Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245.
Activation of the NADPH oxidase of phagocytes in the cell-free system requires the association of several cytosolic components with membrane- bound cytochrome b. In this study we were able to fully reconstitute NADPH oxidase activity in the cell-free system with three recombinant proteins: p67-phox, p47-phox, p21rac1, and pure cytochrome b-245. Activity was dependent upon the concentration of the proteins, with maximal activity observed with roughly equimolar ratios of the cytochrome b and p67-phox (133 and 163 mol/s/ mol, respectively) and concentrations of the other two proteins approximately 1 order of magnitude greater. No activity was observed in the absence of any one of these components. In addition, activation was dependent upon p21rac1 being preloaded with GTP, the cytochrome b being reconstituted with lipid, and the presence of FAD during activation. Half-maximal activity was observed at a concentration of NADPH of approximately 50 microM. These findings confirm our recent description of the membrane-bound cytochrome b as a FAD-containing flavocytochrome b containing the NADPH binding site, and implicate the three cytosolic proteins in its activation.[1]References
- Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245. Abo, A., Boyhan, A., West, I., Thrasher, A.J., Segal, A.W. J. Biol. Chem. (1992) [Pubmed]
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