Abstract：
In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts.
摘要：
在植物中,谷氨酸脱氢酶（GDH）是一种普遍存在的酶，可催化谷氨酸中2-氧戊二酸的可逆胺化。它有助于氨基酸稳态和细胞内铵的管理，它被认为是碳和氮同化途径的关键参与者。迄今为止,有关陆地植物GDH的信息仅指极少数物种。我们专注于属于Marchantiophyta部门的选定物种，提供第一个全景概述的生化和功能特征的GDH。天然电泳分析显示，与拟南芥和其他被子植物相比，同工酶谱的复杂程度要低：存在对应于α-同型的单一同工型，在物种和器官基础上，不同程度地容易发生热灭活，找到了。对氨基酸序列进行的序列分析证实了现代黑草中GDH与拟南芥GDH2蛋白的高度相似性，加强了以下假设：从GDH2产生GDH1同源基因的重复事件发生在分离苔藓植物和气管植物的进化分叉之后。在体外生长的Marchantiapolymorpha和Calypogeiafissa上进行的实验，并与A进行比较。通过凝胶活性检测和一维Western印迹证明，GDH的胺化活性也强烈增强了对紫菜中铵过量的反应，即使与拟南芥和其他维管物种相比程度不同。通过二维WesternBlot的比较分析表明，该酶的调节可能是，至少部分地,从蛋白质翻译后模式中解开。最后,免疫电子显微镜显示，GDH酶在薄壁组织的线粒体和叶绿体中均位于亚细胞水平，并与苔藓的内膜系统特异性相关。
