Enzyme system responsible for the generation of superoxide anion in red tide plankton, Chattonella marina

滨Chattonella marina是一种鞭藻，是红潮中最具毒性的浮游植物之一，对鱼类，特别是对黄尾七尾藻有很高的毒性。虽然查顿菌毒性作用的确切机制尚不清楚，但已经证明，动脉血氧分压的降低是鱼类暴露于查顿菌后最早观察到的生理障碍。暴露于c \marina的鱼类的生理和组织学研究也表明；过多的粘液导致呼吸水流通过鳃片受阻，干扰氧气的转移，导致窒息。已知C. marina在正常生理条件下会产生活性氧（ROS），如超氧阴离子（O2-）、过氧化氢（H2O2）和羟基自由基（- oh）。虽然有几条证据表明活性氧参与了暴露于C. marina的鱼类的死亡，但其机制；C. marina中ROS生成的机制尚不清楚。在本研究中，我们发现从C. marina细胞制备的无细胞上清显示NAD(P) h依赖性的O2生成；这种反应被哺乳动物NADPH氧化酶的特异性抑制剂二苯乙酮（DPI）所抑制。用抗人中性粒细胞细胞色素b558大亚基（91 kDa）的抗体对C. marina的无细胞上清进行免疫印迹分析，检测到约110 kDa的主带。此外，间接免疫荧光法证实了该抗体识别的抗原表位的细胞表面定位。这些结果为C. marina参与类似中性粒细胞NADPH氧化酶的酶系统作为O2生产的来源提供了证据。

Chattonella marina, a raphidophycean flagellate, is one of the most noxious red tide phytoplankton and is highly toxic to fish, especially to yellowtail Seriola quinqueradiata. Although the precise mechanism of the toxic action of Chattonella sp. remains unclear, it has been demonstrated that a decrease in oxygen partial pressure of arterial blood is the earliest physiological disturbance observed in fish after exposure" to C. marina. Physiological and histological studies of fish exposed to C.\marina also'suggested that;the blockade of respiratory water flow through the gill lamellae caused by excessive mucusiinterferes with O2 transfer, resulting in asphyxia. C. marina is known to produce reactive oxygen species (ROS) such as superoxide anion (O2-), hydrogen peroxide (H2O2), and hydroxyl radical (-OH) under normal physiological conditions. Although several lines of evidence suggest that ROS are involved in the mortality of fish exposed to C. marina, the mechanism; of ROS generation in C. marina is remained to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O2- generation; and this response was inhibited by diphenyleneiodonium (DPI), a specific inhibitor of mammalian NADPH oxidase. When the cell-free supernatant of C. marina was analyzed by immunoblotting using antibody raised against the human neutrophil cytochrome b558 large subunit (91 kDa), a main band with approximately 110 kDa was detected. Furthermore, the cell surface localization of the epitope recognized with this antibody was demonstrated in C. marina by indirect immunofluorescence. These results provide evidence for the involvement of enzymatic system analogous to the neutrophil NADPH oxidase as a source of O2- production in C. marina.