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Affinity chromatography of human platelet alpha 2-adrenergic receptors.

Publication ,  Journal Article
Regan, JW; Barden, N; Lefkowitz, RJ; Caron, MG; DeMarinis, RM; Krog, AJ; Holden, KG; Matthews, WD; Hieble, JP
Published in: Proc Natl Acad Sci U S A
December 1982

Catecholamines, such as epinephrine, inhibit the enzyme adenylate cyclase (EC 4.6.1.1) via a specific receptor mechanism involving alpha(2)-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 alpha(2)-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 alpha(2)-adrenergic receptors, can be specifically labeled with [(3)H]yohimbine and can be solubilized with digitonin without loss of their alpha(2)-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 [(3)H]yohimbine binding activity. Adsorption to the affinity gel is blocked by both alpha-adrenergic antagonists (phentolamine >/= yohimbine > prazosin) and by alpha-adrenergic agonists [p-aminoclonidine > (-)-epinephrine > (+)-epinephrine]. Similarly, elution of specific [(3)H]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 alpha-adrenergic receptors, such as (-)-isoproterenol, (-)-alprenolol, atropine, and carbachol, are ineffective for both the blockade of adsorption and the elution of specific [(3)H]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 [(3)H]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 alpha(2)-adrenergic receptor of human platelets.

Duke Scholars

Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

December 1982

Volume

79

Issue

23

Start / End Page

7223 / 7227

Location

United States

Related Subject Headings

  • Stereoisomerism
  • Solubility
  • Receptors, Adrenergic, alpha
  • Receptors, Adrenergic
  • Protein Binding
  • Membrane Proteins
  • Humans
  • Dose-Response Relationship, Drug
  • Chromatography, Affinity
  • Blood Platelets
 

Citation

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Regan, J. W., Barden, N., Lefkowitz, R. J., Caron, M. G., DeMarinis, R. M., Krog, A. J., … Hieble, J. P. (1982). Affinity chromatography of human platelet alpha 2-adrenergic receptors. Proc Natl Acad Sci U S A, 79(23), 7223–7227. https://doi.org/10.1073/pnas.79.23.7223
Regan, J. W., N. Barden, R. J. Lefkowitz, M. G. Caron, R. M. DeMarinis, A. J. Krog, K. G. Holden, W. D. Matthews, and J. P. Hieble. “Affinity chromatography of human platelet alpha 2-adrenergic receptors.Proc Natl Acad Sci U S A 79, no. 23 (December 1982): 7223–27. https://doi.org/10.1073/pnas.79.23.7223.
Regan JW, Barden N, Lefkowitz RJ, Caron MG, DeMarinis RM, Krog AJ, et al. Affinity chromatography of human platelet alpha 2-adrenergic receptors. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7223–7.
Regan, J. W., et al. “Affinity chromatography of human platelet alpha 2-adrenergic receptors.Proc Natl Acad Sci U S A, vol. 79, no. 23, Dec. 1982, pp. 7223–27. Pubmed, doi:10.1073/pnas.79.23.7223.
Regan JW, Barden N, Lefkowitz RJ, Caron MG, DeMarinis RM, Krog AJ, Holden KG, Matthews WD, Hieble JP. Affinity chromatography of human platelet alpha 2-adrenergic receptors. Proc Natl Acad Sci U S A. 1982 Dec;79(23):7223–7227.
Journal cover image

Published In

Proc Natl Acad Sci U S A

DOI

ISSN

0027-8424

Publication Date

December 1982

Volume

79

Issue

23

Start / End Page

7223 / 7227

Location

United States

Related Subject Headings

  • Stereoisomerism
  • Solubility
  • Receptors, Adrenergic, alpha
  • Receptors, Adrenergic
  • Protein Binding
  • Membrane Proteins
  • Humans
  • Dose-Response Relationship, Drug
  • Chromatography, Affinity
  • Blood Platelets