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Nox activator 1 (NoxA1) is a homologue of p67(phox) that acts in conjunction with Nox organizer 1 (NoxO1) to regulate reactive oxygen species (ROS) production by the NADPH oxidase Nox1. The phosphorylation of cytosolic regulatory components by multiple kinases plays important roles in assembly and activity of the phagocyte NADPH oxidase (Nox2) system, but little is known about regulation by phosphorylation in the Nox1 system. Here we identify Ser(172) and Ser(461) of NoxA1 as phosphorylation sites for protein kinase A (PKA). A consequence of this phosphorylation was the enhancement of NoxA1 complex formation with 14-3-3 proteins. Using both a transfected human embryonic kidney 293 cell Nox1 model system and endogenous Nox1 in colon cell lines, we showed that the elevation of cAMP inhibits, whereas the inhibition of PKA enhances, Nox1-dependent ROS production through effects on NoxA1. Inhibition of Nox1 activity was intensified by the availability of 14-3-3zeta protein, and this regulatory interaction was dependent on PKA-phosphorylatable sites at Ser(172) and Ser(461) in NoxA1. We showed that phosphorylation and 14-3-3 binding induce the dissociation of NoxA1 from the Nox1 complex at the plasma membrane, suggesting a mechanism for the inhibitory effect on Nox1 activity. Our data establish that PKA-phosphorylated NoxA1 is a new binding partner of 14-3-3 protein(s) and that this forms the basis of a novel mechanism regulating the formation of ROS by Nox1 and, potentially, other NoxA1-regulated Nox family members.

Here, we report evidence for the production of ozone in human disease. Signature products unique to cholesterol ozonolysis are present within atherosclerotic tissue at the time of carotid endarterectomy, suggesting that ozone production occurred during lesion development. Furthermore, advanced atherosclerotic plaques generate ozone when the leukocytes within the diseased arteries are activated in vitro. The steroids produced by cholesterol ozonolysis cause effects that are thought to be critical to the pathogenesis of atherosclerosis, including cytotoxicity, lipid-loading in macrophages, and deformation of the apolipoprotein B-100 secondary structure. We propose the trivial designation “atheronals” for this previously unrecognized class of steroids.

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} at the expense of NADPH. Extensive phosphorylation of the oxidase subunit p47 PHOX occurs during the activation of the enzyme in intact cells. p47 PHOX carrying certain serine-to-alanine mutations fails to support NADPH oxidase activity in intact cells, suggesting that the phosphorylation of specific serines on p47 PHOX is required for the activation of the oxidase. Earlier studies with both intact cells and a kinase-dependent, cell-free system have suggested that protein kinase C can phosphorylate those serines of p47 PHOX whose phosphorylation is necessary for its activity. Work with inhibitors suggested that a phosphatidylinositol 3-kinase-dependent pathway also can activate the oxidase. Phosphorylation of p47 PHOX by Akt (protein kinase B), whose activation depends on phosphatidylinositol 3-kinase, could be the final step in such a pathway. We now find that Akt activates the oxidase in vitro by phosphorylating serines S304 and S328 of p47 PHOX . These results suggest that Akt could participate in the activation of the leukocyte NADPH oxidase.

Recent studies have suggested that antibodies can catalyze the generation of previously unknown oxidants including dihydrogen trioxide (H 2 O 3 ) and ozone (O 3 ) from singlet oxygen ( 1 O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{*}}}\end{equation*}\end{document} ) and water. Given that neutrophils have the potential both to produce 1 O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{*}}}\end{equation*}\end{document} and to bind antibodies, we considered that these cells could be a biological source of O 3 . We report here further analytical evidence that antibody-coated neutrophils, after activation, produce an oxidant with the chemical signature of O 3 . This process is independent of surface antibody concentration down to 50% of the resting concentration, suggesting that surface IgG is highly efficient at intercepting the neutrophil-generated 1 O \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{*}}}\end{equation*}\end{document} . Vinylbenzoic acid, an orthogonal probe for ozone detection, is oxidized by activated neutrophils to 4-carboxybenzaldehyde in a manner analogous to that obtained for its oxidation by ozone in solution. This discovery of the production of such a powerful oxidant in a biological context raises questions about not only the capacity of O 3 to kill invading microorganisms but also its role in amplification of the inflammatory response by signaling and gene activation.

Neutrophils and macrophages, recruited to the wound site, release reactive oxygen species by respiratory burst. It is commonly understood that oxidants serve mainly to kill bacteria and prevent wound infection. We tested the hypothesis that oxidants generated at the wound site promote dermal wound repair. We observed that H(2)O(2) potently induces vascular endothelial growth factor (VEGF) expression in human keratinocytes. Deletion mutant studies with a VEGF promoter construct revealed that a GC-rich sequence from bp -194 to -50 of the VEGF promoter is responsible for the H(2)O(2) response. It was established that at microm concentrations oxidant induces VEGF expression and that oxidant-induced VEGF expression is independent of hypoxia-inducible factor (HIF)-1 and dependent on Sp1 activation. To test the effect of NADPH oxidase-generated reactive oxygen species on wound healing in vivo, Rac1 gene transfer was performed to dermal excisional wounds left to heal by secondary intention. Rac1 gene transfer accelerated wound contraction and closure. Rac1 overexpression was associated with higher VEGF expression both in vivo as well in human keratinocytes. Interestingly, Rac1 gene therapy was associated with a more well defined hyperproliferative epithelial region, higher cell density, enhanced deposition of connective tissue, and improved histological architecture. Overall, the histological data indicated that Rac1 might be an important stimulator of various aspects of the repair process, eventually enhancing the wound-healing process as a whole. Taken together, the results of this study indicate that wound healing is subject to redox control.

Phosphatidylinositol 3-kinase products play a central role in the regulation of several intracellular pathways via adaptor proteins that share the ability to bind to 3′-phosphoinositides with high affinity and specificity. JFC1 is a C2 domain-containing protein involved in cellular trafficking that has been shown to bind 3′-phosphoinositidesin vitro. In this work, we demonstrate that the C2A domain of JFC1 is the module responsible for its binding to the plasma membrane via 3′-phosphoinositidesin vivo. We show that the C2A domain of JFC1 is the only domain present in this protein that localizes to the plasma membrane in living cells. Moreover, the C2A domain of JFC1 binds 3′-phosphoinositidesin vitrowith similar specificity as that described for full-length JFC1, suggesting that the domain mediates the specific membrane localization of the full-length protein. Furthermore, the C2A domain of JFC1 colocalized with the pleckstrin homology domain of Aktin vivo, and both the JFC1 C2A domain and the full-length JFC1 dissociated from the membrane in the presence of PI 3-kinase specific inhibitors. We also show that the association of the C2A domain to the membrane is modulated by calcium. From these results we analyze possible mechanisms for the role of JFC1 in cellular trafficking.

In this work, we demonstrate that the phosphatidylinositol 3,4,5-trisphosphate-binding protein JFC1 is an ATP-binding protein with magnesium-dependent ATPase activity. We show that JFC1 specifically binds to the ATP analog 8-azido-[α- 32 P]ATP. The affinity of JFC1 for [α- 32 P]ATP was 10× greater than its affinity for [α- 32 P]ADP; the protein did not appear to bind to [α- 32 P]GTP. JFC1 hydrolyzed [α- 32 P]ATP in a Mg 2+ -dependent manner. JFC1, which also hydrolyzed dATP, has a relatively high affinity for ATP, with a K M value of 58 μM, and a k cat value of 2.27 per min. The predicted amino acid sequence of JFC1 denotes a putative nucleotide-binding site similar to those in the GHKL ATPase/kinase superfamily. However, a truncation of JFC1 that contains boxes G2 and G3 but not boxes N and G1 of the Bergerat-binding site showed residual ATPase activity. Secondly, the antitumor ATP-mimetic agent geldanamycin, which inhibits the ATPase activity of Hsp-90, did not affect JFC1 ATPase. Therefore, the characteristics of the ATP-binding site of JFC1 are unique. Phosphatidylinositol 3,4,5-trisphosphate, a high-affinity ligand of JFC1 did not affect its ATPase kinetics parameters, suggesting that the phosphoinositide have a different role in JFC1 function.

We have employed a yeast two-hybrid system to screen a B lymphoblast-derived cDNA library, searching for regulatory components of the NADPH oxidase. Using as bait the C-terminal half of p67(phox), which contains both Src homology 3 domains, we have cloned JFC1, a novel human 62-kDa protein. JFC1 possesses two C2 domains in tandem. The C2A domain shows homology with the C2B domain of synaptotagmins. JFC1 mRNA was abundantly expressed in bone marrow and leukocytes. The expression of JFC1 in neutrophils was restricted to the plasma membrane/secretory vesicle fraction. We confirmed JFC1-p67(phox) association by affinity chromatography. JFC1-containing beads pulled down both p67(phox) and p47(phox) subunits from neutrophil cytosol, but when the recombinant proteins were used, only p67(phox) bound to JFC1, indicating that JFC1 binds to the cytosolic complex via p67(phox) without affecting the interaction between p67(phox) and p47(phox). In contrast to synaptotagmins, JFC1 was unable to bind to inositol 1,3,4,5-tetrakisphosphate but did bind to phosphatidylinositol 3,4,5-trisphosphate and to a lesser extent to phosphatidylinositol 3,4-diphosphate. From the data presented here, it is proposed that JFC1 is acting as an adaptor protein between phosphatidylinositol 3-kinase products and the oxidase cytosolic complex.

Fish oils are known for their anti-inflammatory effects. In this paper we investigated the influence of eicosapentaenoic acid and docosahexaenoic acid (omega-3 fatty acids), as well as docosapentaenoic acid, a metabolic product of omega-3 fatty acid metabolism, on O2(-)-production catalyzed by the NADPH oxidase in whole neutrophils and in a cell-free system consisting of neutrophil membranes and cytosol. As a standard we used arachidonic acid (an omega-6 fatty acid) found in a high proportion in the Western diet, and known as an effective activator of the oxidase in both systems. Our data show that with omega-3 fatty acids, the O2(-)-production in both systems is reduced as compared to the effect of arachidonic acid. The effects are more pronounced with increasing carbon chain length and increasing numbers of double bonds. Our results suggest another mechanism besides the inhibition of eicosanoid and cytokine production to explain the beneficial effects of fish oils in reducing inflammation.

T HE NADPH OXIDASES are a group of plasma membrane–associated enzymes found in a variety of cells of mesodermal origin. The most thoroughly studied of these is the leukocyte NADPH oxidase, which is found in professional phagocytes and B lymphocytes. It catalyzes the production of superoxide (O2

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to superoxide (O-2) at the expense of NADPH in phagocytes and B lymphocytes. The enzyme is dormant in resting cells but becomes active when the cells are exposed to appropriate stimuli. During oxidase activation, the highly basic cytosolic oxidase component p47(PHOX) becomes phosphorylated on several serines and migrates to the plasma membrane. We report here that p47(PHOX)-deficient B lymphoblasts expressing the p47(PHOX) S359A/S370A or p47(PHOX) S359K/S370K double mutation show dramatically reduced levels of enzyme activity and phosphorylation of p47(PHOX) as compared with the same cells expressing wild type p47(PHOX). In addition, these mutant p47(PHOX) proteins fails to translocate to the plasma membrane when the cells are stimulated. In contrast, normal phosphorylation and translocation are seen in mutants containing aspartate or glutamate at positions 359 and 370, but oxidase activity is still greatly reduced. These results imply that a negative charge at position 359 and/or 370 is sufficient to allow the phosphorylation and translocation of p47(PHOX) to take place but that features unique to a phosphorylated hydroxyamino acid are required to support O-2 production. These findings, plus those from an earlier study (Inanami, O., Johnson, J. L., McAdara, J. K., El Benna, J., Faust, L. P., Newburger, P. E., and Babior, B. M. (1998) J. Biol. Chem. 273, 9539-9543), suggest that oxidase activation requires 1) the sequential phosphorylation of at least two serines on p47(PHOX): Ser-359 or Ser-370, followed by Ser-303 or Ser-304; and 2) the translocation of p47(PHOX) to the membrane at some point after the first phosphorylation takes place.

