Activation of the leukocyte NADPH oxidase by protein kinase C in a partially recombinant cell-free system.

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.     

Inhibition of O2-. generating oxidase of neutrophils by iodonium biphenyl in a cell free system: effect of the redox state of the oxidase complex.

The conditions of inhibition of neutrophil O2-. generating oxidase by iodonium biphenyl (IBP) were studied. In a cell free system of oxidase activation consisting of neutrophil membranes and cytosol, GTP-gamma-S, Mg2+ and arachidonic acid, the inhibitory effect of IBP depended on the redox conditions of the medium. Inhibition was observed when the medium was supplemented with dithionite or NADPH. When the cell free system was incubated with IBP in the absence of reducing agents, full oxidase activity was recovered after removal of free IBP by gel filtration. Bovine neutrophil membranes, but not cytosol, contained component(s) sensitive to IBP. Upon treatment of neutrophil membranes by IBP followed by reduction, the spectrum of reduced cytochrome b558 was modified, suggesting that cytochrome b558 is a target site for IBP.     

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.     

Respiratory burst of rabbit peritoneal neutrophils. Transition from an NADPH diaphorase activity to an .O2(-)-generating oxidase activity

Superoxide (.O2-) production by the NADPH oxidase of a membrane fraction derived from rabbit peritoneal neutrophils activated by 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied at 25 degrees C under different conditions, and measured by the superoxide dismutase inhibitable reduction of cytochrome c. Whereas PMA-activated rabbit neutrophils incubated in a glucose-supplemented medium exhibited a substantial rate of production of .O2-, the membranes prepared by sonication of the activated neutrophils were virtually unable to generate .O2- in the presence of NADPH. Instead, they exhibited an NADPH-dependent diaphorase activity, measured by the superoxide-dismutase-insensitive reduction of cytochrome c. Upon addition of arachidonic acid, which is known to elicit oxidase activation, the NADPH diaphorase activity of the rabbit neutrophil membranes vanished and was stoichiometrically replaced by an NADPH oxidase activity. The emerging oxidase activity was fully sensitive to iodonium biphenyl, a potent inhibitor of the respiratory burst, whereas the diaphorase activity was not affected. Addition of 0.1% Triton X-100 or an excess of arachidonic acid, acting as detergent, resulted in the reappearance of the diaphorase activity at the expense of the oxidase activity. These results indicate that the diaphorase-oxidase transition is reversible. When the rabbit neutrophil membranes were supplemented with rabbit neutrophil cytosol, guanosine 5'-[gamma-thio]triphosphate and Mg2+, in addition to arachidonic acid, not only the NADPH diaphorase activity disappeared, but the emerging NADPH oxidase activity was markedly enhanced (about 10 times compared to that of membranes treated with arachidonic acid alone). The diaphorase-oxidase transition was accompanied by a 10-fold increase in the Km for NADPH, suggesting a change of conformation propagated to the NADPH-binding site during the transition. The treatment of PMA-activated rabbit neutrophils with cross-linking reagents, like glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide, prevented the loss of the PMA-elicited oxidase activity upon disruption of the cells by sonication, suggesting that the interactions between the components of the oxidase complex are stabilized by cross-linking.     

Comparison of O2(-)-producing activity of guinea-pig eosinophils and neutrophils in a cell-free system.

1. The NADPH-dependent superoxide (O2-) production in a cell-free system of guinea-pig eosinophils was studied, comparing the eosinophils with neutrophils. 2. Eosinophils produced 2.2-fold more O2- than neutrophils in sonicated and intact cells. 3. The subcellular fractionation experiments showed that the O2- production was dependent on the cooperation between the membrane and cytosol fractions. 4. The cross-mixing experiments indicated that the NADPH oxidase-activating activity of the eosinophil cytosol was about 2-fold greater than that of the neutrophil cytosol. 5. These results suggest that the difference in the O2(-)-producing activity between eosinophils and neutrophils is associated with the difference in cytosolic factors necessary for the activation of NADPH oxidase.     

