【作者】 姜茜；

【导师】 贾凌云；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 硕士

【摘要】 细胞中的蛋白质参与和调控着生物体的各种生理活动,其中,大多数蛋白质是通过与配体分子结合或者是作为一个大的生物复合体的一部分来参与细胞生命活动的。因此,研究并阐明蛋白质间相互作用对于理解细胞代谢过程具有重大意义。在实验室前期研究的基础上,本论文继续探索研究蛋白质间相互作用的方法:基于底物与酶的相互作用,以琼脂糖凝胶为载体、乙二胺和戊二醛为间隔臂,固定化多巴胺小分子,吸附以单胺氧化酶B为主的猪肝细胞蛋白质,对单胺氧化酶B与其相关蛋白质间的相互作用进行初步研究。实验结果表明:(1)通过优化层析材料的配基密度,发现提高多巴胺密度可以增加单胺氧化酶B的吸附量,但其他蛋白量也随之增多,影响单胺氧化酶B纯度。从获得较高的酶比活和酶蛋白纯度的角度出发,配基密度约为36μmol/mL gel的多巴胺层析材料吸附单胺氧化酶B效果比较理想,其洗脱液的酶比活为3723 U/mg,Bandscan软件分析酶蛋白纯度为76%。(2)在采用AKTA凝胶层析对多巴胺层析材料洗脱蛋白进一步分离纯化的过程中,发现单胺氧化酶B(相对分子量约60 KDa)与一个相对分子量约为100 KDa的蛋白质无法完全分离。在进样样品中加入牛血清白蛋白作为参照后,凝胶电泳结果证实单胺氧化酶B与Mr100 KDa蛋白之间存在相互作用。(3)研究比较单胺氧化酶B、猪肝细胞胞质液以及二者混合溶液的酶活性后,发现混合溶液中单胺氧化酶B的酶活性提高了约11倍,初步说明细胞胞质液中含有能够与单胺氧化酶B发生相互作用、促进酶催化活性的物质。以上研究结果表明:利用固定化多巴胺小分子吸附以单胺氧化酶B为主的猪肝细胞蛋白质,再研究单胺氧化酶B与其相关蛋白间相互作用的方法可行,可作为研究蛋白质间相互作用的色谱方法用于功能蛋白质组学的研究。更多还原

【Abstract】 The physiological function of organisms is mainly participated and modulated by proteins in cells. Most proteins combined with ligand or form protein complexes with other proteins, and then execute the process of cell metabolism together. Hence, it is significant to elucidate protein-protein interactions for human life. Accroding to the previous research, the purpose of this study is to unceasingly explore the method for protein-protein interaction. To interact with proteins in porcine liver, such as monoamine Oxidase B, a dopamine molecule was immobilized on agarose beads using ethylenediamine and glutaraldehyde as spacer. The interactions between monoamine Oxidase B and its correlative proteins were investigated.The result of experiments indicated that: (1) It was found that the adsorption capabilities to monoamine Oxidase B were increased by promoting the density of dopamine on support, but the purity of monoamine Oxidase B went up first and then down. As far as higher enzymatic activeness and enzyme purity were concerned, the preferable density of dopamine for purifying monoamine Oxidase B was about 36μmol/mL gel. The activeness of monoamine Oxidase B was 3723 U/mg and its purity was 76% by Bandscan software analyse. (2) After the primary extraction of monoamine Oxidase B from liver crude sample by appropriate density dopamine support, gel filtration was used to progress farther purification. Based on optimization of operating conditions and the control of bovine serum albumin, a protein which may interact with monoamine Oxidase B was found, and its relative molecular weight was about 100000Da. (3) Compared with the purified enzyme eluate, enzymatic activeness of the eluate-cytoplasm mixture increased about eleven times. It primarily showed that some biomolecules in cytoplasm could interact with monoamine Oxidase B and participate in enzymatic catalyze.All these experiments indicated that it is feasible that a dopamine biomolecule is immobilized on support to interact with monoamine Oxidase B, and the interaction between monoamine Oxidase B and its correlative proteins in cells can be investigated. Therefore, it can be applied in the study of protein-protein interaction in functional proteomics as a new chromatographic method.更多还原