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Degradation mechanism of the chloroplast protein in the cell death of the plant

植物细胞死亡中叶绿体蛋白的降解机制

基本信息

批准号：
18570124
负责人：
AMANO Toyoki
金额：
$ 2.5万
依托单位：
Shizuoka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2006
资助国家：
日本
起止时间：
2006 至 2007
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18570124/
关键词：
enzyme protease 品質管理発現系 ATPase プロテアーゼ酵素調節作用機作

项目摘要

Plant FtsH pretence is a membrane-associated protease localized on the thylakoid membrane. Its function is to remove a damaged protein from large protein complex. Major physiological substrate of this protease is Dl protein whose subunit plays a key role in the photosystem II. Knockout mutants of this protease shows variegated phenotype in Arabidopsis, therefore physiological function of this protease is to maintenance the chloroplast proteins. In this study, we constructed over-expression system of ATPase and potease domain in FtsH potease from Arabidopsis and tobacco.In ease of protease domain from tobacco, protein was produced as inclusion body, though them were possible to be activated by urea mediated refolding. Expressed protein showed protease activity on FITC (fluorescein isothiocyanate)-labeled casein. FITC-BSA. (bovine serum albumin) did not digested by this protease, thus substrate specificity was detected on this protease. Biochemical study revealed that protease activity w … More as enhanced by higher concentration of magnesium ion. Optimum pH was determined as alkalescent condition.ATPase domain in FtsH protease from tobacco was constructed over-expression system in E.coli. This system produced the recombinant protein in the soluble fraction. The protein was purified by Ni^(2+) affinity chromatography. The eluted fraction was verified as ATPase domain by western blotting and peptide sequencing. ATPase activity was obviously detected. Using this system, we analyzed the dependencies of pH, divalent cations, ATP concentration, and the effect of detergents.Over-expression system for ATPase domain from Arabidopsis was also constructed. Purification was performed using Ni-NTA column. The quality of this protein was verified by western blotting and peptide sequencing. We examined biochemical analysis on the purified protein. And we revealed pH optimum and dependency of Mg^(2+) concentration. We also investigated effect of inhibitors. Among them, EDTA was the highest inhibitory effect. The results suggest that ATPase domain in the FtsH protease depends on divalent cations. We are trying to determine Km and Vmax, optimum temperature, and effective substrate except for ATP. Less

植物FtsH伪装蛋白是一种定位于类囊体膜上的膜相关蛋白酶。其功能是从大的蛋白质复合物中去除受损的蛋白质。该蛋白酶的主要生理底物是D1蛋白，其亚基在光系统II中起关键作用。该蛋白酶的敲除突变体在拟南芥中表现出多样的表型，因此该蛋白酶的生理功能是维持叶绿体蛋白。本研究构建了拟南芥和烟草中FtsH蛋白酶的ATP酶和POTEASE结构域的过表达系统，烟草中的蛋白酶结构域以包涵体形式表达，但它们可能被尿素介导的复性激活。表达的蛋白对异硫氰酸荧光素标记的酪蛋白具有蛋白酶活性。FITC-BSA（牛血清白蛋白）不被该蛋白酶消化，因此在该蛋白酶上检测到底物特异性。生化研究表明，蛋白酶活性， ...更多信息这通过较高浓度的镁离子而增强。确定了烟草FtsH蛋白酶的最适pH为碱性条件，构建了烟草FtsH蛋白酶ATP酶结构域在大肠杆菌中的高效表达系统。该系统在可溶性级分中产生重组蛋白。经Ni^（2+）亲和层析纯化蛋白。通过蛋白质印迹和肽序列分析证实洗脱的级分为ATP酶结构域。ATP酶活性明显。利用该系统分析了pH、二价阳离子、ATP浓度的依赖性以及去污剂的影响，并构建了拟南芥ATP酶结构域的过表达系统。使用Ni-NTA柱进行纯化。通过蛋白质印迹和肽序列分析验证了该蛋白质的质量。我们对纯化的蛋白进行了生化分析。并揭示了Mg^（2+）浓度的最适pH值和依赖性。我们还研究了抑制剂的作用。其中EDTA的抑制作用最强。结果表明FtsH蛋白酶中的ATP酶结构域依赖于二价阳离子。我们正试图确定Km和Vmax，最佳温度和有效底物（ATP除外）。少