Metabolic Compartmentation of Vertebrate Glutamine Synthetase

该项目的总体目标是阐明在脊椎动物进化过程中导致肝组织中谷氨酰胺合成酶线粒体区室的可能分子事件。这种酶的定位使得尿液作为一种主要的排泄性氮代谢得以发展。大多数线粒体蛋白在细胞核中编码，在细胞质中翻译，并通过n端信号序列靶向线粒体。预酶的较大尺寸使其能够与成熟酶区分开来。我们已经证明，这可能是正确的，对于依丝木鳖肝脏线粒体谷氨酰胺合成酶，我们现在打算表征前酶的进口依丝木鳖肝脏线粒体在体内和体外。我们还发现，在鸟类和短吻鳄中，肝脏中新生的谷氨酰胺合成酶肽和成熟的谷氨酰胺合成酶肽之间没有可检测到的大小差异，尽管其线粒体基质定位于组织中。为了确定禽谷氨酰胺合成酶是否存在内部靶向序列，我们将分别从肝脏和大脑中克隆谷氨酰胺合成酶mRNA cDNA并进行测序。酶留在后一种组织的细胞质中，因此比较推断出的两种氨基酸序列应该可以为肝酶输入信号序列的性质和位置提供一些线索。我们也将得到依山蛛和可能的陆龟的谷氨酰胺前合成酶的推断序列进行比较。这种比较应该给进化过程中可能发生的变化提供一些指示，从原始脊椎动物将酶靶向肝脏线粒体的经典前序机制到高级尿酸脊椎动物中使用的任何机制。​研究酶靶向特定细胞区室的分子机制对细胞生物学和生物化学具有重要意义。酶活性的调控是理解代谢的关键问题。//

The overall goal of this project is an elucidation of the possible molecular events that led to the mitchondrial compartmentation of glutamine synthetase in liver tissue during vertebrate evolution. This localization of the enzyme allowed for the development of uricoteley as a major kind of excretory nitrogen metabolism. Most mitochondrial proteins are coded for in the nucleus, translated in cytosol, and targeted to mitochondria by N-terminal signal sequences. The larger size of the pre-enzyme allows it to be distinguished from the mature enzyme. We have shown this to probably be true for elasmobranch liver mitochondrial glutamine synthetase and we now intend to characterize the import of the pre-enzyme by elasmobranch liver mitochondria in vivo and in vitro. We have also found that in birds and alligators there is no detectable size difference between the nascent and mature glutamine synthetase peptide in liver, despite its mitochondrial matrix localization in tissue. In order to determine if there is an internal targeting sequence in the avian enzyme, we will clone and sequence glutamine synthetase mRNA cDNA from both liver and brain. The enzyme remains in the cytosol in the latter tissue so a comparison of the deduced amino acid sequences for the two should give some clue as to the nature and location of the signal sequence used for import of the liver enzyme. We also will obtain the deduced sequences for elasmobranch and possibly tortoise pre-glutamine synthetase for comparative purposes. This comparison should give some indication of the possible changes that have occured during evolution that led from a classical presequence mechanism for targeting of the enzyme to liver mitochondria in primitive vertebrates to whatever the mechanism used in higher uricotelic vertebrates. This research on the molecular mechanisms of how enzymes are targeted to specific cell compartments is significant to cell biology and biochemistry. The regulation of enzyme activity is a key question for understanding metabolism.//