New insights into the membrane topology of the phagocyte NADPH oxidase: characterization of an anti-gp91-phox conformational monoclonal antibody

Cytochrome b(558) is the catalytic core of the phagocyte NADPH oxidase that mediates the production of bactericidal reactive oxygen species. Cytochrome b(558) is formed by two subunits gp91-phox and p22-phox (1/1), non-covalently associated. Its activation depends on the interaction with cytosolic regulatory proteins (p67-phox, p47-phox, p40-phox and Rac) leading to an electron transfer from NADPH to molecular oxygen and to the release of superoxide anions. Several studies have suggested that the activation process was linked to a change in cytochrome b(558) conformation. Recently, we confirmed this hypothesis by isolating cytochrome b(558) in a constitutively active form. To characterize active and inactive cytochrome b(558) conformations, we produced four novel monoclonal antibodies (7A2, 13B6, 15B12 and 8G11) raised against a mixture of cytochrome b(558) purified from both resting and stimulated neutrophils. The four antibodies labeled gp91-phox and bound to both native and denatured cytochrome b(558). Interestingly, they were specific of extracellular domains of the protein. Phage display mapping combined to the study of recombinant gp91-phox truncated forms allowed the identification of epitope regions. These antibodies were then employed to investigate the NADPH oxidase activation process. In particular, they were shown to inhibit almost completely the NADPH oxidase activity reconstituted in vitro with membrane and cytosol. Moreover, flow cytometry analysis and confocal microscopy performed on stimulated neutrophils pointed out the capacity of the monoclonal antibody 13B6 to bind preferentially to the active form of cytochrome b(558). All these data suggested that the four novel antibodies are potentially powerful tools to detect the expression of cytochrome b(558) in intact cells and to analyze its membrane topology. Moreover, the antibody 13B6 may be conformationally sensitive and used as a probe for identifying the active NADPH oxidase complex in vivo.     

Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity.

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.     

Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein.

Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.     

NADPH oxidase of Epstein-Barr-virus immortalized B lymphocytes. Effect of cytochrome b(558) glycosylation.

The phagocyte NADPH oxidase is known to be expressed in Epstein-Barr virus (EBV) immortalized B lymphocytes. But even if its molecular composition and its catalytic mechanisms are similar, the activity measured in B cells is very low compared to that of neutrophils. This could be explained by the low expression of cytochrome b558, the membrane redox component, but also by a defect in the activation process. This work is focused on gp91-phox glycosylation in B lymphocytes to assess its role in the complex assembly upon activation. Atomic force microscopy (AFM) combined with immunochemical approaches were used to investigate the effect of the glycosylation on the structure of cytochrome b558 inserted into liposomes, on the reconstituted oxidase activity in vitro, and to directly monitor interaction forces between specific antibodies and the hemoprotein in its native or deglycosylated state. The results show that in EBV-B cells, gp91-phox glycosylation is higher than in neutrophils. The interaction force measured between the monoclonal antibody 11C12, known to inhibit O(-2) production in B lymphocytes, and the hemoprotein is increased after deglycosylation. This suggested that the epitope region recognized by this antibody is partly hidden in B cells, and that this region could be involved in the conformational change that occurs in the hemoprotein during the complex assembly. The high glycosylation of gp91-phox in B cells associated with the lipidic environment could lead to additional structural constraints in the membrane-bound hemoprotein that partly blocked the hemoprotein in its inactive state.     

Cell-free translocation of recombinant p47-phox, a component of the neutrophil NADPH oxidase: effects of guanosine 5'-O-(3-thiotriphosphate), diacylglycerol, and an anionic amphiphile.

