A fusion protein between rac and p67phox (1-210) reconstitutes NADPH oxidase with higher activity and stability than the individual components.
Activation of the phagocyte NADPH oxidase, a superoxide-generating enzyme, involves assembly of cytosolic p47(phox), p67(phox), and rac with the membrane-associated cytochrome 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]. In an attempt to stabilize the activity and to investigate the nature of the complex, we have produced fusion proteins between rac and a C-terminal truncated form of p67(phox) (residues 1-210, 67N), which is a minimal active fragment. In a cell-free system, a fusion protein 67N-rac had higher activity and a 3-fold higher affinity than the individual cytosolic proteins, and 67N-Ser3-rac, which has a longer linker, showed a similar activity with the individual proteins. In contrast, rac-67N, a fusion in the opposite orientation, showed considerably lower activity. The enzyme activity reconstituted with 67N-rac showed a 10-fold higher stability and a lower K(m) for NADPH than the individual components. In the absence of p47, 67N-rac fusion protein at a high concentration showed nearly full activation, which was higher than that with the individual components. These results indicate that covalent binding between p67N and rac in the correct order produces a more stable complex than the individual components, suggesting that interactions among the subunits significantly influence the duration of the oxidase activation. On the basis of these findings, we propose a model for the topology among rac, 67N, and cytochrome b(558).
Miyano
K
K
Department of Applied Chemistry, Faculty of Engineering, Ehime University, Matsuyama, Ehime 790-8577, Japan.
Ogasawara
S
S
Han
C H
CH
Fukuda
H
H
Tamura
M
M
D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000235:genetics; D004795:Enzyme Stability / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000378:metabolism*; D013481:Superoxides / Q000378:metabolism; D013997:Time Factors; D020744:rac GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology
D003573:Cytochrome b Group; D010446:Peptide Fragments; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C036888:cytochrome b558; D019255:NADPH Oxidases; D020558:GTP Phosphohydrolases; D020744:rac GTP-Binding Proteins