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研究生:顏岑晏
研究生(外文):Tsen-Yann Yen
論文名稱:NMDAreceptors結合蛋白群之研究
論文名稱(外文):Biochemical Characterization of NMDA receptors Multiprotein Complexes
指導教授:吳宗遠
指導教授(外文):Tzong-Yuan Wu
學位類別:碩士
校院名稱:國立嘉義大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:68
中文關鍵詞:N-甲烷基-D-天冬胺酸受器麩胺酸受器
外文關鍵詞:NMDA receptorglutamate receptor
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摘要
NMDA receptor ( N-methyl-D-aspartate receptor ) 結合蛋白群是一群相當龐大的蛋白質,這群蛋白質分別形成receptor、adaptor、signaling和cytoskeletal等。因為NMDA receptor的離子通道可調控它們的激酶 ( kinases )、去磷酸酵素 ( phosphatase ) 和其他的酵素,進一步調控AMPA receptors ( α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor )、spine cytoskeletal changes、轉譯 ( translation )、轉錄 ( transcription ) 以及其他的改變。這些現象都顯示NMDA receptor連接多種細胞內的訊息傳遞路徑。因此,對於NMDA receptor complexes這一群蛋白之間的生化特性了解越多,就更加清楚了解他們之間的交互作用。
在先前的研究指出,以新鮮豬腦為研究材料,能夠以Triton X-114/KCl選擇性的溶解出AMPA receptor,而NMDA receptor卻不能輕易的以Triton X-114/KCl所選擇性的溶解出來。我們實驗的目的是希望能夠建立一套有效分離純化NMDA receptor以及與其結合蛋白群的系統,並且分析它們的生化特性。
本實驗是以新鮮老鼠腦為材料,先製備成為crude synaptosome,再以Triton X-114/KCl ( 分為不含SDS、含0.25%的SDS、含0.5%的SDS ) 來進行phase separation,結果發現在不含SDS的情況下會有沉澱的現象,而含0.25%和0.5% SDS的情況下沉澱物體積有越來越少,甚至沒有沉澱物的出現。接著以1-dimensional polyacrylamide gel electrophoresis ( 1D-PAGE 一維電泳法 ) 和Western blotting進行分析經phase separation的樣品,結果顯示NMDA receptor和與其結合蛋白群可被Triton X-114/KCl在SDS存在下選擇性的溶解出來。同時在silver staining ( 銀染法 ) 的結果之中,我們也發現一些有趣的現象,在不同條件下,aqueous phase和detergent phase中部分的蛋白質分佈有明顯的變化。經蔗糖梯度離心的分析顯示,NMDA receptor結合蛋白群在0.5% SDS存在下的基本構造單元為NR1+NR2+CaMKⅡ+PSD95。

Abstract
MNDA receptor ( N-methyl-D-aspartate receptor ) associated protein is a large protein. This group of protein forms respectively receptor, adaptor, signaling, cytoskeletal and so on. The ion channel of NMDA receptor may control their kinases, phosphatase and other enzymes. Furthermore, they also control AMPA receptors ( α-amino-3-hydroxy-5methyl-4-isoxazolepropionic acid receptor ), apine cytoskeletal chantes, translation, transcription, as well as other changes. Therefore, the more we understand about the biochemical characteristics of this group of proteins, the more we are clear about their correlations.
Former research pointed out that, by using fresh pig brains as the research material, Triton X-114/KCl might dissolve AMPA receptor, meanwhile, NMDA receptor could not easily be dissolved with Triton X-114/KC1. The objective of our research is to establish a system that effectively isolates and purifies NMDA receptor and its associated protein, and analyzes their biochemical characteristics.
We used fresh mouse’s brain as this research’s material. First of all, we made and prepared it into crude synaptosome, and then we used Triton X-114/KC1 ( including not containing SDS, containing 0.25% of SDS, and containing 0.5% of SDS ) to carry out the phase separation. There are precipitations that under not containing SDS, but less and less, even no precipitation that under 0.25%, 0.5% of SDS. By using 1-dimensional polyacrylamide gel electrophoresis ( 1D-PAGE-electrophoresis ) and Western blotting analysis, the result showed that NMDA receptor and its associated protein were also dissolved with Triton X-114/KC1 and SDS. At the same time, we also found some interesting phenomena from the result of silver stain. Under different conditions, there were significant changes in the distribution of proteins in aqueous phase and detergent phase. Finally, we used sucrose gradient centrifugation, and we found that the basic structure of NMDA receptor associated protein with the existence of 0.5% SDS was NR1+NR2+CaMKII+PSD95.

目錄
英文摘要-----------------------------------------------------1
中文摘要-----------------------------------------------------3
緒論---------------------------------------------------------5
材料和方法--------------------------------------------------11
(一) crude synaptosome的製備----------------------------11
(二) 蛋白質濃度的測定-----------------------------------12
(三) phase separation-----------------------------------14
(四) 電泳分析 ( SDS-PAGE )------------------------------18
(五) 銀染法---------------------------------------------22
(六) 西方轉漬法-----------------------------------------24
(七) Chloroform-methanol protein precipitation----------27
(八) 蔗糖梯度離心---------------------------------------28
結果--------------------------------------------------------30
討論--------------------------------------------------------39
圖表--------------------------------------------------------43
參考文獻----------------------------------------------------64
附錄--------------------------------------------------------68

參考文獻
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13. Stefan Strack and Roger J. Colbran (1998) Autophosphorylation-dependent Tragenting of Calcium/Calmodulin-dependent Protein Kinase Ⅱ by the NR2B Subunit of the N-Methyl-D-aspartate Receptor. The journal of Biological Chemistry 273:20689-30692.
14. S. P. Yu, C.-H. Yeh, U. Strasser, M. Tian, D. W. Choi (1999) NMDA Receptor-Mediated K+ Efflux and Neuronal Apoptosis. SCIENCE 284:336-339.
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16. Ying Lu, Sharon Grady, Michael J. Marks, Marina Picciotto, Jean-Pireer Changeux and Allan C. Collins (1998) Pharmacological Characterization of Nicotinic receptor-stimulated GABA Release From Mouse Brain synaptosome. The Journal of Pharmacology and Experimental Therapeutics 287:648-657.

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