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研究生:蔡侑意
研究生(外文):saprinatsai
論文名稱:豬腦後突觸質密區鋅離子結合蛋白之生化研究
論文名稱(外文):Biochemical study of the zinc binding proteins of the postsynaptic density fromn porcine brain
指導教授:張兗君
學位類別:碩士
校院名稱:國立清華大學
系所名稱:生命科學系
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:英文
論文頁數:77
中文關鍵詞:後突觸質密區鋅離子結合蛋白Zn-NTA column
外文關鍵詞:postsynaptic densityZinc binding proteinsZn-NTA column
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摘要
後突觸質密區postsynaptic density (PSD)為一功能性活化區域,其參與哺乳類中樞神經系統的興奮性神經傳導功能。經過尿素處理可將PSD裂解成較小結構而且這些較小結構並不會自行重組回原來之PSD。我們以感應耦合電漿質譜分析儀定量方法分析得知PSD中有鋅離子存在而且其濃度為4.1 nmol/mg。
我們也發現大部分經由8M 尿素處理並且加以透析之PSD會與Zn-NTA (Zn-Nitrilotriacetic acid) 樹脂結合。我們便利用不同之試劑(Tris-HCl, urea, imidazole, guanidine hydrochloride, SDS, glycerol, 及Triton)來沖洗Zn-NTA column。經過透析處理移除尿素之PSD在被不同試劑沖洗下來後,藉由外加鋅離子我們觀察到用尿素這步驟沖洗下之PSD蛋白質以及與尿素沖下之PSD蛋白質混合之其他試劑沖下之蛋白質這些小結構都會形成不同緻密程度之聚合物。最後利用電子顯微鏡觀察這些聚合物後得知這些聚合物都與原來PSD結構不同。根據以上這些結果可推測,鋅離子可能與由Zn-NTA沖洗步驟中的尿素沖洗下來之鋅離子結合蛋白結合來引發PSD聚合作用。鋅離子結合蛋白可能在PSD形成以及其結構之維持上扮演重要角色

ABSTRACT
The postsynaptic density (PSD) is a functionally active zone involved in excitatory synaptic transmission in mammalian central nerve system. Inductively Coupled Plasma-Mass (ICP-MASS) analyses reveal that the isolated PSD proteins contain zinc at a concentration of 4.1 nmol/mg. A treatment of 8M urea leads to the dissociation of the PSDs into small components and depletion of most of the zinc ions bound to the PSD. Removing urea by dialysis, dissociated PSD proteins do not reassemble into aggregates by themselves.
We also find that the majority of the 8M urea treated and dialyzed PSD proteins are bound to a Zn-Nitrilotriacetic acid (Zn-NTA) column. We then elute the column with different reagents (Tris-HCl, urea, imidazole, guanidine hydrochloride, SDS, glycerol, or Triton). After removing urea by dialysis, the PSD proteins eluted from Zn-NTA column with urea can form aggregates in the presence of zinc ions. The mixtures containing the PSD proteins eluted with urea and other reagents from a Zn-NTA column can also form aggregates with different degrees of compactness. Morphological studies indicate that the resultant zinc-induced aggregates are not identical to the original PSD. Our results suggest that the zinc ions bound to certain zinc binding proteins be eluted with urea from Zn-NTA column. The zinc binding proteins may play important roles in the formation and maintenance of the structure of the PSD.

CONTENTS
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CONTENTS------------------------------------------------1
ACKNOWLEDGEMENT-----------------------------------------2
ABSTRACT------------------------------------------------3-4
INTRODUCTION--------------------------------------------5-14
METHOD AND MATERIAL------------------------------------15-25
RESULT-------------------------------------------------26-32
DISCUSSION---------------------------------------------33-36
REFERENCES---------------------------------------------37-48
TABLES-------------------------------------------------49-51
FIGURES AND FIGURE LEGEND -----------------------------54-77
---------------------------------------------------------------

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