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Localization, Purification, and Characterization of Zn-Binding Protein in the Digestive Tract Tissueof Common Carp, Cyprinus carpio
|Authors: ||Ming-Shyong, Wang|
|Contributors: ||NTOU:Department of Food Science|
Basal lamina;Basolateral plasma membranes;Cell adhesion;Collagenase;Common carp;Extracellular matrix;Laminin;Liposome;RGD peptide;Sulfhydryl groups;Zn;Zn-binding protein
|Issue Date: ||2011-06-30T07:39:57Z
|Abstract: ||在許多哺乳動物組織之鋅濃度，約為10-100 μg/(g wet weight)；而不同種屬間之差異極小。魚類可食部份之鋅濃度平均只有6.5 μg/(g wet weight)，其範圍則在3-24 μg/(g wet weight) 之間。然而，鯉魚消化道組織含有非常高之鋅濃度，其值常在300-500 μg/(g wet weight)。目前己知經次細胞分畫後，鯉魚消化道組織之鋅主要係存在於其nuclei/cell debris fraction中，約佔整體鋅濃度的60-88%;而cytosol、microsome及mitochondria fraction中的鋅只佔整體鋅濃度的12-40%。鯉魚消化道組織之高鋅，主要來自其nuclei/cell debris fraction;而nuclei/cell debris fraction中，與鋅結合物質很可能是一種膜蛋白質。 本研究首先探討鯉魚消化道組織「鋅結合蛋白質」(Zn-binding protein, ZnBP) 之分佈。將鯉魚消化道組織先以次細胞分劃，然後，將各成份以化學及酵素標誌進行確認。以Zn與bound-SH groups作為ZnBP之指標，發現在次細胞分劃後，其nuclei/cell debris fraction中會有多數之ZnBP (79%)，及DNA (85%)、Na+/K+-ATPase (82%)、organic phosphate (90%)；但僅含部份之5’-nucletidase及alkaline phosphatase (<23%)。將nuclei/cell debris fraction以collagenase type I，或type IV處理後，再以蔗糖梯度離心分離；結果發現，以collagenase type IV處理nuclei/cell debris fraction時，大約有50%的ZnBP、Na+/K+-ATPase及organic phosphate會從collagen解離。另以RGD (Arg-Gly-Asp) peptide或tirofiban (模擬RGD peptide構造而合成之藥物) 處理nuclei/cell debris fraction時，亦能將40~60%的ZnBP與basolateral plasma membrane自胞外母質解離。然而，同時以collagenase type IV及tirofiban處理nuclei/cell debris fraction後，發現>80%的ZnBP (與basolateral plasma membrane) 會與胞外母質解離。此結果顯示ZnBP係存在於basolateral plasma membrane之上，而basolateral plasma membrane是藉由multiadhesive protein之RGD序列連結在collagen type IV上。 為分離純化ZnBP，乃將鯉魚消化道組織之nuclei/cell debris fraction，先以collagenase type IV及RGD peptide處理，使plasma membrane自胞外母質解離。所得到之plasma membrane再以清潔劑n-dodecyl-b-D-maltoside抽取ZnBP。清潔劑抽取液再進一步以immobilized metal affinity chromatography (IMAC) 及laminin-Sepharose affinity chromatography進行分離。由上述之層析方法，可成功地分離純化出ZnBP。由SDS-PAGE及autoradiography之分析結果，可知此ZnBP之分子量為43 kDa，可與laminin-Sepharose結合，並可被tirofiban溶離。由wheat germ agglutinin及concanavalin A-Sepharose affinity chromatography的結果顯示ZnBP為一種glycoprotein。此ZnBP約佔 “crude plasma membrane material” 的~0.4-0.8%。由於ZnBP可與laminin-Sepharose結合，並被tirofiban溶離，推測此蛋白質為一種細胞表面受體 (cell surface receptor)，作用於細胞與laminin之黏合。 為了解鯉魚消化道組織ZnBP是否可嵌入細胞膜，乃將純化的ZnBP嵌入liposome中，使其成為ZnBP-liposome。由於ZnBP不溶於水，但可被清潔劑溶解，及可嵌入liposomes的性質，推測其為膜固有蛋白質 (integral membrane protein)。此外，結合在ZnBP上之鋅係存在liposome外。即存在於鯉魚消化道組織細胞膜上之ZnBP，其鋅似乎存在於細胞膜之外。以surface receptor與ligands結合之研究方法，研究ZnBP-liposome與65Zn的結合後，可知ZnBP-liposome與65Zn是特異性的結合。加入trypsin與PCMB作用後，ZnBP-liposome與65Zn即不再結合。在pH 8.0時該特異性結合會達到最高。此外亦進行ZnBP-liposome與65Zn結合之saturation analysis與Scatchard analysis。由其Scatchard plot呈一直線 (r = 0.9992)，推測ZnBP具有一高親和性的binding site，其與65Zn結合之Kd (dissociation constant) 為0.19±0.01 μM；maximum binding sites (Nmax) 為76.7±1.8 pmole/μg protein。以Ca2+、Co2+、Cr2+、Cu2+、Fe2+、Hg2+、Mn2+、Ni2+、及Pb2+ 探討ZnBP-liposome與 65Zn結合之金屬特異性，發現僅有Co2+對於ZnBP與 65Zn的特異性結合有強烈競爭。 本研究亦探討ZnBP-liposome與laminin 結合之特異性與親和性，結果發現ZnBP-liposome可與laminin特異性的結合，其Kd為4.79 μM。然而，ZnBP-liposome並不會與其他胞外蛋白質 (fibronectin、fibrinogen、vitronectin、collagen type I 及 collagen type IV) 有顯著的結合。ZnBP與laminin的結合，只受RGD peptide (RGD或GRGDSPG) 的影響，並不受其他相似peptide (GRGESPG及GRADSPG) 影響。由以上的性質可知，ZnBP應為cell surface receptor，與laminin經由RGD序列專一性的結合。 此外，本研究亦以純化的ZnBP，成功地製成ZnBP多株抗體，並以此抗體發展出來免疫分析方法，可以做為研究ZnBP的指標。此免疫分析方法將來可應用在鯉魚及其他魚類組織中，ZnBP之分佈、特性，及生理功能等相關的研究之上。|
