Rockel Peter, Strube Frank, Rockel Andra, Wildt Juergen, Kaiser Werner M
Forschungszentrum Jülich GmbH, Institut für Biologie des Stoffaustauschs, 52425 Jülich, Germany.
J Exp Bot. 2002 Jan;53(366):103-10.
NO (nitric oxide) production from sunflower plants (Helianthus annuus L.), detached spinach leaves (Spinacia oleracea L.), desalted spinach leaf extracts or commercial maize (Zea mays L.) leaf nitrate reductase (NR, EC 1.6.6.1) was continuously followed as NO emission into the gas phase by chemiluminescence detection, and its response to post-translational NR modulation was examined in vitro and in vivo. NR (purified or in crude extracts) in vitro produced NO at saturating NADH and nitrite concentrations at about 1% of its nitrate reduction capacity. The K(m) for nitrite was relatively high (100 microM) compared to nitrite concentrations in illuminated leaves (10 microM). NO production was competitively inhibited by physiological nitrate concentrations (K(i)=50 microM). Importantly, inactivation of NR in crude extracts by protein phosphorylation with MgATP in the presence of a protein phosphatase inhibitor also inhibited NO production. Nitrate-fertilized plants or leaves emitted NO into purified air. The NO emission was lower in the dark than in the light, but was generally only a small fraction of the total NR activity in the tissue (about 0.01-0.1%). In order to check for a modulation of NO production in vivo, NR was artificially activated by treatments such as anoxia, feeding uncouplers or AICAR (a cell permeant 5'-AMP analogue). Under all these conditions, leaves were accumulating nitrite to concentrations exceeding those in normal illuminated leaves up to 100-fold, and NO production was drastically increased especially in the dark. NO production by leaf extracts or intact leaves was unaffected by nitric oxide synthase inhibitors. It is concluded that in non-elicited leaves NO is produced in variable quantities by NR depending on the total NR activity, the NR activation state and the cytosolic nitrite and nitrate concentration.
通过化学发光检测，持续跟踪向日葵植株（Helianthus annuus L.）、离体菠菜叶片（Spinacia oleracea L.）、脱盐菠菜叶提取物或市售玉米（Zea mays L.）叶片硝酸还原酶（NR，EC 1.6.6.1）产生的一氧化氮（NO）作为气相中的NO排放，并在体外和体内研究其对翻译后NR调节的反应。体外，在饱和NADH和亚硝酸盐浓度下，NR（纯化的或粗提物中的）产生的NO约为其硝酸盐还原能力的1%。与光照叶片中的亚硝酸盐浓度（10 microM）相比，亚硝酸盐的米氏常数（K(m)）相对较高（100 microM）。生理硝酸盐浓度（K(i)=50 microM）对NO产生有竞争性抑制作用。重要的是，在存在蛋白磷酸酶抑制剂的情况下，用MgATP进行蛋白磷酸化使粗提物中的NR失活也抑制了NO的产生。硝酸盐施肥的植物或叶片向净化空气中排放NO。黑暗中的NO排放低于光照下，但通常仅占组织中总NR活性的一小部分（约0.01 - 0.1%）。为了检查体内NO产生的调节情况，通过缺氧、饲喂解偶联剂或AICAR（一种细胞可渗透的5'-AMP类似物）等处理人工激活NR。在所有这些条件下，叶片中亚硝酸盐积累浓度超过正常光照叶片中的浓度达100倍，尤其是在黑暗中NO产生急剧增加。叶片提取物或完整叶片产生的NO不受一氧化氮合酶抑制剂的影响。得出的结论是，在未受刺激的叶片中，NR根据总NR活性、NR激活状态以及胞质亚硝酸盐和硝酸盐浓度产生数量可变的NO。
