Tumor necrosis factor-alpha increases airway smooth muscle oxidants production through a NADPH oxidase-like system to enhance myosin light chain phosphorylation and contractility

Tumor necrosis factor plays a critical role in airway smooth muscle hyperresponsiveness observed in asthma. However, the mechanisms underlying this phenomenon are poorly understood. We investigated if tumor necrosis factor-stimulated airway smooth muscle produced reactive oxygen species, leading to muscular hyperresponsiveness. Tumor necrosis factor increased intracellular and extracellular oxidants production in guinea pig airway smooth muscle cells and tissue homogenates. This production was abolished by inhibitors of NADPH oxidase (diphenylene iodinium or apocynin) and was enhanced by NADPH, whereas inhibitors of mitochondrial respiratory chain, nitric-oxide synthase, cyclooxygenase, and xanthine oxidase had no effect. NADPH oxidase subunits p22(phox) and p47(phox) were detected in smooth muscle cells and tissue homogenates by Western blot, immunohistochemistry, and spectral analysis. Furthermore, oxidants production was significantly reduced by transient transfection of smooth muscle cells with p22(phox) antisense oligonucleotides. Intracellular antioxidants and diphenylene iodinium abolished tumor necrosis factor-induced muscular hyperresponsiveness and increased in phosphorylation of the myosin light chain. Finally, NADPH oxidase subunits p22(phox) and p47(phox) were also detected in human airway smooth muscle. Collectively, these results demonstrate that tumor necrosis factor-stimulated airway smooth muscle produces oxidants through a NADPH oxidase-like system, which plays a pivotal role in muscle hyperresponsiveness and myosin light chain phosphorylation.     

Low extracellular zinc increases neuronal oxidant production through nadph oxidase and nitric oxide synthase activation

A decrease in zinc (Zn) levels increases the production of cell oxidants, affects the oxidant defense system and triggers oxidant sensitive signals in neuronal cells. However, the underlying mechanisms are still unclear. This work tested the hypothesis that the increase in neuronal oxidants that occurs when cellular Zn decreases is mediated by the activation of the NMDA receptor. Differentiated PC12 cells were cultured in control, Zn-deficient or Zn-repleted media. The incubation in Zn deficient media led to a rapid increase in cellular calcium levels, which was prevented by a NMDA receptor antagonist (MK-801). Cellular calcium accumulation was associated with NADPH oxidase and nitric oxide synthase (NOS) activation, an increase in cell oxidant levels, and an associated activation of a redox-sensitive signal (AP-1). In cells incubated in the Zn deficient medium, NADPH oxidase activation was prevented by MK-801 and by a protein kinase C inhibitor. The rise in cell oxidants was prevented by inhibitors of NADPH oxidase, of the NOS and by MK-801. A similar pattern of inhibitor action was observed for zinc deficiency-induced AP-1 activation. Results demonstrate that a decrease in extracellular Zn leads to an increase in neuronal oxidants through the activation of the NMDAR that leads to calcium influx and to a calcium-mediated activation of protein kinase C/NADPH oxidase and NOS. Changes in extracellular Zn concentrations can be sensed by neurons, which using reactive oxygen and nitrogen species as second messengers, can regulate signaling involved in neuronal development and function.     

Initiation of a superoxide-dependent chain oxidation of lactate dehydrogenase-bound NADH by oxidants of low and high reactivity

In cells, NADH and NADPH are mainly bound to dehydrogenases such as lactate dehydrogenase (LDH). In cell-free systems, the binary LDH-NADH complex has been demonstrated to produce reactive oxygen species via a chain oxidation of NADH initiated and propagated by superoxide. We studied here whether this chain radical reaction can be initiated by oxidants other than LDH largely increased the oxidation of NADH (but not of NADPH) by O(2), H(2)O(2) and during the intermediacy of HNO(2). LDH also increased the oxidation of NADH by peroxynitrite. The increases in NADH oxidation were completely prevented by superoxide dismutase (SOD). In contrast, the nitrogen dioxide-dependent oxidation of NADH and NADPH was decreased by LDH in a SOD-independent manner. These experimental data strongly indicate that oxidation of LDH-bound NADH can be induced from reaction of either weak oxidants with LDH-bound NADH or of strong oxidants with free NADH thus yielding which is highly effective to propagate the chain. Our results underline the importance of SOD in terminating superoxide-dependent chain reactions in cells under oxidative stress.     

Assessment of wound-site redox environment and the significance of Rac2 in cutaneous healing

We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.     

