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 sodiu m deoxycholate (2 mg/ml) in the presence of 0.5 M KCl and protease inh ibitors, and bound flunitrazepam with an equilibrium dissociation cons tant (K-d) of 2.7+/-1.2 nM and with 0.40+/-0.04 pmol binding sites per mg protein (B-max). Up to 60% of the benzodiazepine binding sites (av erage 25%) could be reconstituted in lipid vesicles, upon size-exclusi on chromatography of protein-detergent-lipid mixtures on Sephadex G-50 Medium for detergent depletion. The flunitrazepam affinity for the re constituted receptor varied with the lipid composition (K-d 1.4-4 nM), Freezing and thawing increased the size of the small proteoliposomes obtained by chromatographic reconstitution and, on the average, double d the number of operative flunitrazepam binding sites. When the proteo liposomes were stored at -20 degrees C or -80 degrees C or in lyophili zed state, the receptor retained its benzodiazepine binding affinity a nd B-max over a period of 2 months. (C) 1997 Elsevier Science B.V.