Previous studies from this laboratory described the kinetic characteristics of the inhibition by tosylphenylalanine chloromethane (TosPheCH2Cl) on superoxide anion production by human neutrophils (PMN) stimulated with a phorbol ester (PMA). In this study we present further evidence concerning the potential role of the chloromethane target in the normal cellular activation of NADPH oxidase. When PMN are treated with TosPheCH2Cl and subsequently PMA, or with the two reagents in the reverse order, the inhibition of superoxide production by the intact cells is still present in a particulate NADPH oxidase fraction prepared from these cells. Nevertheless, when cells incubated only with the chloromethane and not with PMA are disrupted, both their cytosolic and membrane fractions are fully competent in the cell-free activation assay. Thus, the chloromethane target has a role in NADPH oxidase activation exclusively at the cellular level. This observation constitutes additional evidence in favour of the idea that activation in the cell-free system reflects only partially the events which occur in the cells.
When cells are activated with PMA, their cytosol displays a loss of activating capacity in the cell-free activation assay in the presence of arachidonate, as was shown before with SDS as activator [Ambruso, D. R., Bolsher, B. G. J. M., Stockman, P. M., Verhoeven, A. J. & Roos, D. (1990) J. Biol. Chem. 265, 924–930]. This phenomenon was shown to arise most probably from the translocation of cytosolic factors to the membrane, resulting in a depleted cytosol. When superoxide production was inhibited by cell treatment with TosPheCH2Cl, either before or after activation with PMA, the cytosol from inhibited cells showed a recovery of activation capacity in the cell-free system. This effect probably results from TosPheCH2Cl inhibiting the translocation of the cytosolic factors when added before PMA. This results in an insufficient activation at the membrane level, which was previously considered as an inhibition. The effect of TosPheCH2Cl, when added after PMA, can best be explained again as an inhibition of translocation in the frame of the continuous replenishment-deactivation hypothesis proposed by Akard et al. [Akard, L. P., English, D. & Gabig, T. G. (1988) Blood 72, 322–327]. Thus, TosPheCH2Cl is apparently a promising new tool for studying the activation of NADPH oxidase at the cellular level.
In this study, we have also found that [3H]TosPheCH2Cl-labeled cells show a prominent radiola-beled band of Mr 15000 after SDS/PAGE analysis of the cytosolic proteins from inhibited cells; this species has the same migration as the labeled membrane protein detected previously, but appears much more abundant on a protein/cell basis. The potential role of the TosPheCH2Cl protein target is discussed.