristics. Chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in the adsorption of 70-80% of the initial [3H]yohimbine binding activity. Adsorption to the affinity gel is blocked by both α-adrenergic antagonists (phentolamine ≥ yohimbine > prazosin) and by α-adrenergic agonists [p-aminoclonidine > (-)-epinephrine > (+)-epinephrine]. Similarly, elution of specific [3H]yohimbine binding activity from the affinity gel is effected with the aforementioned agonists and antagonists in the same order of potency. Other drugs that do not interact appreciably with α-adrenergic receptors, such as (-)-isoproterenol, (-)-alprenolol, atropine, and carbachol, are ineffective for both the blockade of adsorption and the elution of specific [3H]yohimbine binding activity from the affinity gel. In addition to the specificity of the interaction, chromatography of solubilized human platelet membrane preparations on the SKF 101253-Sepharose CL-4B affinity gel results in a 40-50% overall yield and an approximately 200-fold increase in the specific binding activity for [3H]yohimbine. The results indicate that the SKF 101253-Sepharose CL-4B affinity adsorbent should provide a powerful tool for the purification of the adenylate cyclase-inhibitory α2-adrenergic receptor of human platelets.

Keywords: [3H]yohimbine binding, benzazepine, adenylate cyclase inhibition

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Caron M. G., Lefkowitz R. J. Solubilization and characterization of the beta-adrenergic receptor binding sites of frog erythrocytes. J Biol Chem. 1976 Apr 25;251(8):2374–2384. [PubMed] [Google Scholar]
  2. Caron M. G., Srinivasan Y., Pitha J., Kociolek K., Lefkowitz R. J. Affinity chromatography of the beta-adrenergic receptor. J Biol Chem. 1979 Apr 25;254(8):2923–2927. [PubMed] [Google Scholar]
  3. De Lean A., Hancock A. A., Lefkowitz R. J. Validation and statistical analysis of a computer modeling method for quantitative analysis of radioligand binding data for mixtures of pharmacological receptor subtypes. Mol Pharmacol. 1982 Jan;21(1):5–16. [PubMed] [Google Scholar]
  4. García-Sevilla J. A., Hollingsworth P. J., Smnith C. B. Alpha 2-adrenoreceptors on human platelets: selective labelling by [3H]clonidine and [3H]yohimbine and competitive inhibition by antidepressant drugs. Eur J Pharmacol. 1981 Sep 24;74(4):329–341. doi: 10.1016/0014-2999(81)90052-2. [DOI] [PubMed] [Google Scholar]
  5. Grant J. A., Scrutton M. C. Novel alpha2-adrenoreceptors primarily responsible for inducing human platelet aggregation. Nature. 1979 Feb 22;277(5698):659–661. doi: 10.1038/277659a0. [DOI] [PubMed] [Google Scholar]
  6. Hieble J. P., Pendleton R. G. Effects of ring substitution on the pre- and postjunctional alpha-adrenergic activity of aryliminoimidazolidines. Naunyn Schmiedebergs Arch Pharmacol. 1979 Nov;309(3):217–224. doi: 10.1007/BF00504753. [DOI] [PubMed] [Google Scholar]
  7. Hoffman B. B., De Lean A., Wood C. L., Schocken D. D., Lefkowitz R. J. Alpha-adrenergic receptor subtypes: quantitative assessment by ligand binding. Life Sci. 1979 May 7;24(19):1739–1745. doi: 10.1016/0024-3205(79)90061-4. [DOI] [PubMed] [Google Scholar]
  8. Hoffman B. B., Dukes D. F., Lefkowitz R. J. Alpha-adrenergic receptors in liver membranes: delineation with subtype selective radioligands. Life Sci. 1981 Jan 19;28(3):265–272. doi: 10.1016/0024-3205(81)90732-3. [DOI] [PubMed] [Google Scholar]
  9. Hoffman B. B., Michel T., Kilpatrick D. M., Lefkowitz R. J., Tolbert M. E., Gilman H., Fain J. N. Agonist versus antagonist binding to alpha-adrenergic receptors. Proc Natl Acad Sci U S A. 1980 Aug;77(8):4569–4573. doi: 10.1073/pnas.77.8.4569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hsu C. Y., Knapp D. R., Halushka P. V. The effects of alpha adrenergic agents on human platelet aggregation. J Pharmacol Exp Ther. 1979 Mar;208(3):366–370. [PubMed] [Google Scholar]
  11. Kafka M. S., Tallman J. F., Smith C. C., Costa J. L. Alpha-adrenergic receptors on human platelets. Life Sci. 1977 Nov 15;21(10):1429–1438. doi: 10.1016/0024-3205(77)90197-7. [DOI] [PubMed] [Google Scholar]
  12. Lynch C. J., Steer M. L. Evidence for high and low affinity alpha 2-receptors. Comparison of [3H]norepinephrine and [3H]phentolamine binding to human platelet membranes. J Biol Chem. 1981 Apr 10;256(7):3298–3303. [PubMed] [Google Scholar]
  13. Michel T., Hoffman B. B., Lefkowitz R. J., Caron M. G. Different sedimentation properties of agonist- and antagonist-labelled platelet alpha 2 adrenergic receptors. Biochem Biophys Res Commun. 1981 Jun 16;100(3):1131–1136. doi: 10.1016/0006-291x(81)91941-0. [DOI] [PubMed] [Google Scholar]
  14. Motulsky H. J., Shattil S. J., Insel P. A. Characterization of alpha 2-adrenergic receptors on human platelets using [3H]yohimbine. Biochem Biophys Res Commun. 1980 Dec 31;97(4):1562–1570. doi: 10.1016/s0006-291x(80)80044-1. [DOI] [PubMed] [Google Scholar]
  15. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  16. Shattil S. J., McDonough M., Turnbull J., Insel P. A. Characterization of alpha-adrenergic receptors in human platelets using [3H]clonidine. Mol Pharmacol. 1981 Jan;19(1):179–183. [PubMed] [Google Scholar]
  17. Shorr R. G., Lefkowitz R. J., Caron M. G. Purification of the beta-adrenergic receptor. Identification of the hormone binding subunit. J Biol Chem. 1981 Jun 10;256(11):5820–5826. [PubMed] [Google Scholar]
  18. Smith S. K., Limbird L. E. Solubilization of human platelet alpha-adrenergic receptors: evidence that agonist occupancy of the receptor stabilizes receptor--effector interactions. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4026–4030. doi: 10.1073/pnas.78.7.4026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Steer M. L., Khorana J., Galgoci B. Quantitation and characterization of human platelet alpha-adrenergic receptors using [3H]phentolamine. Mol Pharmacol. 1979 Nov;16(3):719–728. [PubMed] [Google Scholar]

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