Reconstitution of solubilized delta-opiate receptor binding sites in lipid vesicles

Mark A. Scheideler, R. Suzanne Zukin

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Delta-opiate receptors have been solubilized with the non-ionic bile salt detergent digitonin from NG108-15 cell membranes and reconstituted into lipid vesicles. Specific opiate binding was restored to soluble receptor preparations after supplementation with a brain lipid extract, and dilution below the effective detergent concentration. Saturable and specific opiate binding was measured for both membrane and vesicle preparations; dissociation constants (Kd) obtained from saturation isotherms of [3H]bremazocine binding were 1.3 and 4.2 nM, respectively. Relative affinity (IC50) values of ligand binding measured for subtype-selective agonists confirmed that a delta-opiate binding site interaction was recovered in vesicle preparations. Changes in agonist binding affinity noted for these experiments were explained by dissociation of the GTP-binding protein Gi from the receptor in detergent. The recovery of solubilized opiate receptors was nearly quantitative, and strictly dependent upon the total brain lipid preparation used in the reconstitution. Ligand binding was incompletely recovered after substituting pure, vesicle-forming phospholipid preparations. [3H]Bremazocine binding was also reconstituted after lectin affinity chromatography of solubilized receptor preparations, using conditions which likely effect the removal of endogenous lipid cofactors. A photoaffinity cross-linking methodology was employed to verify recovery of the delta-opiate receptor after its solubilization from membranes and reconstitution. Two membrane-associated proteins (50 and 70 kDa) were covalently tagged with an azido analog of β-endorphin(Leu5) in cell membranes and subsequently identified by immunoblotting with antisera directed against this opioid. Labeling of the 50-kDa polypeptide was prevented by coincubating assay samples with a relative excess of (D-Pen2,5)enkephalin. This opioid binding polypeptide was also present in solubilized/ reconstituted receptor preparations.

Original languageEnglish (US)
Pages (from-to)15176-15182
Number of pages7
JournalJournal of Biological Chemistry
Volume265
Issue number25
StatePublished - Sep 5 1990

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Opiate Alkaloids
delta Opioid Receptor
Opioid Receptors
Binding Sites
Detergents
Lipids
Cell membranes
Opioid Analgesics
Brain
Cell Membrane
Ligands
Endorphins
Membranes
Affinity chromatography
Recovery
Digitonin
Peptides
Enkephalins
Bile Acids and Salts
Affinity Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reconstitution of solubilized delta-opiate receptor binding sites in lipid vesicles. / Scheideler, Mark A.; Zukin, R. Suzanne.

In: Journal of Biological Chemistry, Vol. 265, No. 25, 05.09.1990, p. 15176-15182.

Research output: Contribution to journalArticle

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