Endotoxin priming of neutrophils requires NADPH oxidase-generated oxidants and is regulated by the anion transporter ClC-3

Several soluble mediators, including endotoxin, prime neutrophils for an enhanced respiratory burst in response to subsequent stimulation. Priming of neutrophils occurs in vitro, and primed neutrophils are found in vivo. We previously localized the anion transporter ClC-3 to polymorphonuclear leukocytes (PMN) secretory vesicles and demonstrated that it is required for normal NADPH oxidase activation in response to both particulate and soluble stimuli. We now explore the contribution of the NADPH oxidase and ClC-3 to endotoxin-mediated priming. Lipooligosaccharide (LOS) from Neisseria meningitidis enhances the respiratory burst in response to formyl-Met-Leu-Phe, an effect that was impaired in PMNs lacking functional ClC-3 and under anaerobic conditions. Mobilization of receptors to the cell surface and phosphorylation of p38 MAPK by LOS were both impaired in PMN with the NADPH oxidase chemically inhibited or genetically absent and in cells lacking functional ClC-3. Furthermore, inhibition of the NADPH oxidase or ClC-3 in otherwise unstimulated cells elicited a phenotype similar to that seen after endotoxin priming, suggesting that basal oxidant production helps to maintain cellular quiescence. In summary, NADPH oxidase activation was required for LOS-mediated priming, but basal oxidants kept unstimulated cells from becoming primed. ClC-3 contributes to both of these processes.     

TNFalpha activates c-Jun amino terminal kinase through p47(phox).

Reactive oxygen intermediates have been implicated in the transduction of TNFalpha signals, although the source of such oxidants has not been established. We found that activation of ECV-304 cells by TNFalpha was accompanied by a transient burst of oxidants and activation of JNK, both of which were suppressed by two distinct inhibitors of the phagocyte NADPH oxidase and the thiol antioxidant N-acetyl cysteine (NAC). We cloned partial and full-length cDNA sequences from ECV-304 cells and human umbilical vein endothelial cells (HUVEC), respectively, for p47(phox), demonstrating that these nonphagocytic cells express this adapter protein known to specifically initiate assembly of the NADPH oxidase in professional phagocytes. A mutant p47(phox), defective in the first Src homology 3 (SH3) domain (p47W(193)R), diminished JNK activation by TNFalpha. Surprisingly, p47(phox) resided entirely in the particulate, not cytosolic, fraction of cells. Immunostaining suggested partial colocalization with cytoskeletal elements, and cytoskeletal disrupters decreased both oxidant production and JNK activation by TNFalpha. A p47-GFP fusion protein localized to the cortical cytoskeleton in living cells; further, stimulation of cells with TNFalpha caused a marked concentration of p47-GFP in membrane ruffles, actin-rich structures associated with intense respiratory burst activity in stimulated neutrophils. We conclude that nonphagocytic cells express p47(phox), which appears to localize to the cytoskeleton and participate in TNFalpha signaling. We speculate that this physical targeting may prove important in conferring signal specificity and enhancing signaling efficiency of unstable oxidants.     

The O2Generating NADPH Oxidase of Phagocytes: Structure and Methods of Detection

The NADPH oxidase of phagocytic cells of the immune system plays an important role in the destruction of certain types of microbial pathogens during infections. Its inappropriate activation to release reactive oxidants may also contribute to host tissue damage in inflammatory diseases. In this review, the structure of the NADPH oxidase is described and many of the commonly used methods to detect its activation during the respiratory burst are listed. The advantages and disadvantages of each of these methods are critically discussed.     

Enhanced oxidation of NAD(P)H by oxidants in the presence of dehydrogenases but no evidence for a superoxide-propagated chain oxidation of the bound coenzymes

Recently we demonstrated that lactate dehydrogenase (LDH)-bound NADH is oxidized by O2, H2O2, HNO2 and peroxynitrite predominantly via a chain radical mechanism which is propagated by superoxide. Here we studied both whether other dehydrogenases also increase their coenzymes' reactivity towards these oxidants and whether a chain radical mechanism is operating. Almost all dehydrogenases increased the oxidation of their physiological coenzymes by at least one of the oxidants. The oxidation of NADH or NADPH depended both on the binding dehydrogenase and the applied oxidant and in some cases the reactions were remarkably fast. The highest rate constant (k = 370 M-1 s-1) was found for the reaction of HNO2 with NADH bound to alcohol dehydrogenase. Regardless of the applied oxidant, superoxide dismutase failed to inhibit the oxidation of protein-bound NADH and NADPH. We therefore conclude that several dehydrogenases increase the oxidation of NADH and/or NADPH by the employed set of oxidants in bimolecular reactions, but, unlike LDH, do not mediate a O2*(-) dependent chain radical mechanism.     

Aldosterone increases kidney tubule cell oxidants through calcium-mediated activation of NADPH oxidase and nitric oxide synthase

Chronic hyperaldosteronism has been associated with an increased cancer risk. We recently showed that aldosterone causes an increase in cell oxidants, DNA damage, and NF-κB activation. This study investigated the mechanisms underlying aldosterone-induced increase in cell oxidants in kidney tubule cells. Aldosterone caused an increase in both reactive oxygen and reactive nitrogen (RNS) species. The involvement of the activation of NADPH oxidase in the increase in cellular oxidants was demonstrated by the inhibitory action of the NADPH oxidase inhibitors DPI, apocynin, and VAS2870 and by the migration of the p47 subunit to the membrane. NADPH oxidase activation occurred as a consequence of an increase in cellular calcium levels and was mediated by protein kinase C. The prevention of RNS increase by BAPTA-AM, W-7, and L-NAME indicates a calcium-calmodulin activation of NOS. A similar pattern of effects of the NADPH oxidase and NOS inhibitors was observed for aldosterone-induced DNA damage and NF-κB activation, both central to the pathogenesis of chronic aldosteronism. In summary, this paper demonstrates that aldosterone, via the mineralocorticoid receptor, causes an increase in kidney cell oxidants, DNA damage, and NF-κB activation through a calcium-mediated activation of NADPH oxidase and NOS. Therapies targeting calcium, NOS, and NADPH oxidase could prevent the adverse effects of hyperaldosteronism on kidney function as well as its potential oncogenic action.     

Protein oxidation and proteolysis in RAW264.7 macrophages: effects of PMA activation

Macrophages are stimulable cells able to increase the production of reactive oxygen and nitrogen species dramatically for a short period of time. Free radicals and other oxidants are able to oxidize the intracellular protein pool. These oxidized proteins are selectively recognized and degraded by the intracellular proteasomal system. We used the mouse macrophage-like cell line RAW264.7 to test whether macrophagial cells are able to increase their protein turnover after oxidative stress and whether this is accompanied by an increased protein oxidation. Macrophagial cells are particularly susceptible to bolus additions of hydrogen peroxide and peroxynitrite. In further experiments we activated RAW264.7 cells with PMA to test whether the production of endogenous oxidants has analogous effects. A clear dependence of the protein turnover and protein oxidation on the oxidative burst could be measured. In further experiments the role of the proteasomal system in the selective removal of oxidized proteins could be revealed exploring the proteasome specific inhibitor lactacystin. Therefore, although oxidants are able to attack the intracellular protein pool in macrophages, these cells are able to remove oxidized proteins selectively and protect the intracellular protein pool from oxidation.     

Decrease of NADH in HeLa cells in the presence of transferrin or ferricyanide

The short-term incubation of HeLa cells in the presence of diferric transferrin or ferricyanide, which are reduced externally by the transplasma membrane reductase, produces a stoichiometric decrease in NADH and increase in NAD+, which is stimulated by insulin. The NADP/NADPH ratio does not change during 15 min incubation with the oxidants. The total pyridine nucleotide pool of HeLa cells is not affected. Incubation with apotransferrin and ferrocyanide, which cannot act as oxidants for transmembrane electron transport, does not change the pyridine nucleotide concentrations in the cells. Our results show that NADH can act as the internal electron donor for the reduction of external oxidants by the transmembrane reductase. It appears that oxidation of NADH by the transmembrane electron transport using ferricyanide or iron transferrin as external electron acceptors is sufficient to stimulate growth in HeLa cells.     

Ethylene- and enzyme-mediated superoxide production and cell death in carrot cells grown under carbon starvation

Carrot (Daucus carota L.) suspension cells are known to exhibit acclimation responses, such as fatty acid catabolism, during the first 5 days of carbon starvation. The present study shows that cells start to die once starvation is prolonged beyond this period. We investigated the signals involved in this transition from acclimation to cell death. Significant amounts of superoxide were produced transiently at days 4-5 (after the initiation of starvation). When diphenylene iodonium, an inhibitor of NADPH oxidase and/or other ROS (reactive oxygen species)-generating enzymes, was added to the cell suspension at the beginning of the starvation period, superoxide production and cell death, as measured by internucleosomal cleavage, were effectively inhibited, indicating that NADPH oxidase and/or other ROS-generating enzymes are involved in the production of the superoxide during starvation. Ethylene, which is known to be produced transiently at day 1 in these cells, positively regulated production of the superoxide and cell death, indicating that ethylene is an upstream regulator of NADPH oxidase or other ROS-generating enzymes. We suggest that as the carrot cells are dying under conditions of carbon starvation they put the following signal relays into operation: ethylene production M activation of NADPH oxidase (or other ROS-generating enzymes) M superoxide production M internucleosomal cleavage (cell death).     

