BIOCHEMISTRY OF MACROPHAGE NITRIC OXIDE SYNTHESIS

一氧化氮(NO)的生物合成已成为人体内的一个重要因素 健康和疾病。少量产生的NO似乎起到了 信号面朝下，而过量的生产与 多种疾病，包括脓毒症、免疫型糖尿病、炎症性 肠道疾病，类风湿性关节炎，癌症，多发性硬化症， 和移植排斥反应。免疫刺激诱导A蛋白的表达 一氧化氮合酶异构体(INOS)在许多组织中都有表达，包括肺、肝、肺、肺、肝、肾、肺、肝等组织。 肾脏、心脏、平滑肌和肠道。因为iNOS会产生大量的 NO的数量，它的表达越来越与 上述疾病。 我们正在研究小鼠巨噬细胞诱导型一氧化氮合酶的生物化学，它是高度 与人类诱导型一氧化氮合酶同源。巨噬细胞诱导型一氧化氮合酶是一种双结构域酶 含有FAD、FMN、四氢生物蝶呤、血红素和钙调蛋白。在……里面 巨噬细胞和其他细胞，iNOS表达为单体和 二聚体，单体在没有合成的情况下不活跃。我们 假设iNOS的二聚化是其关键的决定因素 激活，并可用作生理和/或药物控制 细胞内iNOS功能的点。我们将用以下方法来检验这一假设 四个具体的实验目标；第一，我们将确定 二聚化在物理上改变了血红素的环境，从而影响了它的 反应性，或者如果二聚揭示了H4生物蝶呤和L的结合位点- 加氧酶结构域中的精氨酸，从而产生酶活性部位。 技术包括各种分光镜、氧化还原电位法和 放射性配基结合。其次，我们将调查二聚化是否 使iNOS还原酶和加氧酶结构域能够进行通信 以一种富有成效的方式以电子方式。技术包括可见性 诱导型一氧化氮合酶非-二聚体结构的光谱和构筑 在结构域组成或氨基酸方面不同的相同亚基 酸序列。第三，我们将设法鉴定特定的蛋白质 参与亚基二聚体相互作用和辅因子结合的区域 INOS加氧酶结构域。这将涉及到测试加氧酶结构域 H4生物蝶呤、血红素和L与精氨酸结合的多肽和片段， 确定它们形成二聚体或拮抗二聚体的能力； 用抗人氧合酶结构域的单抗定位氧合酶结构域 INOS；检查氧合酶结构域突变体的二聚化，辅因子 结合和催化作用；二聚体的结晶 加氧酶结构域。最后，我们将研究iNOS亚单位是否 二聚化是一种重要的生理或药物控制点。 细胞，通过监测随着时间的推移细胞中二聚体的形成，将其与 被认为促进二聚化的细胞因子水平，以及测试 二聚化是否可以通过药理学来控制。总而言之，这 将提供有关二聚体如何形成的全面图景 激活iNOS，以及如何控制它。

Nitric oxide (NO) biosynthesis has emerged as an important factor in human health and diseae. NO generated in small amounts appears to serve a signaling facedown, whereas production of excessive amounts is linked to numerous diseases including sepsis, immune-type diabetes, inflammatory bowel disease, rheumatoid arthritis, carcinogenesis, multiple sclerosis, and transplant rejection. Immunostimulation induces expression of a distinct NO synthase isoform (iNOS) in many tissues, including lung, liver, kidney, heart, smooth muscle, and intestine. Because iNOS generates large amounts of NO, its expression has become increasingly linked to the diseases noted above. We are studying the biochemistry of mouse macrophage iNOS, which is highly homologous to human iNOS. Macrophage iNOS is a bi-domain enzyme that contains FAD, FMN, tetrahydrobiopterin, heme, and calmodulin. In macrophages and other cells, iNOS is expressed as a mixture of monomers and dimers, with the monomer being inactive regarding NO synthesis. We hypothesize that dimerization of iNOS is a key determinant in its activation, and may serve as a physiologic and/or pharmacologic control point for iNOS function within cells. We will test this hypothesis with four specific experimental objectives; First, we will determine if dimerization physically alters the heme environment, thus affecting its reactivity, or if dimerization unmasks binding sites for H4biopterin and L- arginine in the oxygenase domain, thus creating the enzyme active site. Techniques include a variety of spectroscopies, redox potentiometry, and radioligand binding. Secondly, we will investigate whether dimerization enables the iNOS reductase and oxygenase domains to communicate electronically in a productive manner. Techniques include visible spectroscopy and creation of iNOS heterodimeric structures composed of non- identical subunits that differ with regard to domain composition or amino acid sequence. Thirdly, we will seek to identify the specific protein regions involved in subunit dimeric interaction and cofactor binding within the iNOS oxygenase domain. This will involve testing oxygenase domain peptides and fragments for H4biopterin, heme, and L-arginine binding, determining their ability to form dimers, or antagonize dimerization; mapping the oxygenase domain with monoclonal antibodies raised against iNOS; examining oxygenase domain mutants for dimerization, cofactor binding, and catalytic function; and crystallization of the dimeric oxygenase domain. Lastly, we will investigate whether iNOS subunit dimerization is an important physiologic or pharmacologic control point in cells, by monitoring dimer formation in cells over time, relating it to levels of cellular factors thought to promote dimerization, and testing whether dimerization can be manipulated pharmacologically. Together, this will provide a comprehensive picture regarding how dimer formation activates iNOS, and how it can be controlled.