The leukocyte NADPH oxidase is an enzyme in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen and NADPH. During oxidase activation, serine residues in the C-terminal quarter of the oxidase component p47(PHOX) become extensively phosphorylated, the protein acquiring as many as 9 phosphate residues. In a study of 11 p47(PHOX) mutants, each containing an alanine instead of a serine at a single potential phosphorylation site, we found that all but S379A corrected the defect in O-2 production in Epstein-Barr virus (EBV)-transformed p47(PHOX)-deficient B cells (Faust, L. P., El Benna, J., Babior, B. M., and Chanock, S. J. (1995) J. Clin. Invest. 96, 1499-1505). In particular, O-2 production was restored to these cells by the mutants S303A and S304A. Therefore, apart from serine 379, whose state of phosphorylation in the activated oxidase is unclear, no single potential phosphorylation site appeared to be essential for oxidase activation. We now report that the double mutant p47(PHOX) S303A/S304A was almost completely inactive when expressed in EBV-transformed p47(PHOX)-deficient B cells, even though it was expressed in normal amounts in the transfected cells and was able to translocate to the plasma membrane when the cells were stimulated. In contrast, the double mutant p47(PHOX) S303E/S304E was able to support high levels of O-2 production by EBV-transformed p47(PHOX)-deficient B cells. The surprising discovery that the double mutant S303K/S304K was also able to support considerable O-2 production suggests either that the effect of phosphorylation is related to the increase in hydrophilicity around serines 303 and 304 or that activation involves the formation of a metal bridge between the phosphorylated serines and another region of the protein.

We have previously shown that granulocyte colony-stimulating factor (G-CSF ) delays spontaneous neutrophil apoptosis through activation of the vacuolar proton ATPase (v-ATPase). We have now examined the regulation of the v-ATPase in neutrophils exposed to G-CSF in vitro. When neutrophils were cultivated in the absence of G-CSF, the 57-kD cytosolic B subunit of the v-ATPase disappeared within 1 to 2 hours, its loss preceding the nuclear changes of apoptosis and coinciding with the onset of acidification. By contrast, in neutrophils cultured for 2 hours in the presence of G-CSF, the amount of the 57-kD subunit was similar to that in freshly isolated neutrophils. However, inhibition of protein synthesis with cycloheximide and actinomycin D led to loss of the 57-kD subunit even in the presence of G-CSF. These results indicated that ongoing protein synthesis was required to maintain the v-ATPase, and further suggested that G-CSF acted, at least in part, by maintaining synthesis of the 57-kD cytosolic subunit. G-CSF also promoted the translocation of the 57-and 33-kD cytosolic v-ATPase subunits to the membrane. Our findings suggested two coordinate mechanisms by which the activity of the v-ATPase could be increased by G-CSF: the synthesis of cytosolic v-ATPase subunits and their translocation to the membrane.

The leukocyte NADPH oxidase catalyzes the 1-electron reduction of oxygen to O2− at the expense of NADPH: 2 O2+ NADPH → 2 O2−+ NADP++ H+. The oxidase is dormant in resting cells but acquires activity when the cells are stimulated with a suitable agent. Activation in whole cells is accompanied by extensive phosphorylation of p47PHOX, an oxidase subunit located in the cytosol of resting cells that during oxidase activation migrates to the plasma membrane to complex with cytochrome b558, an oxidase-specific flavohemoprotein. Oxidase activation can be mimicked in a cell-free system using an anionic amphiphile as activating agent. We now report a cell-free system in which the oxidase can be activated in two stages using phosphorylated p47PHOX. The first stage, which effects a change in the membrane, requires ATP and GTP and is blocked by the protein kinase inhibitor GF-109203X, suggesting a protein kinase requirement. The second stage requires phosphorylated p47PHOX and GTP, but no ATP, and is unaffected by GF-109203X; assembly of the oxidase may take place during this stage. Activation is accomplished by p47PHOX phosphorylated by protein kinase C but not protein kinase A or mitogen-activated protein kinase. We believe that activation by phosphorylated p47PHOX is more physiological than activation by amphiphiles, because the mutant p47PHOX S379A, which is inactive in whole cells, is also inactive in this system but works in systems activated by amphiphiles.

Mitochondrial function was examined in Jurkat cells undergoing Fas-mediated apoptosis. With succinate or ascorbate/tetramethylphenylenediamine as substrate, oxygen uptake by digitonin-permeabilized apoptotic mitochondria was greatly decreased as compared with control. Assessment of the function of the cytochrome c-cytochrome oxidase segment of the electron transport chain of apoptotic mitochondria showed that the activity of cytochrome oxidase appeared to be normal, but that of cytochrome c was greatly diminished. A death protease was found to participate in the events leading to the loss of cytochrome c activity, but the cytochrome did not seem to be extensively degraded during the course of apoptosis. Our results suggest that a rapid loss in mitochondrial function due at least in part to the inhibition or inactivation of cytochrome c is a potentially fatal component of the apoptosis program of Jurkat cells.

The mammalian Ras GTPase-activating protein (p120) interacts with activated members of the Ras superfamily of GTP-binding proteins to accelerate their deactivation by sharply increasing their rates of GTP hydrolysis. Among the Ras-family proteins interacting with p120 is Rap1A/Krev1, whose activity is not affected by p120 but which competes with Ras for p120. A second protein that interacts with p120 is p190, which activates the GTPase of guanine nucleotide-binding proteins of the Rho family (including Rac1 and Rac2). Both these p120-binding proteins are of interest in connection with the regulation of the respiratory burst oxidase, Rap1A/Krev1 because it copurifies with cytochrome b, and p190 because it inhibits the Rac2-dependent activation of the respiratory burst oxidase in a cell-free system. Using an 18-mer antisense oligonucleotide, we were able to decrease the expression of p120 in Epstein-Barr virus-transformed B lymphocytes. Under conditions where p120 expression was significantly depressed by antisense oligonucleotides, we observed a 40% increase in protein kinase C-dependent but not receptor-dependent O production. In contrast, sense and scrambled oligonucleotides had no effect on either p120 expression or O production. Our results suggest a role for p120 as a negative regulator in the protein kinase C-mediated activation of the respiratory burst oxidase.

