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首页> 外文期刊>Biochemistry >Blotting Analysis of Native IPP1:A Novel Approach to Distinguish the Different Forms of IRP1 in Cells and Tissues
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Blotting Analysis of Native IPP1:A Novel Approach to Distinguish the Different Forms of IRP1 in Cells and Tissues

机译：天然IPP1印迹分析：区分细胞和组织中不同形式IRP1的新方法

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Irom regulatory protein 1 (TRP 1) is a bifunctional protein,which either has aconitase activity or binds to specific mRNA structures to rgulate the expression of iorn proteinsUsing precombinant human IRP1,we found that the two functional forms are resolved by nondenaturing polyacrylamide gel electrophoresis and that they are distinguished from IRP1/RNA complexes.This allowed us to used specific antibodies to develop a blotting system that recognized the iron-ree and iron-containing IRP1 forms in the soluble fraction and the RNA-bound IRP1 in the high-speed precipitate fraction of cell extracts.The system was used to study IRP1 in HeLa,K562 cells,and monocytes/macrophages before and after treatment with iron salts,iron chelators,or hydrogen peroxide,as well as in stomach and duodenum biopsies.The results showed that iron-bound aconitase IRP1 is by far the prevalent form in most cells and that the major effect of cellular iron modifications is a shift between free and RNA-bound IRP1.The fraction of RNA-bound IRP1 was highly variable among different cells and was often a minor one.Furthermore,blotting showed that electrophoretic mobility shift assay,as commonly used,tends to under-evaluate the amount of total IRP1 and to over-evaluate the actual RNA-binding activity of IRP1.In conclusion,blotting analysis of IRP1 is a new,useful,and convenient method to analyze the amount and conformations of the protein that reveals previously undetected differences in IRP1 compartentalization among various cell types.
机译：Irom调节蛋白1（TRP 1）是一种双功能蛋白，具有乌头酸酶活性或与特定的mRNA结构结合以调节蛋白的表达使用预结合的人IRP1，我们发现这两种功能形式通过非变性聚丙烯酰胺凝胶电泳和从而区别于IRP1 / RNA复合物。这使我们能够使用特异性抗体来开发一种印迹系统，以识别可溶性级分中的铁铁和含铁IRP1形式以及高速沉淀物中的RNA结合IRP1。该系统用于研究铁盐，铁螯合剂或过氧化氢处理前后的HeLa，K562细胞以及单核细胞/巨噬细胞中的IRP1，以及胃和十二指肠活检中的IRP1。迄今为止，铁结合乌头酸酶IRP1是大多数细胞中最普遍的形式，细胞铁修饰的主要作用是游离和RNA结合IRP1之间的转换。 RNA结合的IRP1的分数在不同细胞之间变化很大，并且通常是次要的。印迹显示，通常使用的电泳迁移率变动分析往往会低估总IRP1的数量，而过分评估实际IRP1的数量。总之，IRP1的印迹分析是一种新的，有用的，便捷的方法，可以分析蛋白质的数量和构象，从而揭示出先前未发现的各种细胞类型之间IRP1互补性的差异。

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