BIOCHEMISTRY OF MACROPHAGE NITRIC OXIDE SYNTHESIS

一氧化氮（NO）的生物合成已成为人体内的重要因素， 健康与疾病 少量产生的NO似乎有助于 信号面朝下，而过量的生产与 许多疾病，包括败血症、免疫型糖尿病、炎性 肠道疾病，类风湿性关节炎，致癌作用，多发性硬化症， 和移植排斥反应 免疫刺激诱导a 在许多组织，包括肺，肝， 肾脏、心脏、平滑肌和肠。 因为iNOS产生大量 大量的NO，其表达越来越多地与 上面提到的疾病。 我们正在研究小鼠巨噬细胞iNOS的生物化学， 与人iNOS同源。 巨噬细胞iNOS是一种双结构域酶， 含有FAD、FMN、四氢生物蝶呤、血红素和钙调素。 在 在巨噬细胞和其他细胞中，iNOS表达为单体的混合物， 二聚体，其中单体对于NO合成无活性。 我们 假设iNOS二聚化是其 激活，并可作为生理和/或药理学控制 iNOS在细胞内的功能。 我们将测试这个假设， 四个具体的实验目标;首先，我们将确定， 二聚化在物理上改变了血红素环境，从而影响其 反应性，或者如果二聚化暴露了H4生物蝶呤和L- 精氨酸在加氧酶结构域，从而创建酶的活性位点。 技术包括各种光谱法、氧化还原电位测定法和 放射性配体结合。 其次，我们将研究二聚化是否 使iNOS还原酶和加氧酶结构域能够 以生产性的方式。 技术包括可视 光谱学和由非- 在结构域组成或氨基方面不同的相同亚基 酸性序列 第三，我们将寻求确定特定的蛋白质 参与亚基二聚体相互作用和内辅因子结合的区域 iNOS氧化酶结构域。 这将涉及测试加氧酶域 用于H4生物蝶呤、血红素和L-精氨酸结合的肽和片段， 确定它们形成二聚体或拮抗二聚化的能力; 用单克隆抗体定位加氧酶结构域， 检测加氧酶结构域突变体的二聚化、辅因子 结合和催化功能;以及二聚体的结晶 加氧酶结构域。 最后，我们将研究iNOS亚基是否 二聚化是一个重要的生理学或药理学控制点， 细胞，通过监测细胞中随时间的二聚体形成，将其与 被认为促进二聚化的细胞因子水平， 二聚化是否可以被操纵。 在一起，这 将提供一个全面的图片，关于如何二聚体形成 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.