We reported previously that diacylglycerol (diC8) and GTP gamma S synergize with an anionic amphiphile such as sodium dodecyl sulfate (SDS) to produce high rates of superoxide generation in a cell-free system consisting of neutrophil plasma membrane plus cytosol [Burnham, D. N., Uhlinger, D. J., & Lambeth, J. D. (1990) J. Biol. Chem. 265, 17550-17559]. Here we investigate the effects of these activating factors on the plasma membrane association in an in vitro translated radiolabeled recombinant p47-phox protein. Apparent translocation, assayed by cosedimentation with plasma membranes, required the presence of excess cytosol and an anionic amphiphile, was enhanced by both GTP gamma S and diC8, and was inhibited by high salt, correlating qualitatively with activation; up to 70% cosedimentation was observed with the combination of activators (compared with less than 20% in their absence). Similar results were obtained using heat-inactivated cytosol, wherein another oxidase component, p67-phox, has been inactivated. Unexpectedly, from 50 to 80% of the apparent translocation occurred in the absence of membranes, indicating that protein aggregation accounted for a significant part of the observed translocation. Nevertheless, the percent translocation was increased in all cases by the presence of membranes, indicating some degree of protein-membrane interaction. While a control in vitro translated protein failed to translocate, cosedimentation of p47-phox occurred equally well when red blood cell or neutrophil plasma membranes lacking cytochrome b558 were used. Also, the peptide RGVHFIF, which is contained within the C-terminus of the large subunit of cytochrome b558, failed to inhibit translocation/aggregation of p47-phox, despite its ability to inhibit cell-free activation of the oxidase. The data are consistent with the following: (a) SDS, diC8, and GTP gamma S all act on cytosolic components to alter protein-protein and/or protein-membrane associations, and these changes are necessary (but not sufficient) for activation; (b) these altered associations are likely to function by increasing the local concentration of p47-phox and other components at the plasma membrane; (c) a high background of nonspecific associations in the cell-free activation system is likely to obscure any specific, functionally relevant associations (e.g., with cytochrome b558); and (d) the mechanism of translocation in the cell-free system differs from that seen in intact neutrophils.     

Molecular quality control machinery contributes to the leukocyte NADPH oxidase deficiency in chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited immunodeficiency disease caused by defects in leukocyte NADPH oxidase. Various inherited defects in one of the membrane-bound components of NADPH oxidase, gp91-phox, cause X-linked (X91) CGD. Analysis of three patients with X91 CGD revealed that different mechanisms of molecular quality control lead to the common phenotype of absence of mature membrane-bound NADPH oxidase complex in leukocytes. In the first patient, aberrant intron splicing created a premature stop codon. However, the mutant mRNA was degraded prematurely, which prevented the production of truncated protein. In the second patient, a frameshift mutation with the potential to generate a gp91-phox polypeptide, with an aberrant and elongated C-terminus, led to barely detectable levels of gp91-phox, even though the reported functional domains of the protein appeared unaffected. In the third patient, a point mutation created a single amino acid change in the predicted FAD-binding site of gp91-phox. Although gp91-phox was detectable with Western blotting, no cytochrome b(558) was expressed on the cell surface. These analyses showed that molecular quality control machinery plays an important role in the pathogenesis of CGD, not only in the X910 but also in the X91- form of this X-linked disease.     

Complementation of NADPH oxidase in p67-phox-deficient CGD patients p67-phox/p40-phox interaction.

Chronic granulomatous disease (CGD) is due to a functional defect of the O2- generating NADPH oxidase of phagocytes. Epstein-Barr-virus-immortalized B lymphocytes express all the constituents of oxidase with activity 100 times less than that of neutrophils. As in neutrophils, oxidase activity of Epstein-Barr-virus-immortalized B lymphocytes was shown to be defective in the different forms of CGD; these cells were used as a model for the complementation studies of two p67-phox-deficient CGD patients. Reconstitution of oxidase activity was performed in vitro by using a heterologous cell-free assay consisting of membrane-suspended or solubilized and purified cytochrome b558 that was associated with cytosol or with the isolated cytosolic-activating factors (p67-phox, p47-phox, p40-phox) from healthy or CGD patients. In p67-phox-deficient CGD patients, two cytosolic factors are deficient or missing: p67-phox and p40-phox. Not more than 20% of oxidase activity was recovered by complementing the cytosol of p67-phox-deficient patients with recombinant p67-phox. On the contrary, a complete restoration of oxidase activity was observed when, instead of cytosol, the cytosolic factors were added in the cell-free assay after isolation in combination with cytochrome b558 purified from neutrophil membrane. Moreover, the simultaneous addition of recombinant p67-phox and recombinant p40-phox reversed the previous complementation in a p40-phox dose-dependent process. These results suggest that in the reconstitution of oxidase activity, p67-phox is the limiting factor; the efficiency of complementation depends on the membrane tissue and the cytosolic environment. In vitro, the transition from the resting to the activated state of oxidase, which results from assembling, requires the dissociation of p40-phox from p67-phox for efficient oxidase activity. In the process, p40-phox could function as a negative regulatory factor and stabilize the resting state.     

