Reconstitution of beta-adrenergic receptors into phospholipid vesicles: Restoration of [125I]Iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

J. W. Fleming, E. M. Ross

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

β-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 Å in diameter, and had a lipid content of 6 μmol phospholipid/mg protein. Specific binding of the β-adrenergic ligand [3H]dihydroalprenolol ([3H]DHA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [K(d)=84 pM] and competed appropriately with (+) and (-) isomers of β-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of α-adrenergic receptor function in a defined lipid milieu.

Original languageEnglish (US)
Pages (from-to)407-419
Number of pages13
JournalJournal of Cyclic Nucleotide Research
Volume6
Issue number6
StatePublished - 1980

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Digitonin
Receptors, Adrenergic, beta
Detergents
Restoration
Phospholipids
Adrenergic Receptors
Dihydroalprenolol
Ligands
Dimyristoylphosphatidylcholine
Lipids
Adrenergic Agonists
Adrenergic Antagonists
Ultracentrifugation
Erythrocyte Membrane
Cell membranes
Isomers
Adrenergic Agents
Rats
Proteins
Cell Membrane

ASJC Scopus subject areas

  • Medicine(all)

Cite this

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title = "Reconstitution of beta-adrenergic receptors into phospholipid vesicles: Restoration of [125I]Iodohydroxybenzylpindolol binding to digitonin-solubilized receptors",
abstract = "β-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 {\AA} in diameter, and had a lipid content of 6 μmol phospholipid/mg protein. Specific binding of the β-adrenergic ligand [3H]dihydroalprenolol ([3H]DHA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [K(d)=84 pM] and competed appropriately with (+) and (-) isomers of β-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of α-adrenergic receptor function in a defined lipid milieu.",
author = "Fleming, {J. W.} and Ross, {E. M.}",
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pages = "407--419",
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T2 - Restoration of [125I]Iodohydroxybenzylpindolol binding to digitonin-solubilized receptors

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AU - Ross, E. M.

PY - 1980

Y1 - 1980

N2 - β-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 Å in diameter, and had a lipid content of 6 μmol phospholipid/mg protein. Specific binding of the β-adrenergic ligand [3H]dihydroalprenolol ([3H]DHA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [K(d)=84 pM] and competed appropriately with (+) and (-) isomers of β-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of α-adrenergic receptor function in a defined lipid milieu.

AB - β-adrenergic receptors were solubilized from rat erythrocyte plasma membranes using digitonin. Solubilized receptors were then reconstituted into phospholipid vesicles by the addition of dimyristoylphosphatidylcholine and removal of detergent. Vesicles were separated from residual soluble receptors and detergent by rate-zonal ultracentrifugation. Vesicles were monolamellar, 500-900 Å in diameter, and had a lipid content of 6 μmol phospholipid/mg protein. Specific binding of the β-adrenergic ligand [3H]dihydroalprenolol ([3H]DHA) was 0.9-1.9 pmol/mg protein. Reconstitution of receptors into vesicles restored their ability to bind [125I]iodohydroxybenzylpindolol ([125I]IHYP). This ligand does not bind to detergent-solubilized receptors. [125I]IHYP binding was saturable [K(d)=84 pM] and competed appropriately with (+) and (-) isomers of β-adrenergic agonists and antagonists. These receptor vesicles therefore appear to be an excellent model system for the study of α-adrenergic receptor function in a defined lipid milieu.

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