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 cel:ls contain the redox component of t he O-2(-)-generating NADPH oxidase complex, namely a heterodimeric fla vocytochrome b consisting of an alpha subunit of 22 kDa and a beta sub unit of 85-105 kDa of a glycoprotein nature, The NADPH oxidase is dorm ant in resting neutrophils. When neutrophils are exposed to a variety of particular or soluble stimuli, the oxidase becomes activated, due t o the assembly on the membrane-bound flavocytochrome b of three cytoso lic factors, p47phox, p67phox and Rac 2 (or Rac 1), The effect of phen ylarsine oxide (PAO), which reacts specifically with vicinal and neigh bouring thiol groups in proteins, was assayed on the NADPH oxidase act ivity 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 P AO on the oxidase activation itself was measured independently. PAO pr eferentially inhibited oxidase activation rather than the elicited oxi dase activity, and inhibition resulted from binding of PAO to the memb rane component of the cell-free system. To determine the PAO-binding p rotein responsible for the loss of oxidase activation, we used photoaf finity labeling with a tritated azido derivative of PAO, ido-2-nitroph enyl)amino-[H-3]acetamido]phenylarsin oxide, ([(3)Hlazido-PAO), Phutoi rradiation of plasma membranes from resting neutrophils in the presenc e of [H-3]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the bet a subunit of flavocytochrome b as identified by immunoreaction. Upon d eglycosylation, the photolabeled band at 85105 kDa was shifted to 50-6 0 kDa as was the immunodetected beta subunit. Similar results were obt ained with isolated flavocytochrome b in liposomes, Photoaffinity labe ling of the beta subunit of the membrane bound flavocytochrome b or th e isolated flavocytochrome b in liposomes resulted in abolition of oxi dase activation in the reconstituted cell-free system. Incorporation o f [H-3]azido-PAO into flavocytochrome b was negligible when photoaffin ity labeling was performed on neutrophil membranes that had been pre v iously activated, The results suggest that the beta subunit of flavocy tochrome contains two target sites fur PAO which are accessible in res ting neutrophils, but not in activated neutrophils.