Isolation and cloning the nitric oxide synthase from Arabidopsis

在20世纪80年代末，人们发现一氧化氮在动物和植物中起信号作用，而不是有毒的副产品。在动物系统中已经发现了三种不同的NOS（即nNOS、eNOS和iNOS）。在植物中，情况仍然是模棱两可的。已经提出了三种不同的NO产生源。第一种是通过硝酸还原酶（NR）还原亚硝酸盐，第二种是通过线粒体电子传递产生基于亚硝酸盐的NO，第三种是未知的一氧化氮合酶（NOS）。所有来源都在讨论中（见2.1）。克隆植物NOS已经尝试了几次，但没有成功，因为所使用的探针是异源的，并且没有显示出与拟南芥基因组的任何同源性。因此，我们决定首先对NOS蛋白进行纯化，目的是得到一段可以作为同源探针筛选拟南芥基因组的氨基酸序列，在此我们要完成NOS蛋白的分离。我们建立了一种NOS的测定方法，该方法可重复，以精氨酸为底物，同时产生瓜氨酸和NO（1：1）。我们用ESR测量NO的产生，用2D-TLC或HPLC测量瓜氨酸的产生。我们证明了辅因子的要求，并知道这种酶是显着不同的所有已知的NOS。以该测定为工具，我们建立了将酶活性结合到树脂上的系统，并且可以以400倍的富集活性来富集酶。

In the late 1980s it was detected that nitric oxide acts as a signal rather than being a toxic by-product in animals and plants. In animal systems three different NOS have been found (i.e. nNOS, eNOS and iNOS). In plants the situation is still ambiguous. Three different sources for NO production have been suggested. The first is the reduction of nitrite by nitrate reductase (NR), the second is the nitrite based NO production by the mitochondrial electron transport and the third is an unknown nitric oxide synthase (NOS). All sources are under debate (see 2.1). Cloning the plant NOS has been tried several times without success as the probes used were heterologous and did not show any homology to the Arabidopsis genome. Therefore we decided to first purify the NOS protein with the aim to get an amino acid sequence which can be used as homologous probe to screen the Arabidopsis genome.Here we want to finish the isolation of the NOS protein. We established an assay for NOS which is reproducible producing both, citrulline and NO (1:1) from arginine as substrate. We measured NO production with ESR and the production of citrulline by 2D-TLC or HPLC. We proved the cofactor requirement and know that this enzyme is significantly different to all known NOS. With this assay as a tool we established a system to bind the enzyme activity to resins and can elute the enzyme with 400 fold enriched activity.