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研究生:陳彥樺
論文名稱:鯉魚腎臟、皮及眼睛膜狀組織中之鋅及「與鋅結合物質」之所在與生化特性
論文名稱(外文):Localization and Biochemical Properties of Zinc and Zinc Binding Substances in Kidney, Skin, and Membrane-like Substances of Eyes of Common Carp
指導教授:鄭森雄鄭森雄引用關係
學位類別:碩士
校院名稱:國立海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
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:49
中文關鍵詞:鯉魚皮膚眼睛膜狀組織腎臟基底層
外文關鍵詞:carpskinmembrane-like substance of eyeskidneybasal laminaZinc
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摘要
鯉魚消化道組織中含有較其他魚類高之鋅濃度,此高鋅主要是與膜蛋白質結合;與鋅結合之膜蛋白質係位於basolateral plasma membrane,並且與基底層basal lamina 之collagen type Ⅳ結合。除消化道組織以外,鯉魚腎臟之鋅濃度亦高;而此二組織中,都具有多數之basal lamina存在。為了解鯉魚組織中鋅濃度之高低與其basal lamina之關係,本論文乃研究鯉魚皮及眼睛組織中,鋅之濃度是否亦高,其分佈為何,因鯉魚之皮及眼睛組織中,都具有多數之basal lamina。另外並研討此等組織中,「與鋅結合物質」之生化特性,並與草魚、鰱魚及吳郭魚做一比較。
由實驗結果得知一般市售鯉魚腎臟中之鋅濃度為137∼63 μg/g fresh tissue;但在實驗室中高鋅飼養之鯉魚鋅濃度可達287∼300 μg/g fresh tissue。將鯉魚腎臟經次細胞分劃後,發現腎臟中之鋅不論是市售魚或高鋅魚主要分佈於nuclei/cell debris fraction中。鯉魚腎臟中的總鋅濃度與其nuclei/cell debris fraction中之鋅濃度呈正相關 (r=0.828)。草魚、鰱魚及吳郭魚之腎臟中之鋅濃度為63∼30 μg/g fresh tissue,約為鯉魚腎臟鋅濃度之一半。比較鯉魚與其他三種魚 (草魚、鰱魚及吳郭魚) 腎臟之鋅濃度,可知在cytosol, microsome及mitochondria/lysosome fraction中,4種魚之鋅濃度幾無差異。鯉魚腎臟中鋅濃度較高之理由,乃因其nuclei/cell debris fraction中,有較高之鋅所致。
鯉魚皮膚中之鋅濃度為117∼102 μg/g fresh tissue,鯉魚皮膚經次細胞分劃後,發現其鋅主要分佈於nuclei/cell debris fraction中。鯉魚皮膚中的總鋅濃度與其nuclei/cell debris fraction中之鋅濃度呈正相關 (r=0.726)。草魚、鰱魚及吳郭魚之皮膚中之鋅濃度為25∼38 μg/g fresh tissue ,約為鯉魚皮膚鋅濃度之1/3∼1/4。比較鯉魚與其他三種魚 (草魚、鰱魚及吳郭魚) 皮膚之鋅濃度,可知在cytosol, microsome及mitochondria/lysosome fraction中,4種魚之鋅濃度幾無差異。鯉魚皮膚中鋅濃度較高之理由,乃因其nuclei/cell debris fraction中,有較高之鋅所致。
將鯉魚眼睛粗分為3部份後,發現不管魚體大小,其眼睛之膜狀組織約佔14%左右。將鯉魚眼睛膜狀組織之3部份,分別測定其鋅濃度後,可知鯉魚晶狀體及玻璃體部分之鋅濃度為13∼14 μg/g fresh tissue;而鞏膜軟骨部分鋅濃度為16∼17 μg/g fresh tissue;且左右眼並無明顯差異。然而,膜狀組織之鋅濃度卻高達313±81 μg/g fresh tissue。將鯉魚眼睛之膜狀組織經次細胞分劃後,發現鋅主要亦分佈於nuclei/cell debris fraction中。另外,不同鯉魚眼睛膜狀組織中之總 鋅濃度與其nuclei/cell debris fraction中之鋅濃度呈正相關性 (r=0.858)。將草魚、鰱魚與吳郭魚眼睛之膜狀組織分別測其鋅濃度後,發現此3種魚之鋅濃度亦高達473∼519μg/g fresh tissue。次細胞分劃後,發現其鋅主要亦存在於nuclei/cell debris fraction中 (80∼88%)。
另外,由實驗結果得知,鯉魚腎臟、皮膚及鯉魚、草魚、鰱魚及吳郭魚之眼睛膜狀組織,其nuclei/cell debris fraction中之〝EDTA-extractable Zn〞與Bound-SH groups之濃度,有正相關。由此可推測此等組織中之nuclei/cell debris fraction中之鋅極可能連接於SH group上。然而〝EDTA-extractable Zn〞較少之草魚、鰱魚、及吳郭魚之腎臟與皮膚,其Bound-SH之濃度則較少。測定鯉魚眼睛膜狀組織之Na+/K+ ATPase活性,發現鯉魚眼睛膜狀組織之Na+/K+ ATPase之活性為0.96±0.28 μmole/g fresh tissue/min,主要係存在於其nuclei/cell debris fraction中,約佔總含量之67±9 %。鯉魚消化道組織中之高鋅主要是與膜蛋白質結合,而此與鋅結合之膜蛋白質係位於basolateral plasma membrane。
綜合以上實驗結果,推測鯉魚腎臟、皮膚及鯉魚、草魚、鰱魚及吳郭魚之眼睛膜狀組織中之「與鋅結合物質」,極可能亦是位於basolateral plasma membrane之膜蛋白質,經由SH基而與鋅結合。

Summary
