Fused p47phox and p67phox 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 cytosolic regulatory proteins p47(phox), 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.
Ebisu
K
K
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan.
Nagasawa
T
T
Watanabe
K
K
Kakinuma
K
K
Miyano
K
K
Tamura
M
M
D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli; D005818:Genetic Engineering; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology; D010750:Phosphoproteins / Q000378:metabolism*; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D020744:rac GTP-Binding Proteins / Q000378:metabolism
D003573:Cytochrome b Group; D046911:Macromolecular Substances; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; C036888:cytochrome b558; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D020744:rac GTP-Binding Proteins