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.
Abo
A
A
Department of Medicine, University College London, Rayne Institute, United Kingdom.
Boyhan
A
A
West
I
I
Thrasher
A J
AJ
Segal
A W
AW
D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D003001:Cloning, Molecular; D003573:Cytochrome b Group / Q000097:blood*; D003600:Cytosol / Q000201:enzymology; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000097:blood* / Q000235:genetics; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D010957:Plasmids; D011994:Recombinant Proteins / Q000378:metabolism
D003573:Cytochrome b Group; D011994:Recombinant Proteins; C035130:cytochrome b245; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases