博士 / 國立臺灣海洋大學 / 食品科學系 / 92 / 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.
| Identifer | oai:union.ndltd.org:TW/092NTOU5253008 |
| Date | January 2004 |
| Creators | Ming-Shyong, Wang, 王明雄 |
| Contributors | Sen-Shyong, Jeng, 鄭 森 雄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 195 |
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