P67-phox-mediated NADPH oxidase assembly: imaging of cytochrome b558 liposomes by atomic force microscopy

NADPH oxidase activity depends on the assembly of the cytosolic activating factors, p67-phox, p47-phox, p40-phox, and Rac with cytochrome b(558). The transition from an inactive to an active oxidase complex induces the transfer of electrons from NADPH to oxygen through cytochrome b(558). The assembly of oxidase complex was studied in vitro after reconstitution in a heterologous cell-free assay by using true noncontact mode atomic force microscopy. Cytochrome b(558) was purified from neutrophils and Epstein-Barr virus-immortalized B lymphocytes and incorporated into liposomes. The effect of protein glycosylation on liposome size and oxidase activity was investigated. The liposomes containing the native hemoprotein purified from neutrophils had a diameter of 146 nm, whereas after deglycosylation, the diameter was reduced to 68 nm, although oxidase activity was similar in both cases. Native cytochrome b(558) was used after purification in reconstitution experiments to investigate the topography of NADPH oxidase once it was assembled. For the first time, atomic force microscopy illustrated conformational changes of cytochrome b(558) during the transition from the inactive to the active state of oxidase; height measurements allow the determination of a size of 4 nm for the assembled complex. In the processes that were studied, p67-phox displayed a critical function; it was shown to be involved in both assembly and activation of oxidase complex while p47-phox proceeded as a positive effector and increased the affinity of p67-phox with cytochrome b(558), and p40-phox stabilizes the resting state. The results suggest that although an oligomeric structure of oxidase machinery has not been demonstrated, allosteric regulation mechanisms may be proposed.     

Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components.

Activated human polymorphonuclear neutrophils (PMNs) convert molecular oxygen into superoxide anion, a process known as the respiratory burst, through the activity of a latent multicomponent NADPH-dependent oxidase. Components of this respiratory burst oxidase include the membrane-bound cytochrome b558 and the cytosolic factors p47-phox and p67-phox. We initiated these studies based on three observations: 1) that stimulation of PMN oxidase activity is associated with translocation of the cytosolic oxidase components to the plasma membrane; 2) that p47-phox is phosphorylated during PMN activation and that there is a sequential relationship between phosphorylation of p47-phox in the cytosol and appearance of the phosphoprotein in the membran; and 3) that the predicted amino acid sequences of p47-phox and of p67-phox contain regions of homology to the SH3 or A domain of the src family of tyrosine kinases, a region found in a variety of proteins which interact with the cytoskeleton or the subplasmalemmal cytoskeleton. Thus the purpose of our studies was to examine the role of protein kinase C (PKC)-dependent phosphorylation in the stimulus-induced association of p47-phox and p67-phox with the plasma membrane and the cytoskeleton. Using the PKC activator phorbol myristate acetate (PMA) as the agonist, we found that activation of the respiratory burst oxidase was associated with translocation of cytosolic p47-phox and p67-phox to the plasma membrane as well as redistribution of p47-phox to the Triton-insoluble cytoskeleton. Furthermore, the PKC inhibitor staurosporine inhibited phosphorylation of p47-phox, interrupted the redistribution of cytosolic oxidase factors, and blocked PMA-induced generation of superoxide anion. Taken together these results indicate that PKC-dependent phosphorylation of p47-phox correlates with association of p47-phox with the cytoskeleton and with translocation of p47-phox and p67-phox to the plasma membrane, with the ensuing assembly of an active superoxide-generating NADPH-dependent oxidase.     

Statistical and mutational analysis of chronic granulomatous disease in Japan with special reference to gp91-phox and p22-phox deficiency

Chronic granulomatous disease (CGD) is a group of inherited disorders of host defense caused by a mutation in any of the four components of phagocyte NADPH oxidase, namely gp91-, p22-, p47-, and p67-phox. We have made a precise statistical analysis of 229 registered patients from 195 families in Japan and mutation analysis of 28 and 5 independent patients, respectively, with gp91- and p22-phox deficiency. The gp91- and p22-phox proteins form the membrane cytochrome b558, which plays important roles in the assembly of the active oxidase and electron-transfer reaction, and the lesions in either subunit account for more than 80% of cases. The ratio of male to female patients was 6.6/1, the incidence was calculated to be about 1 out of 220,000 birth, and the life expectancy of the patients born in the 1970s was estimated to be 25-30 years old. For the X-linked gp91-phox deficiency, we found five missense and nine nonsense mutations, seven deletions, three insertions, and four splice site mutations, which included the following novel mutations: four missense, five nonsense, six deletions, one insertion, and two splice site abnormalities. With regard to p22-phox deficiency, two homozygous nonsense mutations and one homozygous deletion, a missense mutation together with a splice site mutation, and two different missense mutations were found. These mutations have not been reported before. Based on the present and reported data from Japan, we discuss the molecular defects of the disease and the difference in statistics between western countries and Japan.     

Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression

Differentiation of myeloid cells is associated with the gradual acquisition of functional capacity to produce a respiratory burst. In our study HL-60 cells were differentiated to the monocyte phenotype with IFN-gamma or 1,25-dihydroxyvitamin D3, or to the neutrophil phenotype with retinoic acid or DMSO to compare the time-course of expression of membrane and cytosolic oxidase components, and to correlate this with the appearance of a functional oxidase. Over a 6-day period of induction the rank order of the ability of these agents to induce expression of PMA-stimulated superoxide production was: IFN-gamma greater than 1,25(OH)2D3 greater than retinoic acid greater than DMSO. Immunoblot analysis of HL-60 membranes and cytosol was used to assess the amount of specific phagocyte oxidase factors (91 and 22 kDa subunits of membrane cytochrome b558 (gp91 and p22), and 47 and 67 kDa cytosol oxidase factors (p47 and p67)). HL-60 cell membranes or cytosol were tested in a cell-free assay of superoxide production by mixing with normal neutrophil cytosol or membranes, respectively. p47 was first detected at 16 h of differentiation, increasing similarly thereafter with all induction regimens and reaching a maximum by 3 to 4 days. The earliest detection of p67 varied from 2 to 6 days depending on the inducing agent and appeared to be the limiting cytosol component. Small amounts of both subunits of cytochrome b558 were detected in uninduced HL-60 membranes, but were sufficient to support substantial superoxide production when combined with normal neutrophil cytosol. Both cytochrome b558 subunit proteins and membrane oxidase activity increased during differentiation in parallel. We conclude that membrane and cytosol components of the NADPH oxidase complex appear at different times and increase differently during HL-60 differentiation. The production of p67 is the major factor limiting the respiratory burst during HL-60 differentiation.     

Functional epitope on human neutrophil flavocytochrome b558

mAb NL7 was raised against purified flavocytochrome b(558), important in host defense and inflammation. NL7 recognized the gp91(phox) flavocytochrome b(558) subunit by immunoblot and bound to permeabilized neutrophils and neutrophil membranes. Epitope mapping by phage display analysis indicated that NL7 binds the (498)EKDVITGLK(506) region of gp91(phox). In a cell-free assay, NL7 inhibited in vitro activation of the NADPH oxidase in a concentration-dependent manner, and had marginal effects on the oxidase substrate Michaelis constant (K(m)). mAb NL7 did not inhibit translocation of p47(phox), p67(phox), or Rac to the plasma membrane, and bound its epitope on gp91(phox) independently of cytosolic factor translocation. However, after assembly of the NADPH oxidase complex, mAb NL7 bound the epitope but did not inhibit the generation of superoxide. Three-dimensional modeling of the C-terminal domain of gp91(phox) on a corn nitrate reductase template suggests close proximity of the NL7 epitope to the proposed NADPH binding site, but significant separation from the proposed p47(phox) binding sites. We conclude that the (498)EKDVITGLK(506) segment resides on the cytosolic surface of gp91(phox) and represents a region important for oxidase function, but not substrate or cytosolic component binding.     

Superoxide production by cytochrome b558 purified from neutrophils in a reconstituted system with an exogenous reductase.

Cytochrome b558, which is considered to be an essential component of the phagocytic superoxide (O2-)-generating system, was highly purified from porcine neutrophils. The isolated cytochrome was resolved into two polypeptides with molecular masses of 60-90 and 19 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For enzymatic reduction of purified cytochrome b558, we utilized hepatic NADPH-cytochrome P450 reductase purified from rat liver microsomes. More than 80% of the cytochrome was reduced by incubation with the reductase and NADPH under the anaerobic condition, and was quickly reoxidized by the air. As indicated by measurement of oxygen consumption, the purified cytochrome catalytically reduced oxygen at a rate equal to approximately 30% of the activity of the phorbol myristate acetate-activated cells on the basis of cytochrome b558 content. Electron paramagnetic resonance study with a spin trapping agent 5, 5-dimethyl-1-pyrroline-1-oxide demonstrated that O2- is the exclusive primary product in the reduction of oxygen by the cytochrome. This gives direct evidence that cytochrome b558 functions as the terminal oxidizing enzyme in the O2- -generating system of neutrophils. This also establishes a new functional class of heme proteins that catalyzes one-electron reduction of molecular oxygen.     

