Presence of cytochrome b-558 in guinea-pig alveolar macrophages-subcellular localization and relationship with NADPH oxidase

The assignment of cytochrome b-558 as a component of the O2- (H2O2) -generating enzyme in guinea-pig alveolar macrophages was investigated. Guinea pig alveolar macrophages contained 76 pmol cytochrome b-558/mg protein, a value very similar to that of neutrophils. The rate of myristic acid-stimulated O2- generation by alveolar macrophages, calculated per cytochrome b-558, was only one-fourth that of neutrophils. An analysis of Percoll density gradient centrifugation profiles showed that the H2O2-generating activity of myristic acid-activated alveolar macrophages was concentrated in a single peak which was consistently associated with 5'-nucleotidase activity, a plasma membrane marker enzyme. A little H2O2-generating activity was seen with unactivated alveolar macrophages. Furthermore, the cytochrome b-558 of both myristic acid-activated and unactivated alveolar macrophages was also predominantly associated with 5'-nucleotidase activity and was found in trace amounts in a peak containing lysozyme activity, a marker of lysosome granules. Only about 6% of the cytochrome b-558 in plasma membranes from myristic acid-activated guinea-pig alveolar macrophages was anaerobically reduced by 0.5 mM NADPH, while under the same conditions about 30% of the heme protein of myristic acid-activated neutrophils was reduced. These results suggest two conclusions: firstly, that in both activated and unactivated alveolar macrophages, cytochrome b-558 is located in the plasma membrane, and the translocation of cytochrome b-558 does not occur during the activation of NADPH oxidase; and secondly, that a smaller part of cytochrome b-558 is associated with the activated NADPH oxidase of guinea pig alveolar macrophages compared with neutrophils.     

Altered oxidative metabolism in selenium-deficient rat granulocytes

Rats fed a selenium-deficient diet for 12 to 15 wk became selenium-depleted, measured by the selenium content of liver and granulocytes. The activity of granulocyte glutathione peroxidase, a selenoenzyme, in deficient rats was 11% of the activity in replete rat granulocytes. When stimulated with an H2O2 generating system, the HMPS activity of the deficient granulocytes was 50% of replete; however, when stimulated with methylene blue, the HMPS activity in deficient and replete granulocytes was the same. When granulocytes were incubated with PMA or OPZ, deficient granulocytes initially had the same O-2-generating activity as replete granulocytes; however, with increasing duration of stimulation, granulocytes from deficient rats generated less O-2 than replete rats. After 20 min in an H2O2-generating system, deficient granulocytes stimulated with PMA or OPZ generated less O-2 than replete granulocytes. These results indicate that deficient granulocytes did not metabolize H2O2 as well as replete granulocytes and that H2O2 caused damage to the O2-generating system. Measurement of O-2 generation in membrane-enriched particles showed the above effects were due to inactivation of the NADPH-dependent O2-generating system. Deficient granulocytes stimulated with OPZ for 20 min had 70% less membrane O-2-generating activity than controls. In addition, when membrane-enriched particles were made from cells that had been stressed with an H2O2-generating system, NADPH-dependent O-2-generating activity in deficient granulocytes was 50% of replete. In selenium-deficient granulocytes with low GSH-Px activity, prolonged incubation with stimulants and prior incubations with an H2O2-generating system caused loss of activity of the membrane-bound, NADPH-dependent, O-2-generating system.     

The inhibition by diphenyleneiodonium and its analogues of superoxide generation by macrophages.

Peritoneal macrophages were elicited in rats by using casein as a stimulus; when stimulated with phorbol 12-myristate 13-acetate (PMA) they produced O2.-. Nearly 60% of the total cytochrome b had a low Em,7.0 of -247 mV, typical of the cytochrome b component found in the NADPH-dependent O2(.-)-generating oxidase of neutrophils. The rate of O2.- generation by macrophages was 1.23 mol of O2.-/s per mol of cytochrome b. Treatment of intact macrophages with diphenyleniodonium (DPI) at 0.9 microM caused 50% inhibition of PMA-induced O2.- generation, with little effect on mitochondrial respiratory activity; KCN inhibited respiratory activity without affecting PMA-induced O2.- generation. A similar specificity of inhibition was found for di-2-thienyliodonium (50% inhibition of O2.- generation at 0.5 microM) and, at higher concentrations, for diphenyl iodonium. When macrophage suspensions were incubated with [125I]DPI followed by autoradiography of SDS/polyacrylamide-gel-electrophoresis-separated polypeptides, radioactivity was most strongly associated with a band of Mr 45,000, similar to that found in neutrophils [Cross & Jones (1986) Biochem. J. 237, 111-116]. The O2(.-)-generating oxidase of macrophages appears to have components in common with the NADPH oxidase of neutrophils, despite differences in activity. Its sensitivity to DPI suggests that selective prevention of radical generation by macrophages in vivo is possible.     

The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavin and cytochrome b in solubilized preparations.

An NADPH-dependent O2.- -generating oxidase was solubilized from phorbol 12-myristate 13-acetate-activated pig neutrophils by using a mixture of detergents. Recovery of oxidase was approx. 40%. The extract contained cytochrome b-245 (331 pmol/mg of protein) and FAD (421 pmol/mg of protein); approx. 30% of each was reduced within 60s when NADPH was added to anaerobic incubations. Three different additives, quinacrine, p-chloromercuribenzoate and cetyltrimethylammonium bromide, strongly inhibited O2.- generation; they also inhibited the reduction by NADPH of cytochrome b at the same low concentrations. In the presence of p-chloromercuribenzoate cytochrome b reduction was strongly inhibited and flavin reduction was less inhibited. A detergent extract prepared from non-stimulated neutrophils also contained flavin and cytochrome b, but its rate of O2.- production was less than 1% of that from activated cells; its initial rate of cytochrome b and flavin reduction was low, although the state of reduction at equilibrium was similar to that of extracts of activated cells. Even in the non-activated cell extract the reduction of flavin and cytochrome was made fast and complete when Methyl Viologen was added to the anaerobic incubations. The oxidase was temperature-sensitive, with a sharp maximum at 25 degrees C; temperatures above this caused loss of O2.- generation, and this coincided with loss of the characteristic cytochrome b spectrum, indicate of denaturation of the cytochrome. The cytochrome b formed a complex with butