Affinity chromatography of the anterior pituitary D2-dopamine receptor.

Published

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Senogles, SE; Amlaiky, N; Johnson, AL; Caron, MG

Published Date

  • February 25, 1986

Published In

Volume / Issue

  • 25 / 4

Start / End Page

  • 749 - 753

PubMed ID

  • 2938619

Pubmed Central ID

  • 2938619

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi00352a002

Language

  • eng

Conference Location

  • United States