Activation of the neutrophil NADPH oxidase occurs via assembly of the cytosolic regulatory proteins p47phox , p67 phox , and Rac with the membrane-associated flavocytochrome b 558 . Following cell-free activation, enzymatic activity is highly labile (Tamura, M., Takeshita, M., Curnutte, J. T., Uhlinger, D. J., and Lambeth, J. D. (1992) J. Biol. Chem. 267, 7529 -7538). To try to stabilize the activity and investigate the nature of the complex, fusion proteins between p47N-(1-286) and p67N-(1-210) were constructed. In a cell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficiency and produced a higher activity than the individual proteins, and also resulted in an 8-fold improved efficiency for Rac and a lowered K m for NADPH. O 2 . generating activity was remarkably stabilized by using p67N-p47N. The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed similar activity and stability as individual proteins, but with a 4-fold improved efficiency compared with the individual cytosolic factors. In the system efficiency for Rac and affinity for NADPH were also higher than those with the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system containing cytochrome b 558 relipidated with phosphatidylinositol-or phosphatidylserine-enriched phospholipid mixtures. From the results we consider multiple roles of anionic amphifiles in a cell-free activation, which could be substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevity of the complex. Based on the findings we propose a model for the topology among p47N, p67N, and cytochrome b 558 in the active complex.The superoxide-generating phagocyte NADPH oxidase (Phox/Nox-2) functions in host defense against microbial infection (1, 2). The enzyme is dormant in resting cells and becomes active upon cell stimulation. The activation is thought to occur via assembly of cytosolic components, p47 phox (p47), 1 p67 phox (p67), and Rac with the membrane-associated flavocytochrome b 558 (cyt. b 558 ), which consists of p22 phox (p22) and gp91 phox (gp91) although the structure of the complex has remained unclear (3-6). Other two factors p40 phox and rap1A are also assumed to be involved in the enzyme regulation although they are not essential for the activation.The activated NADPH oxidase is highly labile, complicating investigations of the subunit structure and preventing isolation of the active enzyme complex (7). In a previous study (8) using a cell-free system consisting of cytosol and plasma membrane (PM) we showed that the stability is dramatically improved by chemical cross-linking, and suggested that the deactivation is caused by dissociation of proteins from the complex. Crosslinking was useful in stabilizing the oxidase, but it was difficult to isolate the active complex because the cross-linked complex resisted solubilization (8). We have also attempted cross-linkin...

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L << p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.

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