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.     

Human neutrophil cytosolic activation factor of the NADPH oxidase. Characterization of activation kinetics

The kinetics of sodium dodecyl sulfate-induced activation of respiratory burst oxidase (NADPH oxidase) in a fully soluble cell-free system from resting (control) or phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system containing solubilized membranes and cytosol fractions (cytosol) derived from control neutrophils (control cell-free system), the values of Km and Vmax for NADPH of the NADPH oxidase from control neutrophils continuously increased with increasing concentrations of cytosol, but with increasing concentrations of solubilized membranes from the control neutrophils, Km values continuously decreased, suggesting cytosolic activation factor-dependent continuous changes in the affinity of NADPH oxidase to NADPH. In a cell-free system containing solubilized membranes and cytosol prepared from PMA-stimulated neutrophils, NADPH oxidase was not activated after the addition of NADPH. However, cytosol from control neutrophils activated the NADPH oxidase of PMA-stimulated neutrophils in a cell-free system. Cytosol from PMA-stimulated neutrophils did not activate the control neutrophil oxidase, although it contained no inhibitors of NADPH oxidase activation. The results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted with an increasing period of time after the stimulation of neutrophils, and that the affinity of PMA-stimulated neutrophil NADPH oxidase to NADPH may almost be the same as that of control neutrophil oxidase. It was concluded that the affinity of NADPH oxidase to NADPH was closely associated with interaction between solubilized membranes and cytosolic activation factors, as indicated by the concentration ratio.     

Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein

Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.     

Purification and characterization of Rac 2. A cytosolic GTP-binding protein that regulates human neutrophil NADPH oxidase.

Human neutrophils and other phagocytes generate superoxide anion (O2-) as a means of destroying ingested microorganisms. O2- is produced by an NADPH-consuming oxidase composed of membrane and cytosolic components. Activation of the NADPH oxidase is absolutely dependent upon GTP, indicating the requirement for a GTP-binding protein in this process. We have utilized a five-step chromatographic procedure to isolate a GTP-binding protein from human neutrophil cytosol which can stimulate NADPH oxidase activity in a cell-free assay. Oxidase enhancing activity was shown to coisolate with this GTP-binding component, which was purified to apparent homogeneity. The GTP-binding protein was identified as Rac 2 by immunological reactivity and amino acid sequencing. Thus, Rac 2 appears to be a third cytosolic component required for human neutrophil NADPH oxidase activation. Recombinant Rac 2 was shown to bind guanine nucleotides in a Mg(2+)-dependent fashion. GDP dissociation rates were determined and shown to be regulated by the free Mg2+ concentration. Rac 2 was found to possess the highest rate of intrinsic GTP hydrolysis of any of the characterized members of the Ras superfamily. The biochemical properties of Rac 2 indicate it is likely to be subject to regulatory cofactors in vivo.     

Activation of NADPH-dependent superoxide production in plasma membrane extracts of pig neutrophils by phosphatidic acid

Phosphatidic acid (PA), a molecule that is rapidly produced by the stimulated turnover of phospholipids in a variety of cells including blood neutrophils, elicited NADPH-dependent superoxide anion (O2-) production in detergent extracts from membranes of resting pig neutrophils. The stimulatory effect of PA was independent of cytosolic factors, differing from arachidonic acid and sodium dodecyl sulfate which, on the contrary, absolutely required the presence of cytosol to elicit the same result. The O2(-)-forming activity of the detergent extract activable by PA, as that by sodium dodecyl sulfate and arachidonic acid plus cytosol, was found in the chromatographic fractions containing cytochrome b558 and presented a chromatographic profile identical to that of the activated NADPH oxidase, which was obtained from neutrophils prestimulated with phorbol 12-myristate 13-acetate. The PA-induced NADPH-dependent O2(-)-forming activity showed kinetic properties and sensitivity to the inhibitors similar to the classical ones of the activated neutrophil NADPH oxidase. The data suggest that, in this cell-free system, PA may stimulate O2- formation by direct interaction with latent NADPH oxidase of neutrophils or with some of its regulatory components.     

Properties of the NADPH dehydrogenase component of the oxidase complex from rabbit peritoneal neutrophils: reconstitution of an oxidase activity with the dehydrogenase component and a membrane extract

A flavin-linked NADPH cytochrome c oxido-reductase of molecular mass 77-kDa was extracted from membranes of rabbit peritoneal neutrophils and purified in the presence of Triton X-100. The redox properties of this enzyme were examined. By some criteria including its high sensitivity to mersalyl, and its relatively high specificity for NADPH compared to NADH, the rabbit neutrophil NADPH cytochrome c reductase resembled NADPH-cytochrome P-450 reductase. Limited proteolysis generated water soluble fragments, with molecular masses of 67-kDa and 57-kDa, which were still endowed with a substantial reductase activity. When added to a lysate of neutrophil membranes in octylglucoside, in the presence of an oxidase activation medium consisting of rabbit neutrophil cytosol, GTP-gamma-S, arachidonic acid and Mg2+, the purified reductase enhanced the production of O2-., suggesting that it forms part of the O2-. generating oxidase.     

Fatty-acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides

Both cis and trans unsaturated fatty acids and sodium dodecyl sulfate activated NADPH oxidase in plasma membranes of human neutrophils in the presence of neutrophil cytosol. In contrast, 5,8,11,14-icosatetraynoic acid, saturated fatty acids, esters, peroxides and 4 beta-phorbol 12-myristate 13-acetate, a potent activator of protein kinase C, were inactive. 5,8,11,14-icosatetraynoic acid inhibited superoxide formation elicited by fatty acids. Guanosine 5'[gamma-thio]triphosphate (GTP[gamma S]), a potent activator of guanine-nucleotide-binding proteins (N-proteins) enhanced superoxide formation elicited by fatty acids up to fourfold, supporting our previous suggestion that NADPH oxidase is regulated by an N-protein [Seifert, R. et al. (1986) FEBS Lett. 205, 161-165]. Cytosols from various tissues, soybean lipoxygenase and protein kinase C, purified from chicken stomach, did not substitute neutrophil cytosol. The activity of neutrophil cytosol was destroyed by heating at 95 degrees C. Superoxide formation was not affected by the inhibitor of protein kinase C 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Removal of cytosolic ATP by preincubation with hexokinase and glucose, dialysis of neutrophil cytosol or chelation of calcium with EGTA did not abolish the stimulatory effect of arachidonic acid and GTP[gamma S]. Thus, the cytosolic cofactor appears to be a neutrophil-specific and heat-labile protein, which is neither a lipoxygenase nor protein kinase C.     

The translocation of respiratory burst oxidase components from cytosol to plasma membrane is regulated by guanine nucleotides and diacylglycerol.

The respiratory burst oxidase is a multimeric enzyme responsible for O2- production by stimulated neutrophils and a few other cell types. In the resting neutrophil, the oxidase is dormant, and its subunits are distributed between the cytosol, in which they appear to exist in the form of a multisubunit complex, and the plasma membrane; but, when the neutrophil is activated, the cytosolic complex translocates to the membrane to assemble the active enzyme. Using a cell-free system in which oxidase activity was elicited with SDS, we examined the effects of GTP gamma S and dioctanoylglycerol (DiC8) on both the activation of O2- production and the transfer of the cytosolic oxidase components p47phox and p67phox to the plasma membrane. GTP (added as undialyzed cytosol) and GTP gamma S augmented the transfer of the oxidase components to the plasma membrane and was essential for the acquisition of O2- producing activity by the oxidase. DiC8 also supported the SDS-mediated transfer of oxidase components to the membrane, but O2- production did not take place unless GTP or GTP gamma S was present. In the presence of these nucleotides, however, DiC8 augmented both translocation and O2- production. We interpreted these results in terms of a mechanism in which 2 membrane-binding sites are created during the activation of the cytosolic complex, one for diacylglycerol and the other for a second site on the membrane. Development of the second membrane-binding site depends upon the action of a G protein and is essential for the expression of oxidase activity. The results further suggested that the priming of the respiratory burst oxidase in intact neutrophils might be due to an increase in membrane diacylglycerol concentration that occurs in response to the priming stimulus. Because of the increased diacylglycerol content, a larger than usual amount of active respiratory burst oxidase could be assembled on the primed plasma membrane when the neutrophil is fully activated.     

