Fused p47(phox) and p67(phox) truncations efficiently reconstitute NADPH oxidase with higher activity and stability than the individual components

Activation of the neutrophil NADPH oxidase occurs via assembly of the cytos olic regulatory proteins p47(phox), p67(phox), and Rac with the membrane-as sociated flavocytochrome b(558). Following cell-free activation, enzymatic activity is highly labile (Tamura, M,, Takeshita, M,, Curnutte, J. T,, Uhli nger, D, J,, and Lambeth, J, D, (1992) J, Biol, Chem, 267, 7529-7538), To t ry to stabilize the activity and investigate the nature of the complex, fus ion proteins between p47N-(1-286) and p67N-(1-210) were constructed. In a c ell-free system, a fusion protein, p67N-p47N, had an 8-fold higher efficien cy and produced a higher activity than the individual proteins, and also re sulted in an 8-fold improved efficiency for Rac and a lowered K, for NADPH, Of, generating activity was remarkably stabilized by using p67N-p47N, The cytosolic proteins fused in the opposite orientation, p47N-p67N, showed sim ilar activity and stability as individual proteins, but with a 4-fold impro ved efficiency compared with the individual cytosolic factors. In the syste m efficiency for Rac and affinity for NADPH were also higher than those wit h the nonfused components. Interestingly, the p67N-p47N showed nearly full activation in the absence of an anionic amphifile in a cell-free system con taining cytochrome b(558) relipidated with phosphatidylinositol- or phospha tidylserine-enriched phospholipid mixtures, From the results we consider mu ltiple roles of anionic amphifiles in a cell-free activation, which could b e substituted by our system. The fact that a fusion produces a more stable complex indicates that interactions among components determine the longevit y 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.