Zn concentration in the digestive tract tissue of common carp is higher than that in other fish. It was found that this is because Zn was attached to a membrane protein, and the「Zn binding membrane protein」(ZnBMP) might be located on the basolateral plasma membrane of the epithelial cells and surrounding muscle cells which are attached to collagen type Ⅳ. Besides the digestive tract tissue, the Zn concentration in kidney of common carp is also high. Both of the digestive tract tissue and kidney have large amount of basal lamina. Besides these two tissues, the eyes and skin also have large amount of basal lamina. In order to understand the relationship between the Zn concentration in tissues of common carp and the amount of basal lamina, the Zn concentration, localization and the biochemical properties of 「Zn binding substance」of these tissues were studied, and compared with those of grass carp, silver carp, and tilapia.
It was found that the Zn concentrations in kidney of common carp bought from market were 137∼63 μg/g fresh tissue, but those fed with high Zn diet (2000 mg/kg) were 287∼300 μg/g fresh tissue. The kidney of common carp was subcellular fractioned, and it was found that the Zn concentration in kidney of common carp either bought from market or fed with high Zn diet (2000 mg/kg) mainly existed in the nuclei/cell debris fraction. High correlation between Zn concentraction in whole tissue and the nuclei/cell debris fraction was observed (r=0.828). The Zn concentrations in kidneys of grass carp, silver carp, and tilapia were 63∼30 μg/g fresh tissue, only about half of that in common carp. It was found that the concentrations of Zn in cytosol, microsome, mitochondria/
lysosome fraction were similar among the four fish species. High concentration of Zn in kidney of common carp mainly existed in the nuclei/cell debris fraction.
The Zn concentrations in skin of common carp were 117∼102 μg/g fresh tissue. The skin of common carp was subcellular fractioned, and it was found that the Zn concentration in skin of common carp mainly existed in the nuclei/cell debris fraction. High correlation between Zn concentration in whole tissue and the nuclei/cell debris fraction was found (r=0.726). The Zn concentrations in skin of grass carp, silver carp, and tilapia were 25∼38 μg/g fresh tissue, only about 1/3∼1/4 of that in common carp. It was found that the concentration of Zn in cytosol, microsome, and mitochondria/lysosome fraction were similar among the four fish species. High concentration of Zn in skin of common carp mainly existed in the nuclei/cell debris fraction.
