Juszczuk Izabela M, Tybura Agnieszka, Rychter Anna M
Department of Plant Bioenergetics, Institute of Experimental Plant Biology, University of Warsaw, Warsaw, Poland.
J Plant Physiol. 2008 Mar 13;165(4):355-65. doi: 10.1016/j.jplph.2007.06.021. Epub 2007 Oct 24.
Reactive oxygen species (ROS) may cause irreversible carbonylation of proteins, resulting in structural and/or functional modifications. Carbonylated proteins were analyzed and compared in tissue extracts or purified mitochondria isolated from the leaves and roots of wild-type (WT) or MSC16 mutant cucumber plants. For analysis of the oxidized protein formation and degradation, several techniques were applied: Western blotting, quantitative, spectrophotometric assay of carbonyl concentration and protease activity measurements. Oxidized proteins were tagged with 2,4-dinitrophenylhydrazine (DNPH) and detected with anti-DNP antibodies. Western blots of 1D gels indicated that, in the leaves of both WT and MSC16 plants, certain oxidized proteins have chloroplastic origin. In MSC16 plants, protein oxidation is probably higher in chloroplasts than in mitochondria. Carbonyl concentration is similar in MSC16 and WT leaf extracts, but this may be the result of twice as high protease activity observed in MSC16 leaf extracts and indicates that chloroplastic proteases may effectively remove the oxidized proteins from chloroplasts. In mitochondria of both WT and MSC16 leaves, the levels of oxidized proteins and protease activity are similar. In MSC16 root extracts, the carbonyl concentration is lower and protease activity is similar as compared to WT plants. Nevertheless, in MSC16 root mitochondria, the 30% lower carbonyl concentration, lower band abundance for oxidized proteins and over 50% higher protease activity indicate that mitochondrial proteases are involved in degradation of the oxidatively damaged proteins. In matrix and membrane subfractions, the levels of oxidized proteins are similar in leaf mitochondria or lower in root mitochondria from MSC16 as compared to WT plants. The results show that the oxidized protein degradation network in MSC16 cucumber mutants is well developed, thus becoming a survival factor for plants with mitochondrial dysfunctions.
活性氧（ROS）可能导致蛋白质发生不可逆的羰基化，从而引起结构和/或功能的改变。对野生型（WT）或MSC16突变体黄瓜植株的叶片和根组织提取物或纯化的线粒体中的羰基化蛋白质进行了分析和比较。为了分析氧化蛋白质的形成和降解，应用了几种技术：蛋白质免疫印迹法、羰基浓度的定量分光光度测定法和蛋白酶活性测定法。氧化蛋白质用2,4-二硝基苯肼（DNPH）标记，并用抗DNP抗体进行检测。一维凝胶的蛋白质免疫印迹表明，在WT和MSC16植株的叶片中，某些氧化蛋白质起源于叶绿体。在MSC16植株中，叶绿体中的蛋白质氧化可能比线粒体中更高。MSC16和WT叶片提取物中的羰基浓度相似，但这可能是由于在MSC16叶片提取物中观察到的蛋白酶活性高两倍的结果，这表明叶绿体蛋白酶可能有效地从叶绿体中去除氧化蛋白质。在WT和MSC16叶片的线粒体中，氧化蛋白质的水平和蛋白酶活性相似。与WT植株相比，在MSC16根提取物中，羰基浓度较低，蛋白酶活性相似。然而，在MSC16根线粒体中，羰基浓度低30%，氧化蛋白质的条带丰度较低，蛋白酶活性高出50%以上，这表明线粒体蛋白酶参与了氧化损伤蛋白质的降解。在基质和膜亚组分中，与WT植株相比，MSC16叶片线粒体中氧化蛋白质的水平相似，或根线粒体中氧化蛋白质的水平较低。结果表明，MSC16黄瓜突变体中的氧化蛋白质降解网络发育良好，因此成为线粒体功能障碍植物的生存因素。
