Size-exclusion chromatographic reconstitution of the bovine brain benzodiazepine receptor. Effects of lipid environment on the binding characteristics.
The benzodiazepine receptor from calf brain was solubilized with sodium deoxycholate (2 mg/ml) in the presence of 0.5 M KCl and protease inhibitors, and bound flunitrazepam with an equilibrium dissociation constant (Kd) of 2.7 +/- 1.2 nM and with 0.40 +/- 0.04 pmol binding sites per mg protein (Bmax). Up to 60% of the benzodiazepine binding sites (average 25%) could be reconstituted in lipid vesicles, upon size-exclusion chromatography of protein-detergent-lipid mixtures on Sephadex G-50 Medium for detergent depletion. The flunitrazepam affinity for the reconstituted receptor varied with the lipid composition (Kd 1.4-4 nM). Freezing and thawing increased the size of the small proteoliposomes obtained by chromatographic reconstitution and, on the average, doubled the number of operative flunitrazepam binding sites. When the proteoliposomes were stored at -20 degrees C or -80 degrees C or in lyophilized state, the receptor retained its benzodiazepine binding affinity and Bmax over a period of 2 months.
Viel
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GT
Groningen Institute for Drug Studies (GIDS), Department of Analytical Chemistry and Toxicology, The Netherlands.
Yang
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Q
Lundahl
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Ensing
K
K
de Zeeuw
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RA
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D002850:Chromatography, Gel; D003840:Deoxycholic Acid / Q000737:chemistry; D003902:Detergents; D005445:Flunitrazepam / Q000378:metabolism; D005615:Freezing; D018757:GABA Modulators / Q000378:metabolism; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry; D011485:Protein Binding; D011963:Receptors, GABA-A / Q000302:isolation & purification* / Q000378:metabolism; D012710:Serum Albumin, Bovine
D003902:Detergents; D018757:GABA Modulators; D008055:Lipids; D008081:Liposomes; D011963:Receptors, GABA-A; D003840:Deoxycholic Acid; D012710:Serum Albumin, Bovine; D005445:Flunitrazepam