Composition of partially purified NADPH oxidase from pig neutrophils.

The superoxide (O2.-)-forming enzyme NADPH oxidase from pig neutrophils was solubilized and partially purified by gel-filtration chromatography. The purification procedure allowed the separation of NADPH oxidase activity from NADH-dependent cytochrome c reductase and 2,6-dichlorophenol-indophenol reductase activities. O2.-forming activity was co-purified with cytochrome b-245 and was associated with phospholipids. However, active fractions endowed with cytochrome b were devoid of ubiquinone and contained only little FAD. The cytochrome b/FAD ratio was 1.13:1 in the crude solubilized extract and increased to 18.95:1 in the partially purified preparations. Most of FAD was associated with fractions containing NADH-dependent oxidoreductases. These results are consistent with the postulated role of cytochrome b in O2.-formation by neutrophil NADPH oxidase, but raise doubts about the participation of flavoproteins in this enzyme activity.     

Guanine nucleotides stimulate NADPH oxidase in membranes of human neutrophils

In the chain of events by which chemotactic peptides stimulate NADPH oxidase-catalyzed superoxide formation in human neutrophils, the involvements of a pertussis toxin-sensitive guanine nucleotide-binding protein (N-protein), mobilization of intracellular calcium and protein kinase C stimulation have been proposed. Superoxide formation was studied in membranes from human neutrophils; NADPH oxidase was stimulated by arachidonic acid in the presence of neutrophil cytosol. Fluoride and stable GTP analogues, such as GTP gamma S and GppNHp, which all activate N-proteins, enhanced NADPH oxidase activity up to 4-fold. GDP beta S inhibited the effect of GTP gamma S. These data suggest that NADPH oxidase is regulated by an N-protein, independent of an elevation of the cytoplasmic calcium concentration.     

Differential expression of cytosolic activation factors for NADPH oxidase in HL-60 leukemic cells

Activation of NADPH oxidase in undifferentiated HL-60 leukemic cells and in HL-60 cells differentiated along the myeloid pathway with dibutyryl cyclic AMP (dbcAMP) or dimethyl sulfoxide (Me2SO) was studied. Upon stimulation with a calcium ionophore, a phorbol ester, arachidonic acid or gamma-hexachlorocyclohexane, Me2SO-differentiated HL-60 cells generated superoxide (O2-) at higher rates than dbcAMP-differentiated cells. Undifferentiated cells generated O2- only at low rates upon stimulation with the above agents. In cell-free systems, NADPH oxidase activity was reconstituted by combining membranes of undifferentiated or dbcAMP- or Me2SO-differentiated HL-60 cells, cytosol of Me2SO-differentiated cells and arachidonic acid. This basal O2- formation was enhanced several-fold by guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]), a potent activator of guanine nucleotide-binding proteins. In contrast, cytosol of dbcAMP-differentiated cells reconstituted O2- formation only in the presence of GTP[gamma S], and cytosol of undifferentiated cells was inactive. Submaximally stimulatory amounts of cytosolic protein of Me2SO- and dbcAMP-differentiated cells synergistically stimulated O2- formation in the presence but not in the absence of GTP[gamma S]. We conclude that differentiations of HL-60 cells with Me2SO and dbcAMP are not equivalent with respect to activation of NADPH oxidase and that two cytosolic activation factors are involved in the regulation of this effector system.     

Changing the conformation state of cytochrome b558 initiates NADPH oxidase activation: MRP8/MRP14 regulation

Phagocyte NADPH oxidase generates O2. for defense mechanisms and cellular signaling. Myeloid-related proteins MRP8 and MRP14 of the S100 family are EF-hand calcium-binding proteins. MRP8 and MRP14 were co-isolated from neutrophils on an anti-p47phox matrix with oxidase cytosolic factors and identified by mass spectrometry. MRP8 and MRP14 are absent from Epstein-Barr virus-immortalized B lymphocytes, and, coincidentally, these cells display weak oxidase activity compared with neutrophils. MRP8/MRP14 that was purified from neutrophils enhanced oxidase turnover of B cells in vitro, suggesting that MRP8/MRP14 is involved in the activation process. This was confirmed ex vivo by co-transfection of Epstein-Barr virus-transformed B lymphocytes with genes encoding MRP8 and MRP14. In a semi-recombinant cell-free assay, recombinant MRP8/MRP14 increased the affinity of p67phox for cytochrome b558 synergistically with p47phox. Moreover, MRP8/MRP14 initiated oxidase activation on its own, through a calcium-dependent specific interaction with cytochrome b558 as shown by atomic force microscopy and a structure-function relationship investigation. The data suggest that the change of conformation in cytochrome b558, which initiates the electron transfer, can be mediated by effectors other than oxidase cytosolic factors p67phox and p47phox. Moreover, MRP8/MRP14 dimer behaves as a positive mediator of phagocyte NADPH oxidase regulation.     

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.     

The superoxide-generating oxidase of phagocytic cells. Physiological, molecular and pathological aspects.

Professional phagocytes (neutrophils, eosinophils, monocytes and macrophages) possess an enzymatic complex, the NADPH oxidase, which is able to catalyze the one-electron reduction of molecular oxygen to superoxide, O2-. The NADPH oxidase is dormant in non-activated phagocytes. It is suddenly activated upon exposure of phagocytes to the appropriate stimuli and thereby contributes to the microbicidal activity of these cells. Oxidase activation in phagocytes involves the assembly, in the plasma membrane, of membrane-bound and cytosolic components of the oxidase complex, which were diassembled in the resting state. One of the membrane-bound components in resting phagocytes has been identified as a low-potential b-type cytochrome, a heterodimer composed of two subunits of 22-kDa and 91-kDa. The link between NADPH and cytochrome b is probably a flavoprotein whose subcellular localization in resting phagocytes remains to be determined. Genetic defects in the cytochrome b subunits and in the cytosolic factors have been shown to be the molecular basis of chronic granulomatous disease, a group of inherited disorders in the host defense, characterized by severe, recurrent bacterial and fungal infections in which phagocytic cells fail to generate O2- upon stimulation. The present review is focused on recent data concerning the signaling pathway which leads to oxidase activation, including specific receptors, the production of second messengers, the organization of the oxidase complex and the molecular defects responsible for granulomatous disease.     

A microtechnique for neutrophil respiratory burst oxidase in a cell-free system--characterization of oxidase activation system

1. A microtechnique for quantitating human neutrophil NADPH oxidase in a cell-free system is described. 2. This spectrophotometric discontinuous (fixed time) method is less material-consuming than existing methods and is more useful for experiments in which superoxide production by neutrophils must be measured in a large number of samples. 3. Measurement of NADPH oxidase using the new method can be accomplished in a final vol of 0.15 ml. 4. In the assay, neutrophil membranes solubilized with deoxycholate were incubated for 3 min with cytosolic fractions, magnesium, sodium dodecyl sulfate, and cytochrome c in the absence of NADPH to preincubate the oxidase before the addition of the reducing agent. 5. The reaction was started by adding NADPH and 2 min later terminated by adding superoxide dismutase. 6. The apparent Km for NADPH obtained by the new method was almost the same as that by the authorized method (39.2 +/- 3.1 SD vs 36.8 +/- 1.6). Activation of neutrophil NADPH oxidase was characterized using the new assay method.