The respiratory burst oxidase is responsible for superoxide (O) production by phagocytes and B lymphocytes. This multicomponent enzyme is dormant in resting cells but is activated on exposure of the cells to an appropriate stimulus. Upon activation, several serine residues on the cytosolic oxidase subunit p47 become phosphorylated. Using two-dimensional tryptic phosphopeptide mapping, we studied the phosphorylation of p47 in P-loaded Epstein-Barr virus-transformed B lymphoblasts expressing wild type p47or any of several p47 Ser Ala mutants. We were able to identify the labeled peptides from wild type p47 as those containing Ser, Ser, Ser, Ser and/or Ser, and Ser; no P-labeled Ser-containing peptide was found. When purified p47 was phosphorylated in vitro by various protein kinases, varying phosphopeptide patterns were observed. Protein kinase C phosphorylated all the peptides except the one containing Ser; protein kinase A phosphorylated the peptide containing Ser and one or both of the peptides containing Ser and Ser; while mitogen-activated protein kinase phophorylated only the peptide containing Ser. These findings suggest that these three kinases play distinct roles in the activation of the respiratory burst oxidase, each of them catalyzing the phosphorylation of a different group of serines in p47.

We have previously shown that in neutrophils deprived of granulocyte colony-stimulating factor, apoptosis is preceded by acidification and that the protection against apoptosis conferred on neutrophils by granulocyte colony-stimulating factor is dependent upon delay of this acidification. To test the hypothesis that acidification could be a general feature of apoptosis, we examined intracellular pH changes in another cell line. Jurkat cells, a T-lymphoblastoid line, were induced to undergo apoptosis with anti-Fas IgM, cycloheximide, or exposure to short-wavelength UV light. We found that acidification occurred in response to treatment with these agents and that acidification preceded DNA fragmentation. Jurkat cells were also found to possess an acid endonuclease that is active below pH 6.8, compatible with a possible role for this enzyme in chromatin digestion during apoptosis. Incubation of the cells with the bases imidazole or chloroquine during treatment with anti-Fas antibody or cycloheximide or after UV exposure decreased apoptosis as assessed by nuclear morphology and DNA content. The alkalinizing effect of imidazole and chloroquine was shown by the demonstration that the percentage of cells with an intracellular pH below 6.8 after treatment with anti-Fas antibody, cycloheximide, or UV was diminished in the presence of base as compared with similarly treated cells incubated in the absence of base. We conclude that acidification is an early event in programmed cell death and may be essential for genome destruction.

The adhesive and signaling functions of integrins are regulated through their cytoplasmic domains. We identified a novel 111 residue polypeptide, designated beta 3-endonexin, that interacted with the cytoplasmic tail of the beta 3 integrin subunit in a yeast two-hybrid system. This interaction is structurally specific, since it was reduced by 64% by a point mutation in the beta 3 cytoplasmic tail (S752-->P) that disrupts integrin signaling. Moreover, this interaction is integrin subunit specific since it was not observed with the cytoplasmic tails of the alpha IIb, beta 1, or beta 2 subunits. beta 3-Endonexin fusion proteins bound selectively to detergent-solubilized beta 3 from platelets and human umbilical vein endothelial cells, and beta 3-endonexin mRNA and protein were detected in platelets and other tissues. A related mRNA encoded a larger polypeptide that failed to bind to beta integrin tails. The apparent specificity of beta 3-endonexin for the beta 3 integrin subunit suggests potential mechanisms for selective modulation of integrin functions.

Human neutrophils are terminally differentiated cells that spontaneously undergo apoptosis in tissue culture. Apoptosis in these cells can be delayed by culture in the presence of granulocyte colony-stimulating factor or other inflammatory mediators. Neutrophils were found to contain an acid endonuclease that appeared to be responsible for the internucleosomal DNA cleavage that accompanies apoptosis. As measured by a plasmid nicking assay, this endonuclease had a molecular weight (M,) of 35,000, a pH optimum of 5.5, and a threshold for activity of pH 6.6 to 6.8. It was weakly inhibited by divalent cations (Ca’’, Mg2+, and Zn2+) and more strongly inhibited by aurintricarboxylic acid EUTROPHILS ARE terminally differentiated cells with a life span of hours to days. In culture, they undergo programmed cell death or apoptosis, an event characterized among other things by the condensation of chromatin and the digestion of genomic DNA into oligonucleosomal fragments (manifested in apoptotic cells as altered nuclear morphology and the appearance of nucleosomal ladders on gel electrophoresis of DNA), whereas energy stores and membrane integrity remain for the most part intact.’ In the absence of cytokines, apoptosis occurs in 30% to 50% of the cells by 24 hours. Apoptosis in neutrophils can be delayed, but not prevented, by cytokines such as granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin- 1 (IL- I), or lipopolysaccharide.*.’ Apoptosis-associated DNA degradation in other systems has been attributed to any of several endonucleases, including a calcium- and magnesium-dependent endonuclease: the calcium-dependent enzyme DNase I,’ and an acidic endonuclease similar or identical to DNase IL6 In the present report we characterize an acid endonuclease as the only DNase detectable in mature human neutrophils and show that, when the intracellular pH of neutrophils is lowered into the range in which this nuclease is active, internucleosomal DNA cleavage is seen. N

Earlier work has suggested that familial Mediterranean fever, an inherited disorder characterized by sporadic episodes of inflammation involving the pleural and peritoneal cavities and the joints, is caused by the lack of a C5a inactivator normally found in serosal fluid. We have purified this inactivator from ascites fluid and obtained a protein of molecular weight 53 to 56 kD with a specific activity 10,000- fold greater than the crude material. On Western blot, an inhibitory antibody recognized a single antigenic species at the same molecular weight. The enzyme had no activity against denatured bovine serum albumin. With recombinant C5a as substrate, the Km and Vm were 3.4 mumol/L and 52 nmol C5a/min/mg protein, respectively.

The respiratory burst oxidase of phagocytes and B lymphocytes catalyzes the reduction of oxygen by NADPH to form O2-, the precursor of a group of reactive oxidants that are employed by phagocytes as microbicidal agents. The enzyme is active in stimulated cells but dominant in resting cells. It molecular weight guanine nucleotide-binding protein. The components p22phox and gp91phox from cytochrome b558, a flavohemoprotein that resides in the cortical cytoskeleton and in the membranes of the specific granules. The other components are found in the cytosol of resting cells, but migrate to the cortical cytoskeleton when the neutrophils are activated, where they assemble the active oxidase. Migration to the cortical cytoskeleton is caused in part by the appearance of a membrane binding site on one or more of the cytosolic subunits, possibly due to the phosphorylation of p47phox that takes place during cell activation.

The respiratory burst oxidase catalyzes the production of O2.- from oxygen and NADPH. It is dormant in resting cells but becomes active when the cells are stimulated. Activation is accompanied by the phosphorylation of multiple serines in the cytosolic oxidase component p47phox, which moves from cytosol to the membrane during oxidase activation. Using immunopurified p47phox isolated from 32Pi-loaded neutrophils activated with phorbol myristate acetate, we showed that all the 32P was in the C-terminal CNBr fragment of the protein, and that in that fragment, Ser-303, Ser-304, Ser-320, Ser-328, Ser-345, and Ser-348 and at least one of the three serines, Ser-359, Ser-370, and Ser-379, were phosphorylated, while Ser-282, Ser-287, Ser-381, and Ser-388 were not. Of the phosphorylated serines, Ser-303, Ser-304, Ser-320, and Ser-328 are located in protein kinase C substrate sequences. Ser-345 and Ser-348, however, are located in sequences recognized by mitogen-activated protein (MAP) kinase (-PXSP-). This finding suggests that MAP kinase or a related proline-directed kinase may participate in the regulation of O2.- production by activated neutrophils. The tryptic peptide map of p47phox phosphopeptides from neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine closely resembled that of p47phox phosphopeptides from phorbol-activated cells, suggesting that the same serines were phosphorylated in response to each agent.

The complement fragment C5a and the cytokine interleukin-8 (IL-8) are proinflammatory peptides with potent chemotactic activity toward neutrophils. We have previously shown that C5a can be inactivated by a protease that is found in normal synovial and peritoneal fluids but is absent from serosal fluids obtained from patients with familial Mediterranean fever (FMF). We report here that serosal fluids can also eliminate the chemotactic activity of IL-8. The agent responsible for IL-8 elimination appears to be the C5a-inactivating protease, because the pure protease can inactivate IL-8, inactivation of IL-8 by normal peritoneal fluid is partly prevented by an antibody raised against the purified C5a-inactivating protease, and IL-8 is not inactivated by peritoneal fluids from patients with FMF. The ability of this protease to inactivate both, early (C5a) and late (IL-8) inflammatory mediators identifies it as a potentially significant regulator of inflammation.

A 35-kbp region of genomic DNA encoding the human muscle phosphofructokinase (HPFK-M) gene including all of the coding exons (1-22) plus 2.2-kbp of 5'-flanking sequence has been cloned. The exon boundaries are the same as has been observed for the rabbit muscle phosphofructokinase (RPFK-M), the human liver phosphofructokinase (HPFK-L), and the mouse liver phosphofructokinase (MPFK-L) genes. Characterization of the structure of the HPFK-M gene and its transcript in Epstein-Barr virus transformed B-cell lines derived from patients with glycogen storage disease type VII (GSDVII or Tarui's disease) demonstrated that this single-copy gene encodes a normal sized 3.0-kb transcript in the four cases examined. This suggests the lesion in these cases represents either a point mutation or possibly a small insertion or deletion resulting in the synthesis of a defective HPFK-M protein. Analysis of the 5'-flanking region demonstrated the presence of a functional promoter located within 114 nucleotides of a proposed transcription initiation site. This promoter was active in the human cervical carcinoma cell line, HeLa S3, the dedifferentiated human hepatoma cell line, HepG2.1, and the mouse myoblast cell line, C2C12, suggesting this promoter has a broad cell-type specificity. In addition, from the known HPFK-M cDNA sequences, this observation indicates that the HPFK-M gene has a second promoter located upstream from the genomic region isolated in this study.

The respiratory burst oxidase is a multicomponent membrane-bound enzyme that uses NADPH to reduce oxygen to O2-. When oxidase-containing membranes from activated neutrophils are treated with 0.3 M KCl, the NADPH-binding component of the oxidase elutes from the membranes in an active form. Treatment of this eluate with [32P]NADPH dialdehyde labels an approximately 32-kDa protein that is absent from eluates obtained from normal resting membranes or from resting or activated membranes from patients with one form of chronic granulomatous disease. We propose that this approximately 32-kDa protein is the NADPH-binding component of the oxidase.

Ethanolamine ammonia-lyase is a bacterial enzyme that catalyzes the adenosylcobalamin-dependent conversion of certain vicinal amino alcohols to oxo compounds and ammonia. Studies of ethanolamine ammonia-lyase from Clostridium sp. and Escherichia coli have suggested that the enzyme is a heterodimer composed of subunits of Mr approximately 55,000 and 35,000. Using a partial Sau3A Salmonella typhimurium library ligated into pBR328 and selecting by complementation of a mutant lacking ethanolamine ammonia-lyase activity, we have cloned the genes for the 2 subunits of the S. typhimurium enzyme. The genes were localized to a 6.5-kilobase fragment of S. typhimurium DNA, from which they could be expressed in E. coli under noninducing conditions. Sequencing of a 2526-base pair portion of this 6.5-kilobase DNA fragment revealed two open reading frames separated by 21 base pairs. The open reading frames encoded proteins of 452 and 286 residues whose derived N-terminal sequences were identical to the N-terminal sequences of the 2 subunits of the E. coli ethanolamine ammonia-lyase, except that residue 16 of the large subunit was asparagine in the E. coli sequence and aspartic acid in the S. typhimurium sequence.

