臺灣博碩士論文加值系統

牡蠣組織之鋅含量常高於100μg/g fresh tissue，為知牡蠣之高鋅與鯉魚消化道組織之「鋅結合蛋白質」是否相同或相似，乃嘗試抽出並探討其鋅及鋅結合物質之分佈。由實驗得知本研究之牡蠣，經熱分解後其總鋅濃度為 91±30μg/g fresh tissue，其中有~80%之鋅可為 EDTA抽取。由此可知牡蠣組織中之鋅約有80％是鬆散地與物質結合，是可游離的鋅；而只有~20％的鋅是與其他物質緊密地結合在一起。經次細胞分畫後，得知牡蠣之鋅主要存在於nuclei/cell debris fraction，其平均鋅濃度為 52±17μg/g fresh tissue，約佔整體鋅含量的80％，與鯉魚消化道組織鋅的分佈情形類似，因此可以推測牡蠣組織中的主要鋅結合物質並非是水溶性的金屬蛋白質。以清潔劑 (lubrol) 抽取牡蠣組織 nuclei/cell debris fraction時，發現可抽出27％之「鋅結合物質」，當清潔劑中添加之 guanidine 達 2 M 以上時，可將抽出率提高至 84％，說明此「鋅結合物質」應為一種膜蛋白質，並且可能與細胞骨架相結合。以guanidine/lubrol extact經過Zn2+-IMAC處理，再進行 SDS-PAGE 電泳，發現在43 kDa的位置附近有一條明顯的band存在，但是immunoblotting卻沒有反應。另外亦發現牡蠣組織之鋅濃度與其 bound SH groups濃度、hydroxyproline濃度並無相關。牡蠣組織之「鋅結合蛋白質」對鯉魚 43 kDa「鋅結合蛋白質」之抗體，不呈免疫反應。綜合上述，可知牡蠣組織中主要與鋅結合之物質似乎亦為43 kDa之膜蛋白質，但是其生化特性與鯉魚消化道組織之 43 kDa「鋅結合蛋白質」似乎不盡相同，有待進一步研究。

The zinc concentration in oyster tissue are usually higher than 100μg/g fresh tissue. In order to understand whether the zinc and zinc-binding protein in oyster tissue were the same or similar to those in common carp’s digestive tract tissue, the distribution and purification of zinc and zinc-binding protein in oyster tissue were studied. It was found that the concentration of zinc was 91μg/(g fresh tissue) in whole oyster, and about ~80% of the zinc in oyster tissue could be extracted by EDTA solution. About ~80% of the zinc in oyster tissue might be bound loosely with some substance. After subcellular fractionation, it was found that 80% of the zinc existed mainly in the nuclei/cell debris fraction. The results suggested that the zinc distribution in oyster tissue was similar to that in common carp’s digestive tract tissue. The main zinc-binding substance in oyster tissue was not metallothionein. The nuclei/cell debris fraction of oyster tissue was extracted with detergent (lubrol), and it was found that only 27% of the zinc could be extracted. However, adding 2 M guanidine in lubrol could improve the extraction rate to 84%. The results showed that the zinc-binding substance in oyster tissue might be a membrane protein, and connected to extracellular matrix. Guanidine/lubrol extract of the nuclei/cell debris fraction of oyster tissue was purified with Zn2+- IMAC, and subjected to SDS-PAGE and immunoblotting. SDS-PAGE showed a clear band in the 43 kDa position, however, no band was observed in immunoblotting. It was also found that the zinc concentration in the nuclei/cell debris fraction of oyster tissue had no relation to its bound SH groups concentration and hydroxyproline concentration. There was no immunoreaction between the antibody of 43 kDa zinc-binding protein from common carp’s digestive tract tissue to that of oyster tissue. The above mentioned results indicated that the zinc-binding protein in oyster was a 43 kDa membrane protein, but its biochemical characteristics might not the same as that in commom carp.

目 錄
中文摘要--------------------------------------------------------------------------II
英文摘要--------------------------------------------------------------------------IV
第一章 研究背景與目的-------------------------------------------------1
一、研究背景------------------------------------------------------------------1
二、研究目的------------------------------------------------------------------2
第二章 文獻整理------------------------------------------------------------3
一、牡蠣之簡介 3
二、鋅的生化功能 4
三、動物體內之鋅含量 4
四、牡蠣之鋅含量-------------------------------------------------------------5
五、鯉魚消化道組織之「鋅結合蛋白質」 5
六、牡蠣組織之「鋅結合物質」 6
第三章 材料與方法 7
一、實驗材料 7
二、牡蠣之次細胞分畫及hydroxyproline之測定 8
三、以熱分解法與EDTA法比較鋅之抽出及水分含量之測定-----10
四、以清潔劑lubrol抽出牡蠣組織中之「鋅結合物質」------------13
五、牡蠣之免疫化學測試---------------------------------------------------13
六、以IMAC (Immobilized metal affinity chromatography)
分離牡蠣組織中之「鋅結合物質」 ---------------------------------15
七、以SDS-Polyacrylamide electrophoresis (SDS-PAGE)
分析蛋白質 --------------------------------------------------------------17
八、Immunoblotting ----------------------------------------------------------18
九、化學測定 -----------------------------------------------------------------20
第四章 實驗結果 ---------------------------------------------------22
一、牡蠣組織之次細胞分畫 -----------------------------------------------22
二、牡蠣組織中鋅之結合型態 --------------------------------------------22
三、牡蠣組織nuclei/cell debris fraction與SH group及collagen之關係 -------------------------------------------------------------------------23
四、以清潔劑 (lubrol) 抽取牡蠣組織之「鋅結合物質」 ---------24
五、牡蠣組織之 lubrol 抽出液對鯉魚43 kDa「鋅結合蛋白質」抗體之免疫化學反應--------------------------------------------------------24
六、牡蠣組織 guanidine/lubrol extract「鋅結合物質」之純化 ---25
第五章 綜合討論 ---------------------------------------------------------27
表 -----------------------------------------------------------------------31
圖 -----------------------------------------------------------------------35
參考文獻 -----------------------------------------------------------------------45

表目錄
表一、牡蠣組織各次細胞區分之鋅濃度 (濕物重) 31
表二、牡蠣組織各次細胞區分之鋅濃度 (乾物重) 32
表三、以 EDTA 及熱分解法測定牡蠣組織之鋅濃度 33


圖目錄
圖一、牡蠣組織之次細胞分畫 35
圖二、鋅在牡蠣組織各次細胞區分之分佈 (濕物重) 36
圖三、鋅在牡蠣組織各次細胞區分之分佈 (乾物重) 37
圖四、牡蠣”核及細胞殘屑”區分之鋅濃度與 bound SH groups 濃度
之關係 38
圖五、牡蠣”核及細胞殘屑”區分之鋅濃度與 hydroxyproline 濃度
之關係 39
圖六、清潔劑 (lubrol) 中，添加不同濃度之urea或guanidine，對牡蠣組織之鋅結合物質之抽出效果 40
圖七、牡蠣組織之lubrol或guanidine/lubrol抽出液之免疫分析
結果 41
圖八、牡蠣組織之lubrol或guanidine/lubrol抽出液之免疫分析
結果 42
圖九、牡蠣組織之guanidine/lubrol抽出液以Zn2+-IMAC分析
之結果 43
圖十、以Zn2+-IMAC純化牡蠣組織之guanidine/lubrol 抽出液後，
進行SDS-PAGE電泳之結果 44

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