The role of kupffer cell oxidant production in early ethanol-induced liver disease

Considerable evidence for a role of Kupffer cells in alcoholic liver disease has accumulated and they have recently been shown to be a predominant source of free radicals. Several approaches including pharmacological agents, knockout mice, and viral gene transfer have been used to fill critical gaps in understanding key mechanisms by which Kupffer cell activation, oxidant formation, and cytokine production lead to liver damage and subsequent pathogenesis. This review highlights new data in support of the hypothesis that Kupffer cells play a pivotal role in hepatotoxicity due to ethanol by producing oxidants via NADPH oxidase.     

Heat shock inactivates cellular antioxidant defenses against hydrogen peroxide: protection by glucose

Hyperthermia is used in cancer treatment and potentiates the cytotoxicity of radiation and certain chemotherapy drugs. The mechanism(s) of heat killing and those involved in heat potentiation of cytotoxic modalities are not understood. This study examines whether heat shock causes a redox imbalance, leading to oxidative changes in Chinese hamster ovary cells. Decreases in the GSH/GSSG ratio reflected an oxidative imbalance in heated (42 degrees C) and in H(2)O(2)-challenged cells. Glucose provided protection against these changes. Glucose also protected cells against cytotoxicity of H(2)O(2) and/or hyperthermia (42 to 43 degrees C). Glucose appears to protect cells against H(2)O(2) and heat shock by providing NADPH through its metabolism via the pentose phosphate cycle (PC). When cells were deprived of glucose, there was a marked decrease in the GSH/GSSG ratio and in NADPH levels, indicating a severe redox imbalance. Glucose deprivation caused cell death, which was consistent with increased accumulation of H(2)O(2), since three distinct H(2)O(2)-detoxifying systems (N-acetyl-L-cysteine, sodium pyruvate, and catalase) rescued cells against cytotoxicity. Nontoxic levels of H(2)O(2) stimulated a corresponding increase in both PC activity and NADPH levels. NADPH levels and basal activity of the PC increased at 42 degrees C. However, the oxidant-stimulated increases in PC activity and NADPH levels were lost in heated cells. Therefore, heat shock inactivates an important cellular defense mechanism against oxidants. These findings suggest that heat shock may enhance the cytotoxicity of oxidants by inhibiting increases in PC activity following oxidative stress. These data are potentially relevant to understanding the potentiation of cytotoxicity of radiation and oxidant-generating drugs by heat shock, used in combined modality cancer treatment.     

The effect of oxidants on biomembranes and cellular metabolism

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na⁺ by oxidizing Na⁺-channels. Increased intracellular Na⁺ leads to an increase in Na⁺/Ca²⁺ exchange. The increased Na⁺ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca²⁺ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.     

Regulation of human neutrophil oxidative burst by pro- and anti-inflammatory cytokines

Human polymorphonuclear neutrophils play a key role in host defenses against invading microorganisms. In response to a variety of stimuli, neutrophils release large quantities of superoxide anion (O2.-) in a phenomenon known as the respiratory burst. O2.- is the precursor of potent oxidants, which are essential for bacterial killing and also potentiate inflammatory reactions. Regulation of this production is therefore critical to kill pathogens without inducing tissue injury. Neutrophil production of O2.- is dependent on the respiratory burst oxidase, or NADPH oxidase, a multicomponent enzyme system that catalyzes NADPH-dependent reduction of oxygen to O2.-. NADPH oxidase is activated and regulated by various neutrophil stimuli at infectious or inflammatory sites. Proinflammatory cytokines such as GM-CSF, TNF and IL-8 modulate NADPH oxidase activity through a priming phenomenon. These cytokines induce a very weak oxidative response by PMN but strongly enhance neutrophil release of reactive oxygen species on exposure to a secondary applied stimulus such as bacterial N-formyl peptides. Priming phenomena are involved in normal innate immune defense and in some inflammatory diseases. The mechanisms underlying the priming process are poorly understood, although some studies have suggested that priming with various agonists is regulated at the receptor and post-receptor levels. Resolution of inflammation involves desensitization phenomena and cytokines are involved in this process by various mechanisms. A better understanding of phenomena involved in the regulation of NADPH oxidase could help to develop novel therapeutic agents for inflammatory diseases involving abnormal neutrophil superoxide production.     

