标题: | 研究生物化学特性和结构特性来探讨黑腹果蝇蛋白质酪氨酸亚硫酸化酵素 Biochemical and structural characterization of Drosophia melanogaster tyrosylprotein sulfotransferase |
作者: | 林资翔 Lin, Tsu-Hsiang 杨裕雄 Yang, Yuh-Shyong 分子医学与生物工程研究所 |
关键字: | 生物化学;结构;黑腹果蝇;蛋白质酪氨酸亚硫酸化酵素;Biochemical;Structural;Drosophia melanogaster;Tyrosylprotein sulfotransferase |
公开日期: | 2009 |
摘要: | 蛋白质酪氨酸亚硫酸化酵素位于细胞内反式高尔基氏网,催化蛋白质中酪氨酸亚硫酸化反应,为一调控细胞外蛋白质交互影响力之重要因子,且调控许多生理上重要功能,例如:发炎作用,人类免疫系统缺乏病毒的入侵及甲状腺机能减退侏儒症。但因缺乏同质性蛋白质酪氨酸亚硫酸化酵素来瞭解其生化上特性,使其在分子层级上之资讯所知甚少。在我的论文研究中,利用一凝血蛋白脢去除融合蛋白- 转录延长因子,首次能够得到同质性黑腹果蝇蛋白质酪氨酸亚硫酸化酵素。藉由此瞭解黑腹果蝇蛋白质酪氨酸亚硫酸化酵素之酵素动力学,蛋白质四级结构,酵素稳定度和受质调控的特性。经分子筛层析法指出于溶液下其具有两种结构,且在盐与甘油的存在下得以稳定,目前已可以将其分离用于日后的研究。在人类与黑腹果蝇蛋白质酪氨酸亚硫酸化酵素上之点突变H269Q,H267Q并不会影响其比活性,但在大肠杆菌内表现量大幅降低导致其总活性随之大幅减少。而相同的点突变被报导在家鼠上会造成侏儒症,推测可能的原因是此点突变会影响酵素的稳定度或表现量。 Protein tyrosine sulfation, mediated by tyrosylprotein sulfotransferase (TPST) that resides in trans-Golgi network, is a key modulator of extracellular protein-protein interactions and consequently regulates various physiological functions including inflammation, HIV infection, and hypothyroidism related dwarfism. Limited information at molecular level is available due to the lack of homogenous TPST for detailed biochemical characterization. In this study, a truncated Drosophila melanogaster TPST (DmTPST) was first prepared following thrombin proteolysis to remove NusA fusion protein. The kinetics, structure, stability, and substrate regulation of DmTPST were characterized. The result of gel filtration indicated that there were two configurations of DmTPST were simultaneously presented in the solution and could be isolated for future studies. DmTPST can be stabilized with salt and glycerol. A hypothyroidism-related mutation in DmTPST and hTPST2 did not cause any loss of specific activity. However, the total TPST activity was significantly decreased following its expression in E. coli. Similar mutation has been reported to cause dwarfism in mouse. It is proposed that such mutation may affect the stability or expression of TPST. |
URI: | http://140.113.39.130/cdrfb3/record/nctu/#GT079650506 http://hdl.handle.net/11536/43258 |
显示于类别: | Thesis |
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