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Phagocytic cells possess a tightly regulated multicomponent enzyme complex, the NADPH oxidase, which produces superoxide, a reactive oxygen molecule that is an essential component of host defense against infection. Upon stimulation, a functional NADPH oxidase is assembled when the cytosolic proteins, Rac, p67phox, p47phox, and possibly p40phox, associate with the gp91phox and p22phox transmembrane proteins. Rac is a GTPase that in the GTP-bound state binds p67phox to activate NADPH oxidase. The function of p40phox is not known; it is believed to have a regulatory function in sequestering p67phox and p47phox in a cytosolic complex. We investigated binding interactions between p40phox, p67phox, and Rac and found that Rac1-GTP displaced p67phox bound to p40phox. In contrast, Cdc42, a GTPase homologous to Rac, did not displace p67phox from p40phox. A synthetic peptide corresponding to p67phox amino acids 170-199, a region identified previously as a Rac binding domain, significantly reduced the ability of Rac1-GTP to disrupt p67phox/p40phox binding. We hypothesize that Rac-GTP binds the p67phox N-terminal domain encompassing amino acids 170-199 that transmits a conformational change which causes p40phox to dissociate from its binding site in the p67phox C-terminus.

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Activation of phagocyte NADPH oxidase requires interaction between p47(phox) and p22(phox). p47(phox) in resting phagocytes does not bind p22(phox). Phosphorylation of serines in the p47(phox) C terminus enables binding to the p22(phox) C terminus by inducing a conformational change in p47(phox) that unmasks the SH3A domain. We report that an arginine/lysine-rich region in the p47(phox) C terminus binds the p47(phox) SH3 domains expressed in tandem (SH3AB) but does not bind the individual N-terminal SH3A and C-terminal SH3B domains. Peptides matching amino acids 301-320 and 314-335 of the p47(phox) arginine/lysine-rich region block the p47(phox) SH3AB/p22(phox) C-terminal and p47(phox) SH3AB/p47(phox) C-terminal binding and inhibit NADPH oxidase activity in vitro. Peptides with phosphoserines substituted for serines 310 and 328 do not block binding and are poor inhibitors of oxidase activity. Mutated full-length p47(phox) with aspartic acid substitutions to mimic the effects of phosphorylations at serines 310 and 328 bind the p22(phox) proline-rich region in contrast to wild-type p47(phox). We conclude that the p47(phox) SH3A domain-binding site is blocked by an interaction between the p47(phox) SH3AB domains and the C-terminal arginine/lysine-rich region. Phosphorylation of serines in the p47(phox) C terminus disrupts this interaction leading to exposure of the SH3A domain, binding to p22(phox), and activation of the NADPH oxidase.

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Rac GTPases regulate activation of the phagocyte NADPH oxidase, a multi-component enzyme complex that produces superoxide in response to host infection. GTP-bound Rac binds to the cytosol protein p67-phox enabling it to participate in oxidase assembly. Details of this interaction are poorly understood. Previous studies showed that Rac/p67-phox binding is GTP-dependent and that several Rac1 mutants lost the ability to activate the oxidase even though they still bound p67-phox. Using two hybrid and blot overlay binding methods, we identified a novel binding site in the p67-phox C-terminus that binds Rac1, Rac2, and Cdc42, a related GTPase which does not activate the oxidase. Binding was independent of the GDP/GTP state. We also showed that GTP-Cdc42 binds p67-phox N-terminus similar to GTP-Rac. Therefore, Rac binding to p67-phox is not synonymous with NADPH oxidase activation, and Rac probably participates in other steps of oxidase activation in addition to binding p67-phox.

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OBJECTIVE: To identify and characterize 4 components of the NADPH oxidase complex, gp91-phox, p22-phox, p67-phox, and p47-phox, in common laboratory animal species. ANIMALS: 2 clinically normal animals from each of the following species: rabbit, sheep, cow, pig, and macaque (Macaca nemistrina). A pool of 8 rats. PROCEDURE: Neutrophils were harvested from blood, Membrane and cytosol fractions were isolated and separated by sodium dodecyl sulfate-polyacrylamide gels. Gels were transferred, and immunoblots were probed with antibodies directed against individual human NADPH oxidase proteins. Human neutrophil membrane and cytosol fractions served as controls. RESULTS: Immunoreactive bands were observed in all species for gp91-phox, p67-phox, and p47-phox proteins. Immunoreactive bands for p22-phox protein were observed in cells from rats, rabbits, pigs, and macaques. CONCLUSIONS: The NADPH oxidase and its component proteins have been highly conserved across mammalian species. Lack of immunoreactivity to p22-phox in sheep and cows can be explained by sequence divergence and epitope variability at the p22-phox C-terminus. CLINICAL RELEVANCE: The high degree of NADPH oxidase protein conservation may allow the existing knowledge of the human neutrophil NADPH oxidase to be applied to the study of animal disease.

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The yeast two-hybrid system is finding increased use in the study of interactions between proteins. In this method, two polypeptides are expressed in yeast as fusion proteins to a transcriptional activator DNA-binding domain (bd) and activating domain (ad), respectively. Interaction between the two polypeptides reconstitutes function of a transactivator which controls expression of reporters. The phagocyte NADPH oxidase is a complex of membrane cytochrome b558 (comprised of subunits p22-phox and gp91-phox) and three cytosol proteins (p47-phox, p67-phox, and p21rac) that translocate to membrane and bind to cytochrome b558. This is the first report to demonstrate that two of cytosolic components of cytochrome b558, p47-phox binding to p67-phox each other. We encountered several methodological problems in the two-hybrid system which are the focus of this report.

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The phagocyte NADPH oxidase complex is an unusual electron transfer system. Its principal component, cytochrome b558, is a heme-containing integral membrane protein consisting of two subunits, gp91-phox and p22-phox. We used a novel method to measure precisely the gp91-phox:p22-phox stoichiometry. Cytochrome b558 was isolated in high purity from human neutrophil membrane preparations using a novel affinity purification method. We performed direct peptide sequencing of purified cytochrome b558 and detected two amino acid sequences which matched predicted sequences for gp91-phox and p22-phox. We quantitated amounts of both amino acids released from p22-phox and gp91-phox in each sequencing cycle. Averaging over 25 cycles, the mean p22-phox:gp91-phox ratio of released amino acids was 0.93 +/- 0.01. To correct for recovery differences between individual amino acids, we measured individual p22-phox:gp91-phox ratios for the eight different amino acids common to both p22-phox and gp91-phox in the first 25 positions. The mean of individual p22-phox:gp91-phox ratios for the eight common amino acids was 0.96 +/- 0.05. The p22-phox:gp91-phox ratios for each of the eight common amino acids varied from 0.81 to 1.20. Taken together, measured ratios for total and individual amino acids are consistent with a predicted ratio of 1.0 for 1:1 p22-phox:gp91-phox stoichiometry in cytochrome b558.

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The phagocyte superoxide-generating NADPH oxidase, a multicomponent, membrane-bound electron transport chain, consists of cytochrome b558, p47-phox, p67-phox, and p21rac1 or p21rac2. The mechanisms of oxidase assembly are poorly understood. In previous studies using a cell-free NADPH oxidase system, we showed that preincubation of neutrophil membrane with neutrophil cytosol containing p47-phox, but not p67-phox, led to formation of a long-lived NADPH oxidase intermediate. This suggested that p47-phox interacted with cytochrome b558 in the early stages of oxidase assembly while p67-phox participated in a later stage. Peptides containing the sequence RGVHFIF (corresponding to amino acids 559-565 of the 91-kDa subunit of cytochrome b558) inhibit NADPH oxidase activity by blocking the early interaction between p47-phox and cytochrome b558. In the present study, we examined whether p21rac facilitated the interaction between p47-phox and cytochrome b558. We preincubated pure recombinant p47-phox with neutrophil membrane containing cytochrome b558 in the cell-free system. Superoxide-generating activity was subsequently reconstituted by adding pure rp67-phox and partially purified p21rac. RGVHFIF inhibited superoxide production if added to the cell-free system during preincubation of rp47-phox with membrane. RGVHFIF was markedly less inhibitory if added to the cell-free system after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox conferred no protection from inhibition of superoxide-generating activity by RGVHFIF added after preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)

