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研究生:陳筑瑄
研究生(外文):Chu-Hsuan Chen
論文名稱:鯉魚腎泌分解之內生性抑制蛋白之純化
論文名稱(外文):Purification of an Endogenous Inhibitor of Carp Nephrosin
指導教授:張震東張震東引用關係
指導教授(外文):Geen-Gong Chang
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:生化科學研究所
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:1999
畢業學年度:87
語文別:中文
論文頁數:60
中文關鍵詞:腎泌分解內生性抑制蛋白鯉魚頭腎醣蛋白間質金屬蛋白間質金屬蛋白組織抑制蛋白複合體
外文關鍵詞:Carp Head KidneyNephrosinNephrosin inhibitorMatrix metalloproteinasetissue inhibitor of matrix metalloproteinaseMMPTIMPComplex
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腎泌分解(Nephrosin)是我們實驗室自鯉魚頭腎萃取液純化到的分泌性金屬耦合蛋白,分子質量約23 kDa,一級結構屬於astacin家族,主要分布於成體鯉魚的頭腎、腎臟及脾臟,是此家族成員中於淋巴造血器官中被發現的首例。腎泌分解可與細胞間質中的肝制凝素(Heparin)等黏多醣類(Glycosaminoglycans)結合而滯留其中。本篇報告中我們以肝制凝素親合力管柱(Heparin affinity column)於鯉魚腎臟純化到腎泌分解與其抑制蛋白之複合體,經酸性處理致使複合體解離,而純化到具活性的腎泌分解抑制蛋白。其分子質量40 kDa,帶有N-linked oligosaccharides,佔總分子量之25%。使用cross-linker可使腎泌分解與其抑制蛋白形成穩定複合體。若以N-glycosidase F去除抑制蛋白的N-linked oligosaccharides,形成複合體及抑制腎泌分解的能力均會減弱。間質金屬耦合蛋白(Matrix metalloproteinase;MMP)與astacin家族蛋白同屬zinc-endopeptidase,主要作用對象是細胞間質蛋白質。MMP的內生性抑制蛋白TIMP(Tissue inhibitor of matrix metalloproteinase)無法取代腎泌分解抑制蛋白對腎泌分解的抑制。由鯉魚組織調查發現游離的腎泌分解與抑制蛋白同時分布於頭腎、腎臟及脾臟,兩者的複合體則大量存在於心臟,顯示腎泌分解及其抑制蛋白除了與造血功能有密切關聯外,也在其他器官執行非造血功能。
Nephrosin is a newly discovered member of the astacin family, a group of secreted or membrane metalloproteinases found in the 1990s. It is the first astacin enzyme found in lymphohematopoietic tissues (kidney, head kidney, and spleen in fish). Carp nephrosin in its secreted form contains 198 amino acid residues whose predicted relative molecular weight is 23k. Four putative heparin binding motifs can be identified by which secreted nephrosin may be localized to the heparan sulphate proteoglycan present in extracellular matrix.
We reported here that a complex formed by nephrosin and its inhibitor was purified from carp kidney extract, by heparin affinity chromatography. By 1% acetic acid treatment, a biologically active inhibitor was released from the complex. Treatment with N-glycosidase F removed the N-linked carbohydrate moiety of nephrosin inhibitor and decreased the apparent molecular mass from 40 kDa to 30 kDa. Deglycosylation also decreased the binding affinity and the inhibitory activity of this inhibitor to nephrosin. The presence of nephrosin inhibitor in different tissues was examined by immunoblotting with a polyclonal antiserum against the purified nephrosin inhibitor. Both nephrosin and the nephrosin inhibitor are present mostly in the head kidney, kidney and spleen. However, immunoreactive proteins of 67 kDa and 72 kDa could also be detected by this antiserum in non-lymphohematopoitic tissues. The findings of an endogenous inhibitor of nephrosin present in a similar distribution as nephrosin suggests that nephrosin is highly regulated by this inhibitor. Therefore regulation of astacin enzymes involves similar mechanisms as in the case of the matrix metalloproteinases.
目錄………………………………………….……………….…..………………...I
中文摘要………………………………………………………………………….III
英文摘要………………………………………………………………………….IV
第一章前言1
第一節魚類頭腎1
第二節腎泌分解2
第三節astacin家族2
第四節間質金屬蛋白(matrix metalloproteinases)與間質金屬蛋白組織抑制蛋白(tissue inhibitor of matrix metalloproteinase)5
第五節尋找腎泌分解的內生性抑制蛋白8
第二章材料與實驗方法11
第一節實驗動物11
第二節實驗藥品11
第三節SDS聚丙烯醯胺凝膠電泳(SDS-PAGE)12
第四節蛋白質膠片染色13
第五節銀染色(Silver-stainig)13
第六節西方轉印法13
第七節Reverse Zymography14
第八節腎泌分解之純化15
第九節腎泌分解內生性抑制蛋白之純化16
第十節對角線雙向式電泳(Diagonal SDS-electrophoresis)18
第十一節蛋白質鍵結(cross-linking)19
第十二節腎泌分解酵素活性測定19
第十三節RCM-BSA之製備20
第十四節製備鯉魚腎泌分解內生性抑制蛋白之多株抗體20
第十五節鯉魚組織萃取液之製備21
第十六節酵素法移除N-link oligosaccharides22
第三章結果23
第一節腎泌分解抑制蛋白的純化23
第二節腎泌分解抑制蛋白的活性測試26
第三節腎泌分解抑制蛋白具有醣類結構27
第四節TIMP1能與腎泌分解結合但不能抑制其活性28
第五節腎泌分解抑制蛋白、腎泌分解與其複合體的抗體製備與組織分布調查28
第四章討論30
第一節腎泌分解抑制蛋白之純化策略30
第二節腎泌分解與其抑制蛋白形成非共價之緊密複合體31
第三節TIMP無法取代腎泌分解抑制蛋白之功能32
第四節腎泌分解可能與抑制蛋白的醣類結構結合33
第五節腎泌分解抑制蛋白及TIMP1的醣類結構可能與heparin類似33
第六節醣類分子對腎泌分解抑制蛋白之活性貢獻34
第七節腎泌分解抑制蛋白、TIMP1、腎泌分解形成homodimer之可能意義35
第八節腎泌分解與腎泌分解抑制蛋白之組織分布36
第九節結語 38
參考資料 40
圖及表 47
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