The genomic DNA encoding human muscle phosphofructokinase (HPFK-M) exons VII to X has been cloned and the coding regions have been sequenced. The intron/exon boundaries are located at the same positions as those identified for the rabbit phosphofructokinase-M gene (Lee, C. -P., Kao, M. -C., French, B. A., Putney, S. D., and Chang, S. H. (1987) J. Biol. Chem. 262, 4195-4199). A HPFK-M cDNA clone lacking the sequences corresponding to exon IX was isolated from a human fibroblast (IMR-90) library, suggesting that the HPFK-M transcript may be alternatively spliced. Exon IX is 93 nucleotides in length, and the absence of this sequence from the HPFK-M transcript would generate an RNA coding for a HPFK-M-related polypeptide lacking 31 amino acids compared with the HPFK-M polypeptide. HPFK-M transcripts approximately 3.0 kilobases in length are expressed in a tissue-specific manner with high levels in cell lines and skeltal muscle tissue and very low levels in peripheral blood mononuclear cells and liver tissue. Characterization of the structure of these HPFK-M transcripts by nuclease S1 and polymerase chain reaction analysis demonstrated that all the cell lines and tissues examined expressed the alternatively spliced transcript in addition to the transcript coding for the enzymatically functional HPFK-M polypeptide.

Most cases of cytosol-defective chronic granulomatous disease are due to the deficiency of a 47-kD protein (p47-phox) whose phosphorylation normally accompanies the activation of the respiratory burst oxidase. Recently, a form of chronic granulomatous disease was described in which the failure of O2- production was associated with the absence of a 67-kD polypeptide (p67-phox) from the cytosol of affected neutrophils. Using neutrophils obtained from a patient with this form of the disease, we examined the function of p67-phox in the activation of the oxidase. Our studies showed that in whole p67-phox-deficient neutrophils, p47-phox was phosphorylated in a normal fashion. In the cell-free oxidase-activating system, the ability of the p67-phox-deficient cytosol to support oxidase activation was partly restored by the addition of p47-phox-deficient cytosol; the p67-phox-deficient cytosol, however, was not complemented by cytosol inactivated with NADPH dialdehyde, an affinity label previously found to block the NADPH-binding component of the oxidase. Despite these differences, the kinetic properties of the p67-phox-deficient cytosol closely resembled those of the p47-phox-deficient cytosol. Taken together with earlier findings, these results suggest that (a) in the neutrophil cytosol, p67-phox is at least partly complexed to p47-phox; (b) it is in the form of this complex that p67-phox participates in oxidase activation; and (c) p47-phox appears to be translocated from the cytosol to the plasma membrane during oxidase activation, but complexation to p67-phox is not necessary for this translocation, nor for the accompanying extra protein phosphorylation.

Sbarra and Karnovsky were the first to present evidence suggesting the presence in phagocytes of a special enzyme designed to generate reactive oxidants for purposes of host defense. In the years since their report appeared, a great deal has been learned about this enzyme, now known as the respiratory burst oxidase. It has been found to be a plasma membrane-bound heme- and flavin-containing enzyme, dormant in resting cells, that catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH: O2 + NADPH----O2- + NADP+ + H+ Its behavior in whole cells and its response to various activating stimuli have been described in detail, although important insights continue to emerge, as for example a very interesting new series of observations on differences in oxidase activation patterns between suspended and adherent cells. The enzyme has been shown by biochemical and genetic studies to consist of at least six components. In the resting cell, three of these components are in the cytosol and three in the plasma membrane, but when the cell passes from its resting to its activated state the cytosolic components are all transferred to the plasma membrane, presumably assembling the oxidase. Of the components initially bound to the membrane, two constitute cytochrome b558, a heme protein characteristic of the respiratory burst oxidase, and the third may represent an oxidase flavoprotein. With regard to the cytosolic components, one is a phosphoprotein and another is the NADPH-binding component, possibly a second oxidase flavoprotein. The nature of the third (p67phox) is a puzzle. Four of the six oxidase components have now been cloned and sequenced. These findings only scratch the surface, however, and many questions remain. How many oxidase components, for example, remain to be discovered, and how do they fit together to form the active enzyme? How is the route of activation of the oxidase integrated into the general signal transduction systems of the cell? How did the oxidase come to be? Could there be a widespread system that generates small amounts of O2- as an intercellular signaling molecule, as recent work is beginning to suggest, and did the ever-destructive respiratory burst oxidase arise from that innocuous system as the creation of some evolutionary Frankenstein--an oxidase from hell? Finally, will it be possible to develop drugs that specifically block the respiratory burst oxidase, and will such drugs prove to be clinically useful as anti-inflammatory agents?(ABSTRACT TRUNCATED AT 400 WORDS)

Oxygen free radical production in hypertension may be associated with elevated arteriolar tone and organ injury. Previous results suggest an enhanced level of oxygen free radical formation in microvascular endothelium and in circulating neutrophils associated with xanthine oxidase activity in the spontaneously hypertensive rats (SHR) compared with their normotensive controls, the Wistar Kyoto rats (WKY). The aim of this study was to gain more detailed understanding of where oxidative enzymes are located in the microcirculation.An approach was developed to delineate the cellular distribution of two selected oxidative enzymes, xanthine oxidase and nicotinamide adenine dinucleotide phosphate (NADPH) dependent oxidase (protein 67-kDa fraction). Immunolabeling with peroxidase substrate was utilized, which permits full delineation of the primary antibody in all microvascular structures of the mesentery.Xanthine oxidase is present in the endothelium of all segments of the microcirculation, in mast cells, and in parenchymal cells of the mesentery. NADPH oxidase can be detected in the endothelium, leukocytes, and mast cells and with lower levels in parenchymal cells. The mesentery of WKY and SHR has similar enzyme distributions with enhancements on the arteriolar and venular side of the microcirculation that coincide with the sites of enhanced free radical production recently reported. Immune label measurements under standardized conditions indicate that both enzymes are significantly enhanced in the SHR. Adrenalectomy, which serves to reduce the blood pressure and free radical production of the SHR to normotensive levels, leads to a reduction of NADPH and xanthine oxidase to normotensive levels, while supplementation of adrenalectomized SHR with dexamethasone significantly increases the oxidase expression in several parts of the microcirculation to levels above the WKY rats.The results indicate that enhanced expression of NADPH and xanthine oxidase in the SHR depends on an adrenal pathway that is detectable in the arteriolar and venular network at high and low pressure regions of the circulation.

