Mechanism of superoxide anion generation in the toxic red tide phytoplankton Chattonella marina: possible involvement of NAD(P)H oxidase.
Red tide phytoplankton Chattonella marina is known to produce reactive oxygen species (ROS), such as superoxide anion (O(2)(-)), hydrogen peroxide (H(2)O(2)) and hydroxyl radical (&z.rad;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 remains to be clarified. In this study, we found that the cell-free supernatant prepared from C. marina cells showed NAD(P)H-dependent O(2)(-) generation, and this response was inhibited by diphenyleneiodonium, an 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 (gp91phox), a main band of approximately 110 kDa was detected. The cell surface localization of the epitope recognized with this antibody was also demonstrated in C. marina by indirect immunofluorescence. Furthermore, Southern blot analysis performed on genomic DNA of C. marina with a probe covering the C-terminal region of gp91phox suggested the presence of a single-copy gene coding for gp91phox homologous protein in C. marina. These results provide evidence for the involvement of an enzymatic system analogous to the neutrophil NADPH oxidase as a source of O(2)(-) production in C. marina.
Kim
D
D
Division of Biochemistry, Faculty of Fisheries, Nagasaki University, N agasaki, Japan.
Nakamura
A
A
Okamoto
T
T
Komatsu
N
N
Oda
T
T
Iida
T
T
Ishimatsu
A
A
Muramatsu
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D004141:Dinoflagellida / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007093:Imidazoles; D008163:Luminescent Measurements; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology; D010933:Plankton / Q000469:parasitology*; D011719:Pyrazines; D017386:Sequence Homology, Amino Acid; D013481:Superoxides / Q000032:analysis / Q000378:metabolism*
D007093:Imidazoles; D008562:Membrane Glycoproteins; D011719:Pyrazines; D013481:Superoxides; C063412:2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo(1,2-alpha)pyrazin-3-one; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases