PURIFIED LEUKOCYTE CYTOCHROME B(558) INCORPORATED INTO LIPOSOMES CATALYZES A CYTOSOLIC FACTOR-DEPENDENT DIAPHORASE ACTIVITY

The leukocyte iodonitrotetrazolium violet (INT) reductase activity of disrupted bovine polymorphonuclear neutrophils is closely associated w ith the activation of the O-2(-)-generating NADPH oxidase in a cell-fr ee system, It is dependent upon NADPH, cytosolic factors, and amphiphi les (such as arachidonate), the same factors required for O-2(-) gener ation. Both O-2(-) generation and INT reductase activity are inhibited by phenylarsine oxide, an inhibitor of the activation of the NADPH ox idase [Li, J., & Guillory, R. J. (1997) J. Biochem. Mob, Blob, Biophys . (in press)]. In this report, the INT diaphorase activity of disrupte d bovine polymorphonuclear neutrophils is shown to be resolved by DEAE -Sepharose chromatography into two fractions: an NADPH-cytochrome c re ductase-containing fraction and a cytochrome b(558)-associated fractio n, The diaphorase activity in the NADPH-cytochrome c reductase-contain ing portion is not dependent upon the presence of an amphiphile or pho spholipid and is not associated with O-2(-) generation. Upon incorpora tion into liposomes, the cytochrome b(558)-containing fraction demonst rates high O-2(-) and INT reductase activities in the presence of cyto solic factors. Both O-2(-) generation and INT reductase activities are SDS and FAD dependent and further stimulated by GTP gamma S. Phenylar sine oxide inhibits both O-2(-) generation and INT reductase activitie s when added prior to activation by SDS. With the cytochrome b-contain ing liposomes, the K-m values (O-2(-) formation) for NADPH and NADH ar e 27.2 mu M and 810 mu M, and for INT reductase the K-m values are 27. 5 mu M and 1017 mu M,, respectively. Under anaerobic conditions and th us in the absence of O-2(-) formation, the NADPH-dependent INT reducta se activity does not change, indicating that the dye reduction is not due to its direct reduction by O-2(-) anion but is an intrinsic proper ty of the superoxide-generating NADPH oxidase. Cytochrome b(558) is th e essential component of the NADPH oxidase and contains all the redox centers necessary for electron flow between NADPH and oxygen. The corr elation of the activation and inhibition patterns for O-2(-) generatio n and INT reduction by cytochrome b(558) incorporated into artificial liposomes strongly indicates that the two activities are associated wi th the same membrane protein, cytochrome b(558).