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谷氨酰胺合成酶线粒体区隔的可能分子事件。这种酶的定位使尿酸钙发展成为一种主要的排泄性氮代谢。大多数线粒体蛋白在细胞核中编码，在胞浆中翻译，并通过N-末端信号序列靶向线粒体。较大的前酶大小使其有别于成熟酶。我们已经证明这一点很可能是真的，对于弹簧鱼肝脏线粒体谷氨酰胺合成酶，我们现在打算在体内和体外表征该酶前酶通过弹簧肌肝脏线粒体的输入。我们还发现，在鸟类和鳄鱼中，尽管线粒体基质定位于组织中，但肝脏中新生的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。为了确定禽类酶中是否存在内靶向序列，我们从肝脏和脑中克隆并测序了谷氨酰胺合成酶mRNA的序列。这种酶仍然留在后一种组织的胞浆中，因此对两者推导的氨基酸序列的比较应该会为用于进口肝酶的信号序列的性质和位置提供一些线索。为了进行比较，我们还将获得甲鱼和甲鱼谷氨酰胺前合成酶的推导序列。这种比较应该为进化过程中可能发生的变化提供一些迹象，这些变化导致原始脊椎动物从经典的将酶定向到肝脏线粒体的前序列机制，到高等尿液脊椎动物使用的任何机制。研究酶如何靶向特定的细胞室的分子机制对细胞生物学和生物化学具有重要意义。酶活性的调节是了解新陈代谢的关键问题。

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.//