Cytoplasmic acidification is now recognized as a feature of apoptosis in a variety of systems. However, its relation to other events in the process of apoptosis is not yet characterized. In this work, we examined the effect of BCL-2 overexpression on acidification mediated by cycloheximide treatment or Fas ligation in Jurkat T-lymphoblasts. We find that BCL-2 overexpression attenuates cytoplasmic acidification and apoptosis detected by annexin V labeling. Acidification and phosphatidylserine externalization were found to occur concurrently. We also examined the requirement for protease activation for cytoplasmic acidification to occur and found that inhibition of interleukin-1β converting enzyme/CED-3 family proteases (using carbobenzoxy-Val-Ala-Asp-fluoromethylketone, an inhibitor of these proteases) prevents acidification and apoptosis mediated by Fas ligation. These studies suggest that BCL-2 acts at a point upstream of acidification and that protease activation is also upstream of acidification.

International audience; The leukocyte NADPH oxidase catalyzes the production of O(2)(-) from oxygen at the expense of NADPH. Activation of the enzyme requires interaction of the cytosolic factors p47(PHOX), p67(PHOX), and Rac2 with the membrane-associated cytochrome b(558). Activation of the oxidase in a semirecombinant cell-free system in the absence of an amphiphilic activator can be achieved by phosphorylation of the cytosolic factor p47(PHOX) by protein kinase C. Another cytosolic factor, p40(PHOX), was recently shown to be phosphorylated on serine and threonine residues upon activation of NADPH oxidase, but both stimulatory and inhibitory roles were reported. In the present study, we demonstrate that the addition of phosphorylated p40(PHOX) to the cell-free system inhibits NADPH oxidase activated by protein kinase C-phosphorylated p47(PHOX), an effect not observed with the unphosphorylated p40(PHOX). Moreover phosphorylated p40(PHOX) inhibits the oxidase if added before or after full activation of the enzyme. Direct mutagenesis of protein kinase C consensus sites enables us to conclude that phosphorylation of threonine 154 is required for the inhibitory effect of p40(PHOX) to occur. Although the phosphorylated mutants and nonphosphorylated mutants are still able to interact with both p47(PHOX) and p67(PHOX) in pull-down assays, their proteolysis pattern upon thrombin treatment suggests a difference in conformation between the phosphorylated and nonphosphorylated mutants. We postulate that phosphorylation of p40(PHOX) on threonine 154 leads to an inhibitory conformation that shifts the balance toward an inhibitory role and blocks oxidase activation.

NADPH oxidase is an enzyme that catalyzes the production of superoxide from oxygen and NADPH. It is a complex enzyme consisting of two membrane-bound components and three components in the cytosol, plus rac 1 or rac 2. Activation of the oxidase involves the phosphorylation of one of the cytosolic components. Recent crystallography data indicate that the tail of this cytosolic component lies in a groove between two Src homology 3 domains and, when phosphorylated, the tail leaves the groove and is replaced by the tail of one of the membrane-bound components. Chronic granulomatous disease is an inherited immune deficiency caused by the absence of one of the components of the oxidase. The most important recent advances in the field have been the crystallographic analysis of the oxidase and the use of antifungal agents in the prophylaxis of chronic granulomatous disease.

The leukocyte NADPH oxidase catalyzes the reduction of oxygen to O2- (superoxide) at the expense of NADPH. The O2- then dismutes to H2O2, which serves to oxidize Cl- to HOCl, a potent microbicidal agent that is used by leukocytes to kill invading microorganisms. This oxidation is catalyzed by myeloperoxidase. O2 is also used to make other microbicidal oxidants, some in reactions with nitric oxide. The oxidase itself is highly complex, consisting of four unique subunits and Rac2. In the resting cell, two of the subunits, p22PHOX and gp91PHOX, are located in the membrane, and the other two, p47PHOX and p67PHOX, are in the cytosol. The electron-carrying components of the oxidase are located in gp91PHOX; the NADPH binding site is generally regarded to be in gp91PHOX as well, but there is some evidence that it may be in p67PHOX. When the oxidase is activated, p47PHOX is phosphorylated at specific sites, and the cytosolic components plus Rac2 migrate to the membrane to assemble the active oxidase.

Recently, we showed that antibodies catalyze the generation of hydrogen peroxide (H 2 O 2 ) from singlet molecular oxygen ( 1 O 2 *) and water. Here, we show that this process can lead to efficient killing of bacteria, regardless of the antigen specificity of the antibody. H 2 O 2 production by antibodies alone was found to be not sufficient for bacterial killing. Our studies suggested that the antibody-catalyzed water-oxidation pathway produced an additional molecular species with a chemical signature similar to that of ozone. This species is also generated during the oxidative burst of activated human neutrophils and during inflammation. These observations suggest that alternative pathways may exist for biological killing of bacteria that are mediated by potent oxidants previously unknown to biology.

The leukocyte NADPH oxidase is regulated chiefly by phosphorylation of the serines of p47(PHOX), one of its cytosolic subunits. Its activity is also regulated, however, by the four cysteines of the same subunit, as indicated by the replacement of those cysteines by alanines.