Antioxidant function of cytosolic sources of NADPH in yeast

The relative antioxidant functions of thiol-dependent mechanisms and of direct catalytic inactivation of H2O2 were examined using a collection of yeast mutants containing disruptions in single or multiple genes encoding two major enzymatic sources of NADPH [glucose-6-phosphate dehydrogenase (ZWF1) and cytosolic NADP+-specific isocitrate dehydrogenase (IDP2)] and in genes encoding two major cellular peroxidases [mitochondrial cytochrome c peroxidase (CCP1) and cytosolic catalase (CTT1)]. Both types of mechanisms were found to be important for growth in the presence of exogenous H2O2. In the absence of exogenous oxidants, however, loss of ZWF1 and IDP2, but not loss of CTT1 and CCP1, was found to be detrimental not only to growth but also to viability of cells shifted to rich medium containing oleate or acetate. The loss in viability correlates with increased levels of intracellular oxidants apparently produced during normal metabolism of these carbon sources. Acute effects in DeltaZWF1DeltaIDP2 mutants following shifts to these nonpermissive media include an increase in the number of cells demonstrating a transient decrease in growth rate and in cells containing apparent nuclear DNA strand breaks. Cumulative effects are reflected in phenotypes, including sensitivity to acetate medium and a reduction in mating efficiency, that become more pronounced with time following disruption of the ZWF1 and IDP2 genes. These results suggest that cellular mechanisms dependent on NADPH are crucial metabolic antioxidants.     

Production of myeloid cell cytosols functionally and immunochemically deficient in the 47 kDa or 67 kDa NADPH oxidase cytosolic factors

Professional phagocytes contain a unique NADPH oxidase responsible for the production of microbicidal oxidants. Activation of this oxidase requires participation of cytosolic and membrane proteins, but the interactions of these components are incompletely understood. Patients with autosomal recessive Chronic Granulomatous Diseases (CGD) are characterized by functional defects in phagocyte oxidase activity resulting from a deficiency of either a 47 kDa (p47) or a 67 kDa (p67) cytosolic oxidase component. Cytosols from such patients are valuable for biochemical studies of the oxidase, but are not generally available because CGD is a rare disorder. The present study illustrates means of producing cytosols functionally and immunochemically deficient in either p47 or p67. Cytosol from monocytes cultured for 6 days is immunochemically deficient in p47 but not p67, while cytosol from HL-60 cells induced with retinoic acid for 3 days is deficient in p67 but not p47. Each of these cytosols fail to generate superoxide when added to neutrophil membranes in a cell-free assay but complement each other when combined. Complementation studies in which these cytosols were mixed in the cell-free assay with p47- or p67- deficient CGD cytosol established the functional characteristics of the experimentally produced cytosols.     

Reactive oxygen intermediate-dependent NF-kappaB activation by interleukin-1beta requires 5-lipoxygenase or NADPH oxidase activity

We previously reported that the role of reactive oxygen intermediates (ROIs) in NF-kappaB activation by proinflammatory cytokines was cell specific. However, the sources for ROIs in various cell types are yet to be determined and might include 5-lipoxygenase (5-LOX) and NADPH oxidase. 5-LOX and 5-LOX activating protein (FLAP) are coexpressed in lymphoid cells but not in monocytic or epithelial cells. Stimulation of lymphoid cells with interleukin-1beta (IL-1beta) led to ROI production and NF-kappaB activation, which could both be blocked by antioxidants or FLAP inhibitors, confirming that 5-LOX was the source of ROIs and was required for NF-kappaB activation in these cells. IL-1beta stimulation of epithelial cells did not generate any ROIs and NF-kappaB induction was not influenced by 5-LOX inhibitors. However, reintroduction of a functional 5-LOX system in these cells allowed ROI production and 5-LOX-dependent NF-kappaB activation. In monocytic cells, IL-1beta treatment led to a production of ROIs which is independent of the 5-LOX enzyme but requires the NADPH oxidase activity. This pathway involves the Rac1 and Cdc42 GTPases, two enzymes which are not required for NF-kappaB activation by IL-1beta in epithelial cells. In conclusion, three different cell-specific pathways lead to NF-kappaB activation by IL-1beta: a pathway dependent on ROI production by 5-LOX in lymphoid cells, an ROI- and 5-LOX-independent pathway in epithelial cells, and a pathway requiring ROI production by NADPH oxidase in monocytic cells.     

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.