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‡, and
- J. Paul Hieble‡
- *Howard Hughes Medical Institute Research Laboratories, Department of Medicine, Duke University Medical Center, Durham, North Carolina 27710
- Howard Hughes Medical Institute Research Laboratories, Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
- †Department of Medicinal Chemistry, Smith Kline & French Laboratories, 1500 Spring Garden Street, Philadelphia, Pennsylvania 19101
- ‡Department of Pharmacology, Smith Kline & French Laboratories, 1500 Spring Garden Street, Philadelphia, Pennsylvania 19101
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.
