Phenylarsine oxide as an inhibitor of the activation of the neutrophil NADPH oxidase--identification of the beta subunit of the flavocytochrome b component of the NADPH oxidase as a target site for phenylarsine oxide by photoaffinity labeling and photoinactivation.
Plasma membranes of neutrophil cells contain the redox component of the O2(-)-generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an alpha subunit of 22 kDa and a beta subunit of 85-105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane-bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell-free system. To determine the PAO-binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4-[N-(4-azido-2-nitrophenyl)amino-[3H]acetamido]phenylarsine oxide, ([3H]azido-PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the beta subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85-105 kDa was shifted to 50-60 kDa as was the immunodetected beta subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the beta subunit of the membrane-bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell-free system. Incorporation of [3H]azido-PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the beta subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.
Doussiere
J
J
Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 314 CEA/CNRS), Département de Biologie Moléculaire et Structurale, CEA/Grenoble, France.
Poinas
A
A
Blais
C
C
Vignais
P V
PV
D000345:Affinity Labels; D000818:Animals; D001152:Arsenicals / Q000494:pharmacology*; D001386:Azides; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000097:blood; D003600:Cytosol / Q000201:enzymology; D019281:Dimerization; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D008970:Molecular Weight; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000097:blood*; D009504:Neutrophils / Q000201:enzymology*; D013481:Superoxides / Q000097:blood
C110916:4-(N-(4-azido-2-nitrophenyl)aminoacetamido)phenylarsine oxide; D000345:Affinity Labels; D001152:Arsenicals; D001386:Azides; D003573:Cytochrome b Group; D004791:Enzyme Inhibitors; D008081:Liposomes; D046911:Macromolecular Substances; C029341:oxophenylarsine; D013481:Superoxides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019255:NADPH Oxidases