Affinity chromatography of the anterior pituitary D2-dopamine receptor.
The D2-dopamine receptor from bovine anterior pituitary has been solubilized with digitonin and purified approximately 1000-fold 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 greater than 2-amino-6,7-dihydroxytetrahydronaphthalene approximately equal to apomorphine greater than dopamine, whereas for antagonists (+)-butaclamol much greater than (-)-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 was 12-15% with specific binding activity of approximately 150 pmol/mg. The reconstituted affinity-purified receptor bound ligands with the expected D2-dopaminergic specificity, stereoselectivity, and rank order of potency.
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D004072:Digitonin; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010903:Pituitary Gland, Anterior / Q000378:metabolism*; D011954:Receptors, Dopamine / Q000302:isolation & purification* / Q000378:metabolism; D017448:Receptors, Dopamine D2; D012995:Solubility; D013134:Spiperone / Q000031:analogs & derivatives / Q000138:chemical synthesis
D011954:Receptors, Dopamine; D017448:Receptors, Dopamine D2; C048620:((carboxymethylene)oximino)spiperone-sepharose 4B; D013134:Spiperone; D004072:Digitonin