Concentrations of Zn in most tissues of several studied mammalian species are in the order of 10-100 mg/(g wet weight), with little variation among species. The average Zn content of the edible portion of finfish is lower, at 6.5 mg/(g wet weight), with a range of 3-24 mg/(g wet weight). However, the common carp Cyprinus carpio has an extraordinarily high Zn concentration of 300-500 mg/(g fresh tissue) in its digestive tract tissue. Subcellular fractionation of the digestive tract tissue has shown that 60-88% of the total cellular Zn was in nuclei/cell debris fraction, and only 12-40% was in all other fractions including cytosol, mitochondria and microsome fractions. The Zn bound to the nuclei/cell debris fraction of digestive tract tissue of common carp was mainly responsible for the high concentration of Zn, and the Zn binding substance was very probably a membrane protein. In this study, the biochemical localization of the Zn-binding protein (ZnBP) in the digestive tract tissue of common carp was studied. The tissue was subjected to subcellular fractionation, and marker enzyme activities were estimated in resultant fractions. Using Zn and Bound-SH groups as indicator for the ZnBP, and the marker enzymes as indicators for subcellular fraction, it was found that the nuclei/cell debris fraction contained most of the ZnBP (79%), DNA (85%), Na+/K+-ATPase (82%), and organic phosphate (90%), but only part of 5’-nucletidase and alkaline phosphatase (<23%). The nuclei/cell debris fraction of the tissue was treated with either collagenase type I or type IV, and subfractionated by sucrose density centrifugation. It was found that the treatment with collagenase type IV could release over 50% of the ZnBP, Na+/K+-ATPase and organic phosphate from collagen. Arg-Gly-Asp (RGD) peptide or tirofiban (a mimic of RGD) was also treated with the nuclei/cell debris fraction, it was found that about 40~60% of the ZnBP together with basolateral plasma membrane was released from the extracellular matrix. However, when the nuclei/cell debris fraction was treated with collagenase type IV and tirofiban, >80% of the ZnBP together with the basolateral plasma membrane were detached from the extracellular matrix. These results suggest that in the digestive tract tissue of common carp, the basolateral plasma membranes are attached to basal laminae through the RGD peptide mediated by the ZnBP. To isolate the ZnBP, the plasma membranes of the digestive tract tissue of common carp were separated from the extracellular matrix by treating the nuclei/cell debris fraction of the tissue with collagenase type IV and RGD peptide. ZnBP was then extracted from the plasma membrane with detergent n-dodecyl-b-D-maltoside. The