Foresi Noelia, Correa-Aragunde Natalia, Santolini Jerome, Lamattina Lorenzo
Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Funes 3250 4to nivel, 7600, Mar del Plata, Argentina.
Laboratoire Stress Oxydant et Détoxication, CNRS, Gif-sur-Yvette, France.
Methods Mol Biol. 2016;1424:149-62. doi: 10.1007/978-1-4939-3600-7_13.
Nitric oxide (NO) functions as a signaling molecule in many biological processes in species belonging to all kingdoms of life. In animal cells, NO is synthesized primarily by NO synthase (NOS), an enzyme that catalyze the NADPH-dependent oxidation of L-arginine to NO and L-citrulline. Three NOS isoforms have been identified, the constitutive neuronal NOS (nNOS) and endothelial NOS (eNOS) and one inducible (iNOS). Plant NO synthesis is complex and is a matter of ongoing investigation and debate. Despite evidence of an Arg-dependent pathway for NO synthesis in plants, no plant NOS homologs to animal forms have been identified to date. In plants, there is also evidence for a nitrate-dependent mechanism of NO synthesis, catalyzed by cytosolic nitrate reductase. The existence of a NOS enzyme in the plant kingdom, from the tiny single-celled green alga Ostreococcus tauri was reported in 2010. O. tauri shares a common ancestor with higher plants and is considered to be part of an early diverging class within the green plant lineage.In this chapter we describe detailed protocols to study the expression and characterization of the enzymatic activity of NOS from O. tauri. The most used methods for the characterization of a canonical NOS are the analysis of spectral properties of the oxyferrous complex in the heme domain, the oxyhemoglobin (oxyHb) and citrulline assays and the NADPH oxidation for in vitro analysis of its activity or the use of fluorescent probes and Griess assay for in vivo NO determination. We further discuss the advantages and drawbacks of each method. Finally, we remark factors associated to the measurement of NOS activity in photosynthetic organisms that can generate misunderstandings in the interpretation of results.
一氧化氮（NO）在所有生命王国的物种的许多生物过程中作为信号分子发挥作用。在动物细胞中，NO主要由一氧化氮合酶（NOS）合成，该酶催化L-精氨酸依赖NADPH氧化生成NO和L-瓜氨酸。已鉴定出三种NOS亚型，即组成型神经元NOS（nNOS）和内皮NOS（eNOS）以及一种诱导型（iNOS）。植物中NO的合成很复杂，是一个正在进行研究和争论的问题。尽管有证据表明植物中存在依赖精氨酸的NO合成途径，但迄今为止尚未鉴定出与动物形式同源的植物NOS。在植物中，也有证据表明存在由细胞质硝酸还原酶催化的依赖硝酸盐的NO合成机制。2010年报道了在微小的单细胞绿藻莱茵衣藻中存在一种植物界的NOS酶。莱茵衣藻与高等植物有共同的祖先，被认为是绿色植物谱系中早期分化类群的一部分。在本章中，我们描述了详细的方案，用于研究莱茵衣藻中NOS的表达及其酶活性的表征。表征典型NOS最常用的方法是分析血红素结构域中氧亚铁复合物的光谱特性、氧合血红蛋白（oxyHb）和瓜氨酸测定以及用于其活性体外分析的NADPH氧化，或使用荧光探针和格里斯测定法进行体内NO测定。我们进一步讨论了每种方法的优缺点。最后，我们指出了与光合生物中NOS活性测量相关的因素，这些因素可能会在结果解释中产生误解。