Activation of the phagocyte NADPH oxidase complex requires assembly of the cytosolic factors p47 PHOX , p67 PHOX , p40 PHOX , and Rac with the membrane-bound cytochrome b 558 . We recently established a direct interaction between p67 PHOX and cytochrome b 558. In the present study, we show that removal of the C-terminal domain of p67 PHOX increased its binding to cytochrome b 558 . Whereas phosphorylated p40 PHOX alone did not bind to cytochrome b 558 , phosphorylated p47 PHOX did, and, moreover, it allowed the binding of p40 PHOX to the cytochrome. Furthermore, both increased the binding of p67 PHOX to the cytochrome. Phosphorylated p47 PHOX thus appears to increase the binding of p67 PHOX to cytochrome b 558 by serving as an adapter, bringing p67 PHOX into proximity with cytochrome b 558 , whereas phosphorylated p40 PHOX may increase the binding by inducing a conformational change that allows p67 PHOX to interact fully with cytochrome b 558 .

The NADPH oxidase of phagocytes catalyzes the conversion of oxygen to O2(-). This multicomponent enzyme complex contains five essential protein components, two in the membrane and three in the cytosol. Unassembled and inactive in resting phagocytes, the oxidase becomes active after translocation of cytosolic components to the membrane to assemble a functional oxidase. Multiple factors regulate its assembly and activity, thus serving to maintain this highly reactive system under spatial and temporal control until recruited for antimicrobial or proinflammatory events. The recent identification of homologs of one of the membrane components in nonphagocytic cells will expand understanding of the biological contexts in which this system may function.

The best known NADPH oxidase is that of phagocytes-neutrophils and monocytes. In these cells, the enzyme manufactures large quantities of O2- and other reactive oxidants that are used for the purpose of killing invading microorganisms. Recent studies, however, have suggested that a number of other tissues contain NADPH oxidases. In contrast to the very vigorous production of oxidants by phagocytes, rates of oxidant production by these other cell types are quite low. Oxidant production by these cells is generally thought to serve a signaling function.

Familial Mediterranean fever is an inherited disorder which causes episodes of severe pain and fever lasting a few days and then recurring shortly afterward. Joints, peritoneal and pleural spaces are primarily affected displaying acute inflammation and an influx of neutrophils into the inflamed area. Keywords: colchicine; amyloid; complement; neutrophil; gene; middle east

The reduction of oxygen by one electron creates superoxide (O2 −), a free radical with surprisingly little reactivity, except with itself. The reactive oxygen is reduced to water through the acceptance of three additional electrons. However, during this process the relatively inert O2 − gives rise to a series of highly reactive oxidants. The production of these oxidants is discussed in the following.

Neutrophils and other phagocytes manufacture O(2)(-) (superoxide) by the one-electron reduction of oxygen at the expense of NADPH. Most of the O(2)(-) reacts with itself to form H(2)O(2) (hydrogen peroxide). From these agents a large number of highly reactive microbicidal oxidants are formed, including HOCl (hypochlorous acid), which is produced by the myeloperoxidase-catalyzed oxidation of Cl(-) by H(2)O(2); OH(*) (hydroxyl radical), produced by the reduction of H(2)O(2) by Fe(++) or Cu(+); ONOO(-) (peroxynitrite), formed by the reaction between O(2)(-) and NO(*); and many others. These reactive oxidants are manufactured for the purpose of killing invading microorganisms, but they also inflict damage on nearby tissues, and are thought to be of pathogenic significance in a large number of diseases. Included among these are emphysema, acute respiratory distress syndrome, atherosclerosis, reperfusion injury, malignancy and rheumatoid arthritis.

The nicotinamide adenine dinucleotide phosphate (NADPH) binding site of the NADPH oxidase complex is believed to be located on the beta, subunit of cytochrome b558. However, our previous studies showed that p67PHOX also contains an NADPH binding site that is essential for normal oxidase activity and that p67PHOX is able to mediate a slow electron transfer from a reduced pyridine nucleotide to an artificial electron acceptor. Using both affinity labeling and fluorescence quenching, we have obtained further evidence that p67PHOX is able to bind NADPH. We have used a number of truncated forms of p67PHOX, including p67PHOX(1-243), p67PHOX(1-210), p67PHOX(1-199), and p67PHOX(244-526) (where the numbers represent the initial and final amino acids in the truncated p67PHOX) in order to localize the binding site. We found that NADPH could bind to p67PHOX(1-243), p67PHOX(1-210), and p67PHOX(1-199) but not to p67PHOX(244-526). The p67PHOX(1-199) fragment consists largely of four tetratricopeptide (TPR) domains. We showed further that Rac2-GTP gamma S and to a lesser extent Rac2-GDP beta S could modulate the binding of NADPH to p67PHOX.

The leukocyte NADPH oxidase is an enzyme present in phagocytes and B lymphocytes that when activated catalyzes the production of O-2 from oxygen at the expense of NADPH. A correlation between the activation of the oxidase and the phosphorylation of p47(PHOX), a cytosolic oxidase component, is well recognized in whole cells, and direct evidence for a relationship between the phosphorylation of this oxidase component and the activation of the oxidase has been obtained in a number of cell-free systems containing neutrophil membrane and cytosol. Using superoxide dismutase-inhibitable cytochrome c reduction to quantify O-2 production, we now show that p47(PHOX) phosphorylated by protein kinase C activates the NADPH oxidase not only in a cell-free system containing neutrophil membrane and cytosol, but also in a system in which the cytosol is replaced by the recombinant proteins p67(PHOX), Rac2, and phosphorylated p47(PHOX), suggesting that neutrophil plasma membrane plus those three cytosolic proteins are both necessary and sufficient for oxidase activation. In both the cytosol-containing and recombinant cell-free systems, however, activation by SDS yielded greater rates of O-2 production than activation by protein kinase C-phosphorylated p47(PHOX), indicating that a system that employs protein kinase C-phosphorylated p47(PHOX) as the sole activating agent, although more physiological than the SDS-activated system, is nevertheless incomplete.

The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.