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.
