Solubilization and partial purification of GABAB receptor from bovine brain.
gamma-Aminobutyric acid (GABA)B receptor has been solubilized and partially purified by an affinity column chromatography. GABAB receptor was solubilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) in the presence of asolectin. The solubilized GABAB receptor was adsorbed on baclofen-coupled epoxy-activated Sepharose 6B. The affinity matrix adsorbed 80% of the solubilized [3H]GABA binding activity to GABAB receptor, and approximately 75% of the adsorbed activity could be eluted with 1 M KC1. GABAB receptor binding in the fraction eluted from affinity column was displaced by GABA, baclofen and 2-hydroxy saclofen in a dose-dependent manner. Furthermore, the purified GABAB receptor showed approximately 2800-fold purification as compared with the original solubilized fraction and possessed the specific binding activity of 17.68 p mol/mg of protein. This binding consisted of a single binding site with a dissociation constant of 64.4 nM. The present results indicate that affinity column chromatographic procedures using baclofen-coupled epoxy-activated Sepharose 6B are suitable for the partial purification of GABAB receptor from cerebral tissues.
Ohmori
Y
Y
Department of Pharmacology, Kyoto Prefectural University of Medicine, Japan.
Kuriyama
K
K
D000818:Animals; D001418:Baclofen / Q000494:pharmacology; D001667:Binding, Competitive; D002417:Cattle; D002540:Cerebral Cortex / Q000378:metabolism*; D002846:Chromatography, Affinity; D007700:Kinetics; D011963:Receptors, GABA-A / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility; D013570:Synaptic Membranes / Q000378:metabolism*; D005680:gamma-Aminobutyric Acid / Q000378:metabolism
D011963:Receptors, GABA-A; D005680:gamma-Aminobutyric Acid; D001418:Baclofen