Affinity chromatography of the anterior pituitary D2-dopamine receptor

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Abstract
The D2-dopamine receptor from bovine anterior pituitary has been solubilized with digitonin and purified .apprx. 1000-bold by affinity chromatography on a new affinity support. This support consists of a (carboxymethylene)oximino derivative of the D2-selective antagonist spiperone (CMOS) covalently attached to Sepharose 4B through a long side chain. The interaction of the solubilized receptor activity with the affinity gel was biospecific. Dopaminergic drugs blocked adsorption of solubilized receptor activity to the CMOS-Sepharose with the appropriate D2-dopaminergic potency and stereoselectivity. For agonists, (-)-N-n-propylnorapomorphine > 2-amino-6,7-dihydroxytetrahydronaphthalene .simeq. apomorphine > dopamine, whereas for antagonists (+)-butaclamol >> (-)-butaclamol. The same D2-dopaminergic specificity was observed for elution of receptor activity from the gel. To observe eluted receptor binding activity, reconstitution of the eluted material into phospholipid vesicles was necessary. Typically, 70-80% of the solubilized receptor was adsorbed by CMOS-Sepharose, and 40-50% of the adsorbed activity could be recovered after reconstitution of the eluted material. The overall recovery of D2-receptor activity from bovine anterior pituitary membranes 12-15% with specific binding activity of .apprx. 150 pmol/mg. The reconstituted affinity-purified receptor bound ligands with the expected D2-dopaminergic specificity, stereoselectivity, and rank order of potency.