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标题：Superoxide Generation by Nox1 in Guinea Pig Gastric Mucosal Cells Involves a Component with p67phox-Ability
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作者：Lucia Satiko Yoshida ; Satoshi Nishida ; Takashi Shimoyama 等
期刊名称：Biological and Pharmaceutical Bulletin
印刷版ISSN：0918-6158
电子版ISSN：1347-5215
出版年度：2004
卷号：27
期号：2
页码：147-155
DOI：10.1248/bpb.27.147
出版社：The Pharmaceutical Society of Japan
摘要：Nox1, a homologue of gp91 ^phox subunit of the phagocyte NADPH oxidase, is responsible for spontaneous superoxide (O₂⁻) generation in guinea pig gastric mucosal cells (GMC), but involvement of regulatory components (p67 ^phox , p47 ^phox , and Rac) which are essential in phagocytes remains unknown. Here, we aimed to figure out how Nox1 of GMC achieves an active oxidase status. GMC in primary culture show low O₂⁻ generation but acquire a 9-fold higher activity when cultured with Helicobacter pylori lipopolysaccharide (LPS), in correlation with a far increased Nox1 expression. Investigation into the O₂⁻-generating ability of LPS-induced Nox1 in cell-free reconstitution assays showed that: 1) Nox1 is unable to generate O₂⁻ per se ; 2) the combination of Nox1 with GMC cytosol is insufficient for a significant O₂⁻ generation; 3) the combination with neutrophil cytosol enables Nox1 to act like gp91 ^phox , i.e. , to produce O₂⁻ appreciably in response to myristate stimulation; 4) Nox1 prefers NADPH to NADH under the in vitro assay with neutrophil cytosol plus myristate ( K _m=10.4 μ M ); 5) substitution of neutrophil cytosol by a set of recombinant cytosolic components (rp67 ^phox , rp47 ^phox , Rac2) is, however, ineffective and still requires GMC cytosol. Thus, Nox1 probably requires an additional cytosolic factor(s). In contrast, GMC cytosol enables cytochrome b ₅₅₈ to generate plenty of O₂⁻, on condition that rp47 ^phox is added. This result suggests that GMC cytosol contains a component with p67 ^phox -ability, and also Rac, but lacks p47 ^phox . These data indicate that GMC Nox1 requires at least a p67 ^phox counterpart and Rac to acquire NADPH oxidase activity.
关键词：Nox1;gp91phox;cytochrome b558;NADPH oxidase;neutrophils;cell-free reconstitution assay