Metabolic Compartmentation of Vertebrate Glutamine Synthetase

在拟议的项目期间将实现两个具体目标。首先，将包含禽肝谷氨酰胺合成酶编码信息的全长克隆pCGS11N在pKK223或其衍生物等表达载体上表达。需要特别关注的是表达产物的表征，尤其是它的构象。初步结果表明，表达产物可能聚集。由于这很可能使其无法导入，因此将建立防止聚集并保持蛋白质处于未展开结构的条件。第二个主要目标将是建立一个体外系统，其中谷氨酰胺合成酶被导入到分离的线粒体。如有必要，将使用原始的将分离线粒体与新翻译的合成酶相结合的技术，尽管使用纯表达产物现在更可取，并且需要建立物种和组织特异性的细胞质输入因子。75%的陆地脊椎动物利用尿液类型的机制来解毒在肝脏氨基酸分解代谢过程中形成的氨。氨，通过谷氨酸脱氢酶反应形成，被谷氨酰胺合成酶转化为谷氨酰胺的酰胺功能，然后进入细胞质，在那里它被用于尿酸合成。在哺乳动物和一些成年两栖动物利用的输尿管机制中，氨在线粒体中转化为瓜氨酸，瓜氨酸随后进入细胞质转化为尿素。这两种途径的共同之处在于，在每种情况下，氨解毒机制都是将线粒体基质中形成的氨转化为一种在线粒体内膜运输过程中不与质子结合的形式，因此不会解耦氧化磷酸化。特别有趣的是，在具有输尿管功能的脊椎动物（如哺乳动物）中，谷氨酰胺合成酶只存在于细胞质中，从不存在于线粒体中，而在具有输尿管功能的蜥目脊椎动物（爬行动物和鸟类）中，谷氨酰胺合成酶定位于肝脏的线粒体，但存在于其他组织（如神经组织）的细胞质中。已经确定了鸟类酶中假定的线粒体靶向序列，而不存在于哺乳动物酶中。然而，鸟类肝脏（线粒体）和神经组织（细胞质）形式的初级氨基酸序列是相同的，这就提出了如何发生差异靶向的问题。本项目旨在了解该酶在蜥脚类动物中靶向的分子事件。从生物化学、细胞生物学、生理学和进化的角度来看，这项工作具有相当大的意义。

Two specific objectives will be pursued during the proposed project period. The first will be expression of the full length clone, pCGS11N, which contains the coding information for avian liver glutamine synthetase, in expression vectors such as pKK223 or various derivatives thereof. Of special concern will be a characterization of the expression product, especially its conformation. Preliminary results indicate the expression product may aggregate. Since this is likely to render it import- incompetent, conditions that prevent aggregation and also keep the protein in an unfolded configuration will be established. The second major objective will be to establish an in vitro system in which glutamine synthetase is imported into isolated mitochondria. If necessary, the original technique of presenting isolated mitochondria with freshly translated synthetase will be used, although the use of the pure expression product is now preferable and is required to establish species- and tissue-specific cytosolic import factors. %%% Seventy-five percent of land vertebrates utilize a uricotelic-type mechanism for detoxifying ammonia formed during hepatic amino acid catabolism. Ammonia, formed via the glutamate dehydrogenase reaction, is converted by glutamine synthetase to the amide function of glutamine which then exits to the cytosol where it is utilized for uric acid synthesis. In the ureotelic mechanism, utilized by mammals and some adult amphibians, ammonia is converted to citrulline in the mitochondria, which then exits to the cytosol to be converted to urea. What these two pathways have in common is that in each case, the ammonia-detoxifying mechanisms are directed toward converting ammonia formed in the mitochondrial matrix to a form that does not bind protons during transit of the inner mitochondrial membrane and therefore does not uncouple oxidative phosphorylation. What is particularly interesting is that, in ureotelic vertebrates (e.g., mammals), the enzyme glutamine synthetase is only found in the cytosol, never in the mitochondria, whereas in the uricotelic sauropsid vertebrates (reptiles and birds) the enzyme is localized to mitochondria in the liver but is cytosolic in other tissues such as neural tissue. A putative mitochondrial targeting sequence in the avian enzyme, not present in the mammalian enzyme, has been identified. However, the primary amino acid sequences of the avian liver (mitochondrial) and neural tissue (cytosolic) forms are identical, raising the question of how the differential targeting occurs. This project aims to understand the molecular events responsible for targeting of the enzyme in sauropsids. The work is of considerable interest from biochemical, cell biological, physiological, and evolutionary perspectives.