Affinity chromatography of human platelet alpha 2-adrenergic receptors.
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.
D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D001552:Benzazepines; D001792:Blood Platelets / Q000032:analysis*; D002846:Chromatography, Affinity; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification; D011485:Protein Binding / Q000187:drug effects; D011941:Receptors, Adrenergic / Q000302:isolation & purification*; D011942:Receptors, Adrenergic, alpha / Q000302:isolation & purification*; D012995:Solubility; D013237:Stereoisomerism
D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D001552:Benzazepines; D008565:Membrane Proteins; D011941:Receptors, Adrenergic; D011942:Receptors, Adrenergic, alpha; C036606:9-allyloxy-6-chloro-3-methyl-2,3,4,5-tetrahydro-1H-benzazepine