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Solubilization and characterization of σ-receptors from guinea pig brain membranes

Solubilization and characterization of σ-receptors from guinea pig brain membranes
Solubilization and characterization of σ-receptors from guinea pig brain membranes
The σ-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for σ-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective ρ-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 ± 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(l-propyl)piperidine {(+)-[3H]3-PPP} (KD = 36 ± 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a ρ-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the σ-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macro-molecular complex.
0022-3042
1575-1580
Kavanaugh, M.P.
6713d148-1581-499e-b488-e95c6044eefb
Parker, Joel D.
31d7a4f1-e316-43da-9d09-c183b5388eb5
Bobker, D.H.
478ae36b-b573-4afb-bbaa-753491f232c5
Keana, J.F.W.
038f6329-91b0-4078-a9a1-4497c4f5fb0d
Weber, E.
989e8ee2-b080-4c36-972b-c6928a6f1516
Kavanaugh, M.P.
6713d148-1581-499e-b488-e95c6044eefb
Parker, Joel D.
31d7a4f1-e316-43da-9d09-c183b5388eb5
Bobker, D.H.
478ae36b-b573-4afb-bbaa-753491f232c5
Keana, J.F.W.
038f6329-91b0-4078-a9a1-4497c4f5fb0d
Weber, E.
989e8ee2-b080-4c36-972b-c6928a6f1516

Kavanaugh, M.P., Parker, Joel D., Bobker, D.H., Keana, J.F.W. and Weber, E. (1989) Solubilization and characterization of σ-receptors from guinea pig brain membranes. Journal of Neurochemistry, 53 (5), 1575-1580. (doi:10.1111/j.1471-4159.1989.tb08554.x).

Record type: Article

Abstract

The σ-receptor, a distinct binding site in brain tissue that may mediate some of the psychotomimetic properties of benzomorphan opiates and phencyclidine, has been solubilized using the ionic detergent sodium cholate. Binding assays were performed with the solubilized receptor using vacuum filtration over polyethyleneimine-treated glass fiber filters. The pharmacological specificity of the solubilized binding site for σ-receptor ligands is nearly identical to the membrane-bound form of the receptor, with the order of potencies for displacement of the selective ρ-ligand [3H]di-o-tolylguanidine ([3H]DTG) closely correlated. The stereoselectivity for (+)-benzomorphan opiate enantiomers was retained by the solubilized receptor. The soluble receptor retained high affinity for binding of [3H]DTG (KD = 28 ± 0.5 nM) and (+)-[3H]3-(3-hydroxyphenyl)-N-(l-propyl)piperidine {(+)-[3H]3-PPP} (KD = 36 ± 2 nM). Photoaffinity labeling of the solubilized receptor by [3H]p-azido-DTG, a ρ-selective photoaffinity label, resulted in labeling of a 29-kilodalton polypeptide identical in size to that labeled in intact membranes. Estimation of the Stokes radius of the [3H]DTG binding site was obtained by Sepharose CL-6B chromatography in the presence of 20 mM cholate and calculated to be 8.7 nm. This value was identical to the molecular size found for the binding sites of the σ-selective ligands (+)-[3H]3-PPP and (+)-[3H]SKF-10,047, supporting the hypothesis that all three ligands bind to the same macro-molecular complex.

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Published date: November 1989
Organisations: Biological Sciences

Identifiers

Local EPrints ID: 143689
URI: http://eprints.soton.ac.uk/id/eprint/143689
ISSN: 0022-3042
PURE UUID: 06a721b6-4c5f-46e7-89be-48b06772dcbf

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Date deposited: 21 Jul 2010 10:47
Last modified: 14 Mar 2024 00:44

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Contributors

Author: M.P. Kavanaugh
Author: Joel D. Parker
Author: D.H. Bobker
Author: J.F.W. Keana
Author: E. Weber

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