Partial purification of nicotinic cholinergic acetylcholine receptor in rat brain

• Published in: Japanese Journal of Pharmacology 1988, Vol.46 (suppl), p.104-104
• Main Authors: Nakayama, Hitoshi, Shirase, Masako, Nakashima, Toshikatsu, Kurogochi, Yutaka
• Format: Article
• Language: English
• ISSN: 0021-5198; 1347-3506
• DOI: 10.1016/S0021-5198(19)57156-6

The nicotinic cholinergic acetylcholine receptor (nAChR) in chick and rat brains have been reported to be purified by monoclonal antibody affinity chromatography. To purify the receptor by ligand affinity chromatography, we have investigated the assay conditions of solubilized nAChR and adsorption conditions of the receptor on DE-52, hydroxylapatite, concanavaline-A-Sepharose, carbachol-Affi-Gel and bromoacetylcholine-Affi-Gel columns. Bromoacetylcholine-Affi-Gel was prepared from Affi-Gel 401 and bromoacetylcholine. Acetylcholine binding sites in the membrane fraction of rat brains were solubilized with 1.5% Lubrol PX. After the solubilized membrane fraction of rat brains was incubated with ^^3 H-ACh in the presence of atropine and eserine ^^3 H-ACh bound to the solubilized components was assayed by filtration through Whatman GF/B glass filter presoaked in 0.3% polyethylenimine. Adsorption of ACh binding sites on concanavaline-A-Sepharose was found to decrese the binding of ^^3 H-ACh on the binding sites. ACh binding sites were adsorbed on DE-52, hydroxylapatite, concanavaline-A-Sepharose and bromoacetylcholine-Affi-Gel but not carbachol-Affi-Gel. ACh binding sites adsorbed on bromoacetylcholine-Affi-Gel was eluted with 0.1 M carbachol. Bromoacetyl-choline-Affi-Gel column chromatography is useful to purify nAChR in the rat brain.