电鳐(Narcine timlei)乙酰胆硷受体的分离纯化  

ISOLATION AND PURIFICATION OF THE ACETYLCHOLINE RECEPTOR FROM NARCINE TIMLEI

作　　者：龚潮梁 孙欣 潘淑英 何其伟[1] 宋世廉 Gong Chao-liang;Sun Xin;Pan Shu-ying;He Qi-wei;Song Shi-lian(Kunming Institute of Zoology,Academia Sinica)

机构地区：[1]中国科学院昆明动物研究所

出　　处：《Zoological Research》1981年第S01期51-57,共7页动物学研究（英文）

摘　　要：丁氏双鳍电鳐(Narcine timlei)新鲜电器官的匀浆,经冷冻干燥制成冻干粉(每克冻干粉相当于17.5克新鲜电器官)。10克冻干粉的细胞膜碎片用含1%Triton X-100的溶液提取,此悬浮液经100,000g离心,取其上清液对Sepharose-4B-眼镜蛇神经毒素进行亲和吸附。每毫升压缩的亲和胶含有华南眼镜蛇(Naja naja atra)的神经毒素0.25mg。吸附后的亲和凝胶用几种缓冲液洗去非特异性吸附的蛋白,最后用含1M氨基甲酰胆硷的溶液解脱乙酰胆硷受体(AchR)。有含AchR的氨基甲酰胆硷解脱液对含0.02%去氧胆酸钠的磷酸缓冲液充分透析,以除去氨基甲酰胆硷。从10克冻干粉中最后得到约10mg纯化的AchR。 提纯的AchR能与^(131)I-眼镜蛇神经毒素结合,生成的AchR-^(131)I-眼镜蛇神经毒素复合物用Sephadex G-200凝胶过滤分开后,求得其比结合力为4.7nmol/mg蛋白。此种结合能为眼镜蛇神经毒素、d-筒箭毒和十甲鎓所抑制,当这些竞争性抑制剂的浓度为^(131)I-眼镜蛇神经毒素浓度的300倍时,其抑制率分别为88%、48%和55%。提纯的AchR在含有0.1%Triton X-100的聚丙烯酰胺凝胶电泳时,显示单一的区带;乙酰胆硷酯酶活力很低,其比活力仅为0.05OD_(412)/分·mg蛋白。Fresh electric organ of Narcine timid was homogenized and then lyophi-lized ( 1 g of the lyophilized pellet epuals 17.5g of Narcine timid electro-plax).The cell membrane fragmemts of 10g lyophilized pellet were extracted in a solution containing 1% Triton X-100. This suspension was then centrifuged at 100,000g. The supernatant was adsorbed to Sepharose-4B-cobra neurotoxin affinity gel. The concentration of the neurotoxin fron Naja naja aira venom was 0.25 mg per ml packed affinity gel. The adsorbed affinity gel was eluted with several buffers, to remove nonspecifically bound proteins. Finally, the acetylcholine receptor (AchR) was desorbed with a solution containg 1M carba-chol. The solution containing AchR was dialysed extensively in phosphate buffer containing 0.02% sodium deoxycholate to remove carbachol. The solution containing AchR was then concentrated. About 10 rng of purified AchR was obtained from lOg lyophilized pellet. The purified AchR was able to bind with 131I-cobra neurotoxin. When the AchR-131I-cobra neurotoxin complex was separated by gel filtration on Sepha-dex G-200, the specific binding activity was calculated to be 4.7nmol/mg protein. This binding capacity was blockaded by cobra neurotoxin, d-tubocu-raine or decamethonium. The inhibition ratio was 88%, 48%, and 55% respectively when the concentration of these competitive inhibitors were 300-fold over that of 131I-cobra neurotoxin. The purified AchR shows a single band on polyacrylamide gel (containing 0.1% Triton X-100) electrophoresis. Acctyl-cholinesterase activity of the purified AchR is very low, the specific activity is 0.05 OD412nm/min. mg protein.

关 键 词：眼镜蛇神经毒素 乙酰胆硷 氨基甲酰 竞争性抑制剂 去氧胆酸钠 非特异性吸附 ACHR 冻干粉 

分 类 号：R74[医药卫生—神经病学与精神病学]