Isolation and cloning the nitric oxide synthase from Arabidopsis

点击翻译按钮获取中文摘要

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.