The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum.

E F Kirkness, A J Turner

The Biochemical journal 1986 Jan 1

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The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid.

Citation

E F Kirkness, A J Turner. The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum. The Biochemical journal. 1986 Jan 1;233(1):265-70