Nitric oxide synthase(NOS) like enzyme mediated nitric oxide(NO) generation by harmful red tide phytoplankton, Chattonella marina

已知单细胞海洋浮游植物查顿菌具有强大的杀鱼活性。以往的研究表明，红鲌可产生活性氧（ROS），并提出了活性氧介导的鱼毒性机制。事实上，一些研究已经证明查顿菌可以产生活性氧（ROS）。此外，我们利用电子自旋共振（ESR）光谱和自旋阱5,5-二甲基-1-吡咯- n -氧化物和n -t-丁基-a-苯基硝基酮的研究表明，C. marina产生羟基自由基（OH）。由于活性氧通常被认为对生物体具有毒性，因此查顿菌产生的活性氧可能至少部分参与了鱼类死亡机制。此外，我们还发现C. marina对一种海洋细菌溶藻弧菌具有ros介导的毒性作用。除查托纳菌外，另一种鞭毛虫赤石异虫对虹鳟鱼也表现出ros介质毒性。因此，ROS的产生似乎是鞭藻类的共同特征。然而，确切的毒性机制仍然存在争议。在本研究中，我们获得了C. marina在正常生长条件下产生一氧化氮（NO）的证据。利用NO与鲁米诺- h_2o_2的化学发光（CL）反应检测C. marina细胞悬浮液中的NO。在本实验中，在C. marina中观察到明显的CL以细胞数量依赖的方式存在，并且通过添加羧基ptio（一种特异性NO清除剂）来减少这种情况。采用重氮反应阳性物质（NO_x）测定的分光光度法和采用高特异性NO荧光指示剂的荧光法也证实了C. marina产生NO。特异性NO合成酶（NOS）抑制剂L-NAME显著降低了C. marina的NO水平。添加l -精氨酸使NO水平升高，而添加NaNO_2对NO水平无影响。这些结果表明，一种类似nos的酶主要负责C. marina中NO的生成。少

The unicellular marine phytoplankton Chattonella marina is known to exhibit potent fish-killing activity. Previous studies have demonstrated that C. marina produces reactive oxygen species (ROS), and ROS-mediated ichthyotoxic mechanism has been postulated. In fact, several studies have demonstrated that Chattonella spp. generate reactive oxygen species (ROS). Furthermore, our studies using electron spin resonance (ESR) spectroscopy with the spin traps 5,5-dimethyl-1-pyrroline-N-oxide and N-t-butyl-a-phenylnitrone showed that C. marina generates hydroxyl radical (OH). Since ROS are generally considered to be toxic against living organisms, the ROS generated by Chattonella spp. may at least partly involve in the fish-kill mechanism. Furthermore, we have found that C. marina exhibited ROS-mediated toxic effect on a marine bacterium, Vibrio alginolyticus. In addition to Chattonella spp., it has been reported that another raphidophycean flagellate, Heterosigma akashiwo also showed ROS-media … More ted toxicity on rainbow trout. Thus, it seems likely that the production of ROS is a common feature of raphidophycean flagellates. However, the exact toxic mechanism is still controversial. In this study, we obtained evidence that C. marina produces nitric oxide (NO)under normal growth conditions. We utilized chemiluminescence (CL)reaction between NO and luminol-H_2O_2 to detect NO in C. marina cell suspensions. In this assay, significant CL was observed in C. marina in a cell-number dependent manner, and this was diminished by the addition of carboxy-PTIO, a specific NO scavenger. The NO generation by C. marina was also confirmed by a spectrophotometric assay based on the measurement of the diazo-reaction positive substances (NO_x)and by fluorometric assay using highly specific fluorescent indicator of NO. The NO level in C. marina was significantly decreased by L-NAME, a specific NO synthase (NOS)inhibitor. The addition of L-arginine resulted in the increase in NO level, whereas NaNO_2 had no effect. These results suggest that a NOS-like enzyme is mainly responsible for NO generation in C. marina. Less