Abstract
Recombinant mouse 18 kDa peripheral-type benzodiazepine receptor (PBR) protein was overexpressed in Escherichia coli and isolated using a His. Bind metal chelation resin. Recombinant PBR protein was purified with sodium dodecyl sulfate and reincorporated into liposomes using Bio-Beads SM2 as a detergent removing agent. Negative staining of the reconstituted PBR samples, examined by electron microscopy, showed the formation of proteoliposomes. Freeze-fracture of these proteoliposomes revealed the presence of transmembranous particles of an average size of 3.5 +/- 0.25 nm, consistent with the presence of a monomeric form of the recombinant PBR protein. The reconstituted protein exhibited the ability to bind both the PBR drug ligand isoquinoline carboxamide PK 11195 and cholesterol with nanomolar affinities. These data suggest that a PBR monomer is the minimal functional unit, binding drug ligands and cholesterol.
Copyright 2001 Academic Press.
Publication types
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Benzodiazepinones / metabolism
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Cholesterol / metabolism
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Chromatography
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Detergents / chemistry
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Escherichia coli
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Freeze Fracturing
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Isoquinolines / metabolism
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Ligands
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Lipid Bilayers / chemistry
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Mice
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Particle Size
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Porins / metabolism
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Protein Binding / physiology
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Protein Isoforms / chemistry
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Protein Isoforms / metabolism
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Protein Isoforms / ultrastructure
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Proteolipids / chemistry
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Proteolipids / ultrastructure
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Radioligand Assay
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Receptors, GABA-A / chemistry*
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Receptors, GABA-A / metabolism
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Receptors, GABA-A / ultrastructure
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / metabolism
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Recombinant Fusion Proteins / ultrastructure
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Sodium Dodecyl Sulfate / chemistry
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Voltage-Dependent Anion Channels
Substances
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Benzodiazepinones
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Detergents
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Isoquinolines
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Ligands
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Lipid Bilayers
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Porins
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Protein Isoforms
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Proteolipids
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Receptors, GABA-A
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Recombinant Fusion Proteins
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Voltage-Dependent Anion Channels
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proteoliposomes
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4'-chlorodiazepam
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Sodium Dodecyl Sulfate
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Cholesterol
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PK 11195