Affinity chromatography of human platelet alpha 2-adrenergic receptors.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Regan, JW; Barden, N; Lefkowitz, RJ; Caron, MG; DeMarinis, RM; Krog, AJ; Holden, KG; Matthews, WD; Hieble, JP

Published Date

  • December 1, 1982

Published In

Volume / Issue

  • 79 / 23

Start / End Page

  • 7223 - 7227

PubMed ID

  • 6130523

Pubmed Central ID

  • 6130523

International Standard Serial Number (ISSN)

  • 0027-8424

Digital Object Identifier (DOI)

  • 10.1073/pnas.79.23.7223

Language

  • eng

Conference Location

  • United States