ZnBP was isolated from the detergent extract by immobilized metal affinity chromatography, and affinity chromatography on laminin-Sepharose. A 43 kDa protein was bound by the laminin-Sepharose and specifically eluted with tirofiban. Affinity chromatography on wheat germ agglutinin and concanavalin A-Sepharose showed that the ZnBP is a glycoprotein. The 43 kDa protein represents ~0.4-0.8% of the “crude plasma membrane material”, as determined by protein analysis. The binding of the ZnBP to the laminin-Sepharose and its specific elution with the tirofiban strongly suggest that the ZnBP might function as a cell surface receptor involved in the adhesion of cells to laminin. Since such a receptor would be likely to be integrated into the plasma membrane, the capacity of the ZnBP to become incorporated into liposome model membrane was examined. The ZnBP was incorporated into liposomes at a high efficiency. Both the insolubility of the ZnBP in aqueous buffers without detergents and its ability to insert into liposomes support the notion that it may be an integral membrane protein. It was found when ZnBP was inserted into liposome, Zn was on the outside part of the ZnBP-liposome. Zn binding activity of the ZnBP-liposome was characterized. Specific binding of 65Zn to the ZnBP-liposome was detected. Specific binding was eliminated by protease or PCMB treatment. Highest level of specific binding was observed at pH 8.0. Binding was inhibited by both low and high pH. The saturation and Scatchard analyses of the ZnBP-liposome with 65Zn as ligand were performed. Linearity of the Scatchard plot (r＝0.9992) was strongly suggestive of the presence of a single high-affinity binding site. The binding parameter of Zn to the ZnBP was found to be: Nmax, maximum binding site, 76.7±1.8 pmole/μg protein; and Kd, equilibrium dissociation constant, 0.19±0.01 μM. The specificity of the ZnBP-liposome for binding Zn was studied by introducing the following cation: Ca2+, Co2+, Cr2+, Cu2+, Fe2+, Hg2+, Mn2+, Ni2+, and Pb2+. Only Co2+ competed significantly with the binding of Zn to the protein. The binding affinity and specificity of the ZnBP-liposome to laminin was also studied. It was found that ZnBP-liposome bound to laminin specifically, and the Kd was 4.79 mM. In contrast, no significant binding of ZnBP-liposome to other extracellular proteins (fibronectin、fibrinogen、vitronectin、collagen type I and collagen type IV) was observed. In addition, the binding was specifically inhibited by the RGD peptide (RGD or GRGDSPG). The RGD peptide analogues (GRGESPG and GRADSPG) were without any effect at the same concentration. These properties suggested that the ZnBP is a cell surface receptor involved in the adhesion of cells to laminin mediated by the RGD sequence. The polyclonal antibody against ZnBP was produced, and a novel immunoassay method for detection and quantification of the ZnBP in the tissue of common carp and other species with the ZnBP antibody was also developed.
|Appears in Collections:||[食品科學系] 博碩士論文|
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