Diphenylene iodonium as an inhibitor of the NADPH oxidase complex of bovine neutrophils. Factors controlling the inhibitory potency of diphenylene iodonium in a cell-free system of oxidase activation.

Diphenylene iodonium (Ph2I), a lipophilic reagent, is an efficient inhibitor of the production of O2- by the activated NADPH oxidase of bovine neutrophils. In a cell-free system of NADPH oxidase activation consisting of neutrophil membranes and cytosol from resting cells, supplemented with guanosine 5'-[gamma-thio]triphosphate, MgCl2 and arachidonic acid, or in membranes isolated from neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate, addition of a reducing agent, e.g. NADPH or sodium dithionite, markedly enhanced inhibition of the NADPH oxidase by Ph2I. The membrane fraction was found to contain the Ph2I-sensitive component(s). In the presence of a concentration of Ph2I sufficient to fully inhibit O2- production (around 10 nmol/mg membrane protein), addition of catalytic amounts of the redox mediator dichloroindophenol (Cl2Ind) resulted in a by-pass of the electron flow to cytochrome c, the rate of which was about half of that determined in non-inhibited oxidase. A marked increase in the efficiency of this by-pass was achieved by addition of sodium deoxycholate. The Cl2-Ind-mediated cytochrome c reduction was negligible in membranes isolated from resting neutrophils. At a higher concentration of Ph2I (100 nmol/mg membrane protein), the Cl2Ind-mediated cytochrome c reductase activity was only half inhibited, which indicated that, in the NADPH oxidase complex, there are at least two Ph2I sensitive components, differing by their sensitivity to the inhibitor. At low concentrations of Ph2I (less than 10 nmol/mg protein), the spectrum of reduced cytochrome b558 in isolated neutrophil membranes was modified, suggesting that the component sensitive to low concentrations of Ph2I is the heme binding component of cytochrome b558. Higher concentrations of Ph2I were found to inhibit the isolated NADPH dehydrogenase component of the oxidase complex. A number of membrane and cytosolic proteins were labeled by [125I]Ph2I. However, the radiolabeling of a membrane-bound 24-kDa protein, which might be the small subunit of cytochrome b558, responded more specifically to the conditions of activation and reduction which are required for inhibition of O2- production by Ph2I. The O2(-)-generating form of xanthine oxidase was also inhibited by Ph2I. Inhibition of xanthine oxidase, a non-heme iron flavoprotein, by Ph2I had a number of features in common with that of the neutrophil NADPH oxidase, namely the requirement of reducing conditions for inhibition of O2- production by Ph2I and the induction of a by-pass of electron flow to cytochrome c by Cl2Ind in the inhibited enzyme, suggesting some similarity in the molecular organization of the two enzymes.     

The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.     

The phagocyte 47-kilodalton cytosolic oxidase protein is an early reactant in activation of the respiratory burst

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.     

Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity.

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.     

Regulation of growth and apoptosis of cultured guinea pig gastric mucosal cells by mitogenic oxidase 1

We previously reported that primary cultures of guinea pig gastric pit cells expressed all of the phagocyte NADPH oxidase components (gp91-, p22-, p67-, p47-, and p40-phox) and could spontaneously release superoxide anion (O(2)(-)). We demonstrate here that pit cells express a nonphagocyte-specific gp91-phox homolog (Mox1) but not gp91-phox. Inclusion of catalase significantly inhibited [(3)H]thymidine uptake during the initial 2 days of culture. Pit cells, matured on day 2, slowly underwent spontaneous apoptosis. Scavenging O(2)(-) and related oxidants by superoxide dismutase plus catalase or N-acetyl cysteine (NAC) and inhibiting Mox1 oxidase by diphenylene iodonium activated caspase 3-like proteases and markedly enhanced chromatin condensation and DNA fragmentation. This accelerated apoptosis was completely blocked by a caspase inhibitor, z-Val-Ala-Asp-CH(2)F. Mox1-derived reactive oxygen intermediates constitutively activated nuclear factor-kappaB, and inhibition of this activity by nuclear factor-kappaB decoy oligodeoxynucleotide accelerated their spontaneous apoptosis. These results suggest that O(2)(-) produced by the pit cell Mox1 oxidase may play a crucial role in the regulation of their spontaneous apoptosis as well as cell proliferation.