Regulation of Mitochondrial Nitric-oxide Synthase

描述（由申请人提供）：我们的研究首次为纯化线粒体生产一氧化氮提供了证据。该分子的产生通过氧合肌红蛋白的氧化、电子自旋共振光谱与自旋捕获技术和l -瓜氨酸的产生来证明。这些技术连同纯化的、完整的线粒体（来自大鼠肝脏或肝细胞）的使用，为这些细胞器产生一氧化氮提供了实验支持。进一步的证据提供了线粒体一氧化氮合酶的分离与其他同工异构体的特征。线粒体产生一氧化氮调节了线粒体的耗氧量和ATP的产生。这是通过竞争性抑制细胞色素氧化酶来实现的。因此，一氧化氮表现出的作用不一定是通过激活可溶性鸟苷酸环化酶介导的，它指向不同的靶点，在这种情况下，是细胞色素氧化酶。鉴于这种一氧化氮的产生可能为一种新的调控途径奠定基础——考虑到线粒体在组织中的普遍分布以及这种分子在能量代谢、氧气消耗和氧自由基产生方面的相关作用——研究线粒体中负责这种产生的酶的生物化学变得势在必行。该提案旨在研究通过翻译后修饰对酶的调节，以及这些修饰如何改变或调节酶的靶向和/或其活性。研究mtNOS的生物化学将有助于更好地理解一氧化氮在细胞-线粒体相互作用中与生理状况相关的作用。

DESCRIPTION (provided by applicant): Our studies constituted the first ones providing evidence for a production of nitric oxide by purified mitochondria. The production of this molecule was demonstrated by the oxidation of oxymyoglobin, electron spin resonance spectroscopy with the spin trapping technique, and L-citrulline production. These techniques along with the use of purified, intact mitochondria (from rat liver or from hepatocytes) provided experimental support for a production of nitric oxide by these organelles. Further evidence was furnished by the isolation of a mitochondrial nitric-oxide synthase with characteristics to other isoforms. The production of nitric oxide by mitochondria modulated the oxygen consumption and ATP production of mitochondria. This was accomplished through the competitive inhibition of cytochrome oxidase. Thus, nitric oxide exhibits effects not necessarily mediated via the activation of the soluble guanylate cyclase, pointing at a different target site, in this case, cytochrome oxidase. Given that this production of nitric oxide may establish the basis for a novel regulatory pathway--considering the ubiquitous distribution of mitochondria in tissues and the relevant role this molecule may have on energy metabolism, oxygen consumption, and oxygen free radical production--, it becomes imperative to study the biochemistry of the enzyme responsible for this production in mitochondria. This proposal is aimed at studying the regulation of the enzyme by posttranslational modifications and how these modifications alter or modulate the targeting of the enzyme and/or its activity. Studying the biochemistry of mtNOS will provide a better understanding of the role of nitric oxide in the cell-mitochondria interactions relevant to physiological situations.