Search for proteins as target sof cellular polyamines

具有基团的多胺类似物的合成和作为细胞多胺作用靶点的蛋白质的检测：系统地合成了生物素化多胺。Western blot分析发现，它们对大鼠肝癌HTC细胞有结构特异性标记。采用生物素化多胺，建立了组织转谷氨酰胺酶微滴板检测方法。根据该方法所鉴定的酶的底物特异性，可以推测其活性位点结构。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。采用荧光标记多胺法合成精氨酸基转移酶人工底物，建立了精氨酸基转移酶的检测方法。分析多胺耗竭和积累对细胞功能的影响：多胺抑制剂处理的多胺耗竭的HTC细胞，较长时间的多胺生物合成，容易积累给药的亚精胺，细胞内亚精胺的异常积累导致细胞凋亡等异常死亡。为了研究细胞死亡的机制，我们寻找在死亡过程中细胞水平发生变化的蛋白。观察到细胞组蛋白水平在此过程中下降，其降解先于细胞死亡。丝氨酸蛋白酶抑制剂能部分抑制亚精胺的降解和细胞死亡，提示组蛋白降解可能参与了亚精胺积累的细胞死亡过程。提出了用超滤法测定玉米油细胞质组分中细胞多胺的游离浓度，并应用于HTC细胞培养体系。结果表明，该方法可用于估计游离细胞多胺水平，并且多胺的消耗或积累会影响HTC细胞的细胞体积。建立了一种无需分离步骤的质谱法测定多胺的方法。多胺类似物的合成及其作用的研究：合成了长链多胺类似物，考察了其对NMDA受体、DNA三螺旋结构等的影响。少

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