The existence of NADPH oxidase in fish neutrophils

在呼吸爆发期间，鳗鱼中性粒细胞消耗氧气，并产生活性氧。超氧化物生产相当于氧气消耗，过氧化氢生产大约是氧气消耗或超氧化物生产的一半。这种关系在化学计量上与NADPH氧化酶在哺乳动物吞噬细胞呼吸爆发时引起的氧代谢相同，表明鱼类中性粒细胞中存在与哺乳动物相似的氧自由基机制。刺激诱导的中性粒细胞消耗的细胞色素和产生的氧自由基显着高于未诱导的中性粒细胞。细胞色素b558是存在于哺乳动物吞噬细胞中的NADPH氧化酶的膜结合组分，由大亚基和小亚基组成。制备了针对人细胞色素b558大亚基羧基末端合成肽的抗Li抗体。用日本细胞中性粒细胞裂解物进行Western blotting，该抗体在80-110 kDa与人中性粒细胞样本均能特异性地检测到一条宽带。荧光抗体技术表明，只有当细胞被洗涤剂渗透后，抗体才能识别存在于鳗鱼中性粒细胞中的表位。这些结果表明，鱼类中性粒细胞中存在细胞色素b558的大亚基，其羧基末端暴露在细胞质中，提示NADPH氧化酶在不同物种的中性粒细胞中是保守的。

During respiratory burst, eel neutrophils consumed oxygen, and produced active oxigens. Superoxide production was equivalent to oxygen consumption, and hydrogen peroxide production was approximately half of oxygen consumption or superoxide production. This correlation is stoichiometrically identical to that of oxygen metabolism caused by NADPH oxidase during the respiratory burst in mammalian phagocytes, demonstrating the existence of the similar mechanism of oxygen radicals in fish neutrophils to mammals. The amounts of ocygen consumed and oxygen radicals produced in irritant-elicited neutrophils were significantly higher than those in unelicited neutrophils. This indicates that elicited neutrophils which possess high bactericidal activity were released from hematopoietic tissue to inactivate an exclude foreign irritants.Cytochrome b558 is a membrance-bound component of NADPH oxidase present in mammalian phagocytes, and consisted of large and small subunits. Anti-Li antigocy against the synthetic peptide corresponding to the carboxyl terminal of human cytochrome b558 large subunit was produced. When western blotting was performed against Japanese ell neutrophil lysate, a broad band was specifically detected with the antibody at 80-110 kDa as well as human neutrophil smaple. Fluorescent antibody technique shows that the antibody recognized the epitope existed in eel neutrophils only after the cells were permeabilized with detergent. These results demonstrate tha the large subunit of cytochrome b558 exists in fish neutrophils and the caboxyl terminal is exposed to cytoplasmic sede, and suggest that NADPH oxidase is conserved in neutrophils across species.