The interaction of cytosolic components of neutrophil NADPH oxidase with phosphoinositides

Abstract

The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system consisting of cell membranes, cytosol and an activating detergent (e.g. arachidonate or SDS). It has previously been reported [Aviram and Sharabani (1989) Biochem. Biophys. Res. Commun. 161, 712-719] that a mixture of phosphoinositides (PPIs), as well as the individual inositol lipids, interfere with the activation process. In the present study it is shown that exposure of the cytosol to PPI results in a progressive (t1/2 = 30 s) loss of its oxidase-supporting activity and that Mg2+ ions eliminate this inactivation. Neomycin, previously described as an inhibitor of cell-free activation, counteracted the effect of PPI and vice versa. Fractionation experiments implicated the p67-phox cytosolic component of the oxidase in the association with PPI. PPI blocked activity of recombinant p67-phox also and quenched the fluorescence intensity of its tryptophan residues. It is suggested that PPIs may mediate the interaction of the oxidase with the cytoskeleton and/or with the membrane.

The assembly of neutrophil NADPH oxidase: effects of mastoparan and its synthetic analogues

“…Human neutrophils were isolated from fresh buffy coats by standard procedures [19]. Cells (108/ml) in 10 mM potassium phosphate-buffered saline (pH 7.0) (PBS) containing 1 mM EGTA, 3.5 mM PMSF and leupeptin (15 ,ug/ml) were disrupted by sonication and fractionated into cytosolic and light membrane fractions as described elsewhere [19].…”

“…The proteins were detected by enhanced chemiluminescence (ECL). In some experiments pre-activated pellets were resuspended in the buffer and their superoxide generation was determined separately [19].…”

“…The resin-treated cytosol (the last flowthrough of the column) was used in the NADPH oxidase activation assay and analysed by Western blotting using equal amounts (in protein) ofuntreated cytosol as a control. An aliquot of the cytosol-treated resin was washed with the column equilibration buffer, combined with 25 ,1 of SDS/PAGE sample buffer and analysed by immunoblotting with antibodies to p47-phox and p67-phox [19].…”