臺灣博碩士論文加值系統

目前已知台灣牡蠣之鋅濃度約為 22~283 μg/g fresh tissue 左右，但世界不同地區的牡蠣鋅濃度差異大，為了解其中鋅如何分佈、結合之蛋白質為何，乃將台灣市售牡蠣進行次細胞分劃。分析 14 批不同牡蠣後，發現牡蠣中的鋅濃度為 35~147 μg/g fresh tissue，且個體差異甚大。牡蠣組織中水溶區分之鋅濃度大致不變，為 29±6 μg/g fresh tissue；然而，水不溶區分之鋅濃度為 56±34 μg/g fresh tissue，變化極大，其範圍為 3~106 μg/g fresh tissue。由此得知牡蠣之所以有不同高低之鋅，乃由於水不溶區分之鋅而來。生物組織中之水不溶蛋白質，主要為膜蛋白質，及結締組織中之蛋白質，清潔劑常被用來抽取膜蛋白質，而加入 guanidine 可切斷膜蛋白質與胞外母質 (matrix) 之間的連結。本實驗以清潔劑 (lubrol buffer) 抽取牡蠣組織的水不溶區分，發現僅能抽取 21% 之鋅；然而，在含 4 M guanidine 之 lubrol (Gn/Lub buffer) 可以抽取另外 38% 之鋅。結果表明，牡蠣組織中約 21% 的鋅結合物質可能是膜蛋白質，另外 38% 可能是與胞外母質相連之膜蛋白。藉由 Zn2+-IMAC 純化牡蠣組織水不溶區分之的 Lubrol ext 及Gn/Lub ext，再以電泳鑑定。SDS-PAGE 顯示，Lubrol ext 在 62 kDa、56 kDa、48 kDa、43 kDa 位置具有較明顯的 band；Gn/Lub ext 則在 48 kDa 和 43 kDa 位置具有明顯的 band。牡蠣組織中與鋅結合之主要蛋白質似乎為 48 kDa 及 43 kDa 蛋白質。此二種蛋白質可能即為牡蠣中會持續累積鋅之蛋白質。本報告另以ZnSO4、牡蠣水溶區分及水不溶區分投餵缺鋅小鼠，發現此三種不同來源的鋅化合物，皆可使缺鋅小鼠之血漿鋅濃度恢復至與對照組相近數值，但是牡蠣水不溶區分對於平均體重之恢復，似乎較有效果，牡蠣水溶區分次之。綜合上述結果，牡蠣主要與鋅結合之物質似乎為 48 kDa及 43 kDa 之膜蛋白質，而以牡蠣水不溶區分作為飼料之鋅來源似乎對缺鋅小鼠之體重回復有較佳之效果。

It was reported previously that oyster in Taiwan has a zinc concentration of 22~283 μg/g fresh tissue. To know whether this zinc is bound by protein and its distribution, 14 different lots of oyster from market was subjected to subcellular fractionation. It was found that the zinc concentration in the oyster ranged from 35 to 147 μg/g fresh tissue, and there was a great individual difference. However, the zinc level in the water-soluble fraction was relatively constant, approximately 29±6 μg/g fresh tissue; in the water-insoluble fraction was 56±6 μg/g fresh tissue, with a range from 3 to 106 μg/g fresh tissue. It is the water-insoluble fraction made the difference. In biological tissues, the main kind of water-insoluble protein is membrane protein and connective tissues proteins. Detergents are often used to extract membrane protein, and the addition of guanidine can cleave the link between membrane protein and extracellular matrix. In this report, the water-insoluble fraction of oyster tissue was extracted with detergent (lubrol buffer), and it was found that only 21% of the zinc could be extracted. However, adding 4 M guanidine in lubrol (Gn/Lub buffer) could extract another 38% of zinc. The results showed that about 21% of the zinc-binding substance in oyster tissue might be membrane protein, and another 38% might be membrane protein connected to extracellular matrix. Lubrol extract and guanidine/lubrol extract of the water-insoluble fraction of oyster tissue was purified by Zn2+- IMAC, and the eluates were identified by electrophoresis. SDS-PAGE showed lubrol extract had bands at 62 kDa, 56 kDa, 48 kDa and 43 kDa position. Gn/Lub extract had a clear band at the 48 kDa and 43 kDa position. The main zinc binding protein in the oyster tissue seems to be the 48 and 43 kDa proteins, which may be the proteins that functioned to accumulate zinc in oyster. In this report, ZnSO4, oyster water-soluble fraction and water-insoluble fraction of oyster meat were fed to zinc-deficient mice. It was found that the zinc compounds from these three different sources could recover the zinc level in plasma of zinc-deficient group to the same value as the control group. However, the water-insoluble fraction of oyster meat seems to be more effective for the recovery of the average body weight, followed by the water-soluble fraction of oyster meat. The above mentioned results indicated that the mainly zinc-binding substance in oyster seems to be 48 kDa and 43 kDa membrane protein. The zinc source from the water-soluble fraction of oyster meat as feed seems to be more effective for the recovery of the average body weight of zinc-deficient mice.

摘要....I
Abstract....II
目次....IV
圖目次....VI
表目次....VII
第一章、研究背景與目的....1
一、研究背景....1
二、研究目的....1
第二章、文獻整理....2
一、鋅的生理功能....2
二、牡蠣之簡介....2
三、牡蠣之鋅含量....3
四、鋅結合蛋白質之簡介....3
五、鯉魚消化道組織之鋅結合蛋白質....3
六、牡蠣組織之鋅結合蛋白質....4
七、牡蠣鋅的分佈....4
八、抽取牡蠣鋅結合物質之方法....4
九、鋅鹽補充劑與鋅結合蛋白質之吸收....5
十、體內鋅變化之指標....5
第三章、鋅在牡蠣組織「水溶」及「水不溶」區分之分布與「鋅結合物質」
之鑑定....7
一、前言....7
二、實驗材料與試藥....7
三、實驗方法....8
(一) 牡蠣之次細胞分劃....8
(二) 熱分解法....9
(三) EDTA 法....10
(四) 以清潔劑 lubrol 抽出牡蠣組織中之鋅結合蛋白質....10
(五) 鋅濃度測定....11
(六) 利用 IMAC 分離牡蠣組織中之鋅結合蛋白質....11
(七) 以 SDS - Polyacrylamide gel electrophoresis分析蛋白質 ....13
(八) 以 BCA 法測定蛋白質含量....16
四、實驗結果....17
第四章、缺鋅小鼠對牡蠣組織「水溶」與「水不溶」區分之鋅的吸收....19
一、前言....19
二、實驗材料與試藥....19
三、實驗方法....20
(一) 小鼠飼養方式與分析....20
(二) 製備飼料....20
(三) 血液學數值測定....21
(四) 血液鋅濃度測定....22
(五) 肝臟、腎臟、脾臟之鋅濃度測定....23
四、實驗結果....24
第五章、綜合討論....26
參考文獻....28
圖....33
表....40
謝辭....48

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