Nox1, a homologue of gp91
phox subunit of the phagocyte NADPH oxidase, is responsible for spontaneous superoxide (O
2−) 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 O
2− 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 O
2−-generating ability of LPS-induced Nox1 in cell-free reconstitution assays showed that: 1) Nox1 is unable to generate O
2− per se; 2) the combination of Nox1 with GMC cytosol is insufficient for a significant O
2− generation; 3) the combination with neutrophil cytosol enables Nox1 to act like gp91
phox,
i.e., to produce O
2− appreciably in response to myristate stimulation; 4) Nox1 prefers NADPH to NADH under the
in vitro assay with neutrophil cytosol plus myristate (
Km=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
b558 to generate plenty of O
2−, 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.
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