Affinity Chromatography of the Anterior Pituitary D2-Dopamine Receptor

Susan Senogles, Nourdine Amlaiky, Anne L. Johnson, Marc G. Caron

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

The D2-dopamine receptor from bovine anterior pituitary has been solubilized with digitonin and purified ~ 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 > 2-amino-6,7-dihydroxytetrahydronaphthalene = 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 was 12-15% with specific binding activity of ~ 150 pmol/mg. The reconstituted affinity-purified receptor bound ligands with the expected D2-dopaminergic specificity, stereoselectivity, and rank order of potency.

Original languageEnglish (US)
Pages (from-to)749-753
Number of pages5
JournalBiochemistry
Volume25
Issue number4
DOIs
StatePublished - Jan 1 1986

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Affinity chromatography
Dopamine D2 Receptors
Butaclamol
Affinity Chromatography
Sepharose
Stereoselectivity
Gels
Spiperone
Digitonin
Dopamine Agents
Dopamine Antagonists
Apomorphine
Adsorption
Phospholipids
Ligands
Derivatives
Membranes
Recovery

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Affinity Chromatography of the Anterior Pituitary D2-Dopamine Receptor. / Senogles, Susan; Amlaiky, Nourdine; Johnson, Anne L.; Caron, Marc G.

In: Biochemistry, Vol. 25, No. 4, 01.01.1986, p. 749-753.

Research output: Contribution to journalArticle

Senogles, Susan ; Amlaiky, Nourdine ; Johnson, Anne L. ; Caron, Marc G. / Affinity Chromatography of the Anterior Pituitary D2-Dopamine Receptor. In: Biochemistry. 1986 ; Vol. 25, No. 4. pp. 749-753.
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