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Search for proteins as target sof cellular polyamines

寻找作为细胞多胺靶标的蛋白质

基本信息

批准号：
09672285
负责人：
SHIRAHATA Akira
金额：
$ 2.3万
依托单位：
Josai University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Synthesis of polyamine analogs having groups and detection of proteins as targets of cellular polymine actions: Biotinylated polyamines were systematically synthesized. When they were administered to rat hepatoma HTC cells, their structure specific labeling of cellular proteins were found by Western blot analysis. Microtiter plate assay method of tissue transglutaminase was newly developed using biotinylated polyamine. From the substrate specifisity of the enzyme elucidated by using the method, its active site structure could be proposed. A biotinylated and azidophenylated analog was usefull for detecting cell membrane protein having affinity to physiological polyamine. Artificial substrate of arginyltransferase were synthesized by using fluorescence labeled polyamines, and the assay method of the enzyme was established with the substrate.Analysis cell function which are affected by depletion and accumulation of polyamine : Polyamine-depleted HTC cells, treated with inhibitor of polyam … More ine biosynthesis for relatively long period, easily accumulate administered spermidine and the abnormal accumulation of spermidine in the cells leads to abnormal cell death like apoptosis. In order to investigate the mechanism of the cell death, the protein whose cellular level changed during the death process was searched. It was observed that the level of cellular histon decreased during the process and its degradation preceded the cell death. The degradation and the cell death were partially suppressed by the administration of serine protease inhibitor, suggesting the histon degradation would be involved in the cell death accompanied by the accumulation of spermidine. Ultrafiltration method for cytosol fraction in corn oil was proposed to determine the free concentration of cellular polyamine, and applied to HTC cell culture system. It was found that the method was useful for the estimation of free cellular polyamine level, and that cell volume of HTC cells was affected by the depletion or accumulation of polyamine. A new determination method of polyamine which does not have separation steps was developed by using mass- spectrometry.Syntheis of polyamine analogs and examination of their action : Polyamine analogs including ones having long chain lenghth were synthesized and their effect on NMDA receptor, DNA triple helix formation and so on were examined. Less

具有基团的多胺类似物的合成和作为细胞多胺作用靶标的蛋白质的检测：系统地合成了生物素化的多胺。将其应用于大鼠肝癌HTC细胞，通过Western印迹分析发现其具有细胞蛋白的结构特异性标记。用生物素化多胺建立了组织转谷氨酰胺酶的微量平板法。根据该方法确定的底物特异性，可以推测该酶的活性部位结构。生物素化和叠氮苯化类似物可用于检测与生理多胺有亲和力的细胞膜蛋白。利用荧光标记的多胺合成了精氨酸基转移酶的人工底物，并建立了该酶的测定方法。分析了多胺耗竭和积累对细胞功能的影响：多胺耗竭的HTC细胞，用多胺…的抑制剂处理较长时间内较多的亚精胺生物合成，容易积聚给药的亚精胺，亚精胺在细胞内的异常蓄积会导致细胞的异常死亡，如细胞凋亡。为了探讨细胞死亡的机制，寻找在细胞死亡过程中细胞水平发生变化的蛋白质。观察到在此过程中，细胞组蛋白水平下降，其降解先于细胞死亡。给予丝氨酸蛋白酶抑制剂可部分抑制细胞的降解和死亡，提示组蛋白的降解可能参与了伴随亚精胺积聚的细胞死亡。提出了一种测定细胞内多胺游离浓度的超滤法，并将其应用于HTC细胞培养体系。结果表明，该方法可用于细胞内游离多胺水平的估计，细胞体积受细胞内多胺的耗尽或积聚的影响。建立了一种新的无分离步骤的多胺类化合物的测定方法，合成了多胺类似物并研究了它们的作用：合成了包括长链多胺类似物在内的多胺类似物，考察了它们对NMDA受体、DNA三螺旋形成等的影响。较少