Simultaneous presence of p47(phox) and flavocytochrome b-245 are required for the activation of NADPH oxidase by anionic amphiphiles. Evidence for an intermediate state of oxidase activation.
We have examined the kinetics of NADPH oxidase activation induced by arachidonic acid or SDS in a cell-free system using mixtures of recombinant Phox proteins and purified flavocytochrome b-245. Activation of oxidase activity required the simultaneous presence of p47(phox), flavocytochrome b-245, and the anionic amphiphile. The activation of electron transfer reactions was much more rapid when iodonitrotetrazolium violet was used as electron acceptor than when oxygen alone was the acceptor. We propose that this difference represents an intermediate activation state of NADPH oxidase in which electron flow can proceed from NADPH to enzyme flavin (and hence to iodonitrotetrazolium violet) but not from flavin to heme (or not between the hemes). A model for NADPH oxidase activation is presented that is consistent with these observations.
Cross
A R
AR
Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California 92037, USA. scross@scripps.edu
Erickson
R W
RW
Curnutte
J T
JT
D000838:Anions / Q000494:pharmacology; D016718:Arachidonic Acid / Q000494:pharmacology; D003573:Cytochrome b Group / Q000378:metabolism*; D004579:Electron Transport; D004789:Enzyme Activation / Q000187:drug effects; D005415:Flavins; D005420:Flavoproteins / Q000378:metabolism; D006418:Heme; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism; D013501:Surface-Active Agents / Q000494:pharmacology*; D013778:Tetrazolium Salts
D000838:Anions; D003573:Cytochrome b Group; D005415:Flavins; D005420:Flavoproteins; D010750:Phosphoproteins; D013501:Surface-Active Agents; D013778:Tetrazolium Salts; C035130:cytochrome b245; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; C013574:iodonitrotetrazolium; D016718:Arachidonic Acid; D012967:Sodium Dodecyl Sulfate; D006418:Heme; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C028796:iodophenyl-nitrophenyl-phenyltetrazolium reductase; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1