AFM观察胆碱类底物对AChE四聚体亚分子结构的影响  

AFM observation of the effects of choline analogic substrates on the submolecular structure of acetylcholinesterase tetramer

作　　者：姜爽[1] 张英鸽[1] 张飒[2] 

机构地区：[1]军事医学科学院毒物药物研究所 国家纳米科学中心协作实验室,北京100850 [2]国家生物医学分析中心,北京100850

出　　处：《军事医学科学院院刊》2009年第3期208-212,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家重点基础研究计划(“973”计划)资助项目(2006CB933304);国家高技术研究发展计划(“863”计划)资助项目(2006AA03Z333);北京市科委重点计划项目(D07010201160703)

摘　　要：目的:为探索乙酰胆碱酯酶(AChE)活性中心狭隙与其水解底物高效性相适应的可能机制,研究胆碱类底物对AChE四聚体(AChE G4)亚分子结构的影响。方法:冰浴超声法制备磷脂膜,囊泡融合技术将AChE重组到以云母为支撑的磷脂膜上,原子力显微技术(AFM)观察AChE分别与乙酰胆碱(ACh)和丁酰胆碱(BCh)作用前后的亚分子结构。结果:单个AChE G4颗粒是表面光滑、中间突出的椭圆形结构,亚基排列紧密,平均大小为[(89±7)nm×(68±9)nm×(6±3)nm,长×宽×高,n=100]。与底物反应后,可清楚地看到亚基排列松散,酶蛋白是由4个亚基组成,亚基之间形成一个无阻碍空间,这与X线晶体学和分子动力学研究结果相一致。与S-ACh反应后,单个AChE G4颗粒平均大小为[(104±7)nm×(91±5)nm×(8±2)nm,长×宽×高,n=100],无阻碍空间大小为[(60±5)nm×(51±9)nm,长×宽,n=30];与S-BCh反应后,酶的大小为[(100±5)nm×(87±6)nm×(7±3)nm,长×宽×高,n=100],无阻碍空间大小为[(38±4)nm×(35±2)nm,长×宽,n=30]。结论:胆碱类底物对AChE G4亚分子结构具有明显影响,酶亚分子结构变化与其水解底物的高效性是一致的。Objective：To explore the possible mechanism of adaptation of the active center gorge of acetylcholinesterases （ACHE） to the high efficiency of its hydrolyzsis substrates, and study the effects of choline analogic substrates on the submolecular structures of G4 AChEs. Methods ： Ice-bath ultrasound was used to prepare phospholipid membrane. Ves-fusion technique was applied to the reconstitution of G4 AChE in the phospholipid membrane on mica. The submolecular structures of G4 AChEs reconstituted in the phospholipid membrane were imaged with atomic force microscope （AFM） before and after reacted with S-acetylcholine （S-ACh） or S-butyrocholine （S-BCh）. Results： Single G4 AChE particle was ellipsoid in shape, and had smooth surface with a central projection and clear border, with a mean size of （89 ± 7 ） nm long, （68 ± 9） nm wide and （6 ± 3 ）nm height ,n = 100. The sizes of enzymes after reacted with substrates were larger than those before reaction, with mean sizes of （ 104 ± 7） nm long, （ 91 ± 5 ） nm wide and （ 8 ± 2 ） nm height （ after reacted with S-ACh） and （ 100 ±5）nm long, （87 -＋6）nm wide and（7 ±3）nm height （after reacted with S-BCh）, n = 100. The arrangement of subunits of single G4 AChE was loose after reacted with substrate, the enzyme protein composed of four subunits was seen under AFM and there was an apparent free space between the subunits of the enzyme, which was consistent with the results of the X-ray diffraction crystallography and molecular dynamics study. The protein particles after reacted with S-ACh were mainly distributed in the defects of membranes, and the apparent free space in the center of G4 AChE was （60 ± 5 ）nm long and （51 ± 9） nm wide （ n = 30）, while the protein particles after reacted with S-BCh remain to be distributed in the membranes, the apparent free space in the center of G4 AChE was （ 38 ± 4 ）nm long and （ 35 ± 2） nm wide （ n = 30）. Conclusion ：There are obvious effects of

关 键 词：乙酰胆碱酯酶四聚体 原子力显微技术 囊泡融合 

分 类 号：Q556[生物学—生物化学]