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The phagocyte NADPH oxidase is a multicomponent membrane-bound electron transport chain that catalyzes the reduction of O2 to superoxide. Cytochrome b558, the terminal electron donor to O2, is an integral membrane heterodimer containing 91- and 22-kDa subunits (gp91-phox and p22-phox, respectively). Synthetic peptides, whose amino acid sequences correspond to a gp91-phox carboxyl-terminal domain, inhibit superoxide production by blocking assembly of the oxidase from membrane and cytosol components. In this study, we examined the amino acid sequence requirements of a series of synthetic truncated gp91-phox peptides for inhibition of human neutrophil NADPH oxidase activation. RGVHFIF, corresponding to gp91-phox residues 559-565, was the minimum sequence capable of inhibiting superoxide generation. Contributions of individual amino acids to overall RGVHFIF inhibitory activity were determined by comparing the abilities of alanine-substituted RGVHFIF peptides to inhibit superoxide production. Substitution of alanine for arginine, valine, isoleucine, or either of the phenylalanines (but not glycine or histidine) within RGVHFIF resulted in loss of inhibitory activity. Synthetic gp91-phox carboxyl-terminal peptides are likely to be competitive inhibitors of the corresponding carboxyl-terminal domain of native gp91-phox by virtue of amino acid identity. We conclude that properties of arginine valine, isoleucine, and phenylalanine side chains within an RGVHFIF-containing domain of gp91-phox contribute significantly to cytochrome b558-mediated activation of the oxidase.

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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.

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Cytoplasmic domains of transmembrane proteins play a critical role in cellular processes involving interactions between membrane and cytosolic components. Activation of the phagocytic cell respiratory burst oxidase, the electron transport chain responsible for superoxide anion (O2-.) production, requires membrane components including cytochrome b558 and several cytosolic proteins; but the biochemical interactions of these components are poorly understood. Cytochrome b558 is an electron transport component of the oxidase. A role for cytochrome b558 in the organization or integration of other oxidase components has also been hypothesized. Antibodies binding the cytoplasmic carboxyl-terminal tail of the transmembrane 91-kDa subunit of cytochrome b558 specifically inhibited an amphiphile-activated cell-free O2-.-generating system that requires neutrophil membranes and cytosol. Synthetic peptides encompassing a 7-amino acid carboxyl-terminal sequence (RGVHFIF) within the same region of the 91-kDa subunit blocked activation of the oxidase by arachidonate, but did not affect activity of the assembled oxidase when added after arachidonate to the cell-free O2-.-generating system. The same peptides inhibited activation of the respiratory burst when allowed to diffuse into electrically permeabilized neutrophils before stimulation with formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These studies define a functional cytoplasmic domain of the transmembrane 91-kDa subunit of cytochrome b558 which may mediate interactions with other cellular proteins essential to activation of the phagocyte respiratory burst.

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Cytochrome b558, an essential component of the respiratory burst of phagocytic cells, is the terminal electron donor to molecular oxygen that results in the formation of superoxide anion (O2-.). It is an integral membrane heterodimer that in neutrophils consists of a 22-kDa small subunit and a highly glycosylated 91-kDa large subunit. Identical core proteins often differ in glycosylation in different cell types and with some membrane glycoproteins, the glycosylation state may markedly affect function. In the present study, antisera reactive with cytochrome b558 large subunit was used for immunoblot analysis of the glycosylation pattern of this subunit from different types of phagocytic cells. Striking variability in the apparent m.w. of this broadly banding subunit was detected in five different phagocytic cell types (neutrophils 78,000 to 93,000; eosinophils 74,000 to 115,000; monocytes 82,000 to 99,000; dibutyryl cyclic AMP-induced HL-60 cells 79,000 to 103,000; dimethyl sulfoxide-induced HL-60 cells 77,000 to 110,000). However, after complete cleavage of N-linked oligosaccharides with endoglycosidase F, the core peptide of cytochrome b558 large subunit from these different cell types had the same Mr (58,000). Inhibition of N-glycosylation with tunicamycin in differentiating HL-60 cells resulted in the synthesis of immunoreactive protein of the same m.w. and banding pattern as seen after endoglycosidase F cleavage. These tunicamycin treated cells retained some capacity to generate superoxide anion when stimulated with PMA. We conclude that the identity of the N-linked oligosaccharides of the cytochrome b558 large subunit differ in various phagocytic cells. All N-linked glycans on cytochrome b558 in all cell types examined were of the complex type as defined by resistance to endoglycosidase H cleavage. N-linked glycosylation of the cytochrome b558 large subunit may not be essential for activation of the respiratory burst.

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Nystatin forms two types of channels in sterol-containing planar bilayer membranes. One type is formed when it is added to only one side of the membrane; the other is formed when it is added to both sides of the membrane. The relative permeability of these channels to nonelectrolytes (urea and glycerol) is identical. The sensitivity of membranes to the one-sided action of nystatin is critically dependent on their thickness; in particular, membranes made from monoglycerides with more than 18 carbon atoms in their acyl chain are insensitive to nystatin's one-sided action. These data are consistent with a model in which the two types of channels formed by nystatin have essentially identical structures, except that the channel formed by its two-sided action is twice the length of that formed by its one-sided action, because it is a tail-to-tail dimer of the latter.

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