The eyes of common carp were separated into three parts, and the Zn concentration was measured. It was found that the Zn concentration in lens and vitreous of eyes of common carp was 13∼14 μg/g fresh tissue, and in scelera cartilage was 13∼14 μg/g fresh tissue, and there was no difference between right eye and left eye. However, the Zn concentration in membrane-like substances of eyes of common carp reached to 339±66 μg/g fresh tissue. The membrane-like substances of eyes of common carp was subcellular fractionated, and it was found that the nuclei/cell debris fraction contained most of the Zn. A high correlation between Zn concentration in whole tissue and the nuclei/cell debris fraction was observed (r=0.858). The Zn concentration in membrane-like substances of eyes of grass carp, silver carp, and tilapia was found to be 473∼519 μg/g fresh tissue. After subcellular fractionation, it was found that the nuclei/cell debris fraction contained most of the Zn (80∼88%).
The experiments also show that there was a high correlation between the concentration of Bound-SH groups and 〝EDTA-extractable Zn〞 in the nuclei/cell debris fraction of kidney and skin of common carp; and the membrane-like substances of eyes of common carp, grass carp, silver carp, and tilapia. It is proposed that the Zn in nuclei/cell debris fraction of these tissues might be bound by SH group. However, the kidney and skin of grass carp, silver carp, and tilapia which had low 〝EDTA-extractable Zn〞, also had low concentration of Bound-SH groups. The Na+/K+ ATPase activity in membrane-like substances of eyes of common carp was found to be 0.96±0.28 μmole/g fresh tissue/min, and it mainly existed in the nuclei/cell debris fraction (67±9%).
Based on the experiments, we propose that the「Zn binding substances」 in the kidney and skin of common carp; and the membrane-like substances of eyes of common carp, grass carp, silver carp, and tilapia are very probable to be a membrane protein located on the basolateral plasma, and they are attached to Zn by SH group.

目 錄
頁數
摘要 i
Summary iv
第一章 研究目的及文獻整理
第一節 研究背景及目的…………………….……………….. 1
一. 研究背景…………………………………………….. 1
(一) 鯉魚內臟高濃度鋅之發現…………………………. 1
(二) 鯉魚內臟中鋅之分布………………………………. 1
(三) 鯉魚皮膚及眼睛組織中之鋅含量…………………. 1
(四) 鯉魚消化道組織含鋅物質之生化特性……………. 2
(五) 鯉魚消化道組織中之「與鋅結合之膜蛋白質」…. 2
二. 研究目的…………………………………………….. 4
第二節 文獻整理……………………………………………... 5
(一) 鋅之生理機能……………………………………… 5
(二) basal lamina之分布及組成………………………… 5
(三) 腎臟之basal lamina……………………………….. 6
(四) 生物眼睛組織中之高濃度鋅……………………… 6
(五) 老鼠腎臟brush border membrane中存在之
「與鋅結合蛋白質」……………………………… 7
附圖…………………………………………………………… 8
第二章 鯉魚、草魚、鰱魚及吳郭魚之腎臟、皮及眼睛
膜狀組織中鋅之分布
第一節 前言………………………………………………… 10
第二節 材料與方法………………………………………… 10
第三節 結果………………………………………………… 13
一. 鯉魚、草魚、鰱魚及吳郭魚腎臟組織中之鋅濃度
及分布……………………………………………… 13
二 . 鯉魚、草魚、鰱魚及吳郭魚皮膚組織中之鋅濃度
及分布……………………………………………… 13
三. 鯉魚、草魚、鰱魚及吳郭魚眼睛組織中之鋅濃度
及分布……………………………………………… 14
第四節 討論………………………………………………….. 15
附圖與附表…………………………………………………… 16
第三章 鯉魚、草魚、鰱魚及吳郭魚nuclei/cell debris fraction中
與鋅結合物質之生化特性
第一節 前言………………………………………………….. 27
第二節 材料與方法………………………………………….. 27
第三節 結果………………………………………………….. 30
一. 〝EDTA- extractable Zn〞與硫氫基之關係…………. 30
二. 鯉魚眼睛膜狀組織經次細胞分劃後,Na+/K+ ATPase
之分佈………………………………………………. 30
第四節 討論…………………………………………………... 31
附圖與附表……………………………………………………. 32
第四章 綜合討論…………………………………………………. 40
參考文獻…………………………………………………………… 44
謝辭………………………………………………………………… 49

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