Abstract
Activation of the phagocyte NADPH oxidase requires participation of membrane-bound cytochrome b558 and cytosol proteins of 47 kDa (p47) and 67 kDa (p67). We examined the sequence of participation of p47 and p67 in activation of the oxidase using an arachidonate-activated cell-free superoxidase (O2-) generating assay requiring phagocyte membrane and cytosol. Neutrophil cytosol from patients with certain forms of autosomal recessive chronic granulomatous disease (CGD) lack either p47 or p67. Initial incubation of membrane and arachidonate with CGD cytosol deficient in either p47 or p67 fails to generate superoxide in the cell-free assay until addition of complementary cytosol. CGD cytosol was incubated with arachidonate and membrane for 5-15 min and the lag time of O2- generation was measured after addition of complementary CGD cytosol. The lag time is shortened when p47, but not p67, is present in the initial incubation. We have previously shown that the peptide, RGVHFIF, corresponding to a cytoplasmic carboxyl-terminal domain of the large subunit of cytochrome b558, inhibits activation of NADPH oxidase in the cell-free assay, but does not affect the enzyme activity of fully assembled oxidase. Experiments with sequential addition of complementary CGD cytosols were performed as above, except that RGVHFIF was added after the initial incubation. The peptide failed to inhibit when added after initial incubation if p47 was present during that incubation. In contrast, the peptide markedly inhibited oxidase activity if p47 was absent during the initial incubation. These results suggest that p47, but not p67, is a participant with membrane and/or other cytosol components in early arachidonate-dependent reactions. In the absence of p67, these reactions culminate in the irreversible formation of a metastable activation intermediate that is insensitive to inhibition by RGVHFIF. After addition of p67, this activation intermediate subsequently reacts to form the active NADPH oxidase.
Highlights
&a and cytosol proteins of 47 kDa (~47) and 67 kDa (~67)
From analysis of the kinetics of activation of the NADPH oxidase and inhibition of its formation by RGVHFIF, we demonstrate that p47 is a participant in a reaction that leads to the irreversible, early formation of a metastable activation intermediate which precedes the requirement for p67 in formation of the active NADPH oxidase
We examined the influence of ~47 and p67 on susceptibility of NADPH oxidase activation to inhibition by one of these peptides, RGVHFIF
Summary
&a and cytosol proteins of 47 kDa (~47) and 67 kDa (~67). We examined the sequence of participation of p47 and p67 in activation of the oxidase using an arachidonate-activated cell-free superoxide (0;) generating assay requiring phagocyte membrane and cytosol. Initial incubation of membrane and arachidonate with CGD cytosol deficient in either p47 or p67 fails to generate superoxide in the cell-free assay until addition of complementary cytosol. The peptide markedly inhibited oxidase activity if p47 was absent during the initial incubation These results suggest that ~47, but not ~67, is a participant with membrane and/or other cytosol components in early arachidonate-dependent reactions. The oxidase is dormant in resting neutrophils and becomes activated on stimulation, resulting in greatly augmented cellular oxygen consumption This “respiratory burst” is deficient in phagocytes of patients with chronic granulomatous disease of childhood (CGD),’ a genetically heterogeneous group of disorders characterized by defective microbicidal activity and recurrent infections [2, 3]. Despite evidence for the requirement of ~47, ~67, and cytochrome bSS8for oxidase activity, the exact roles of these components in activation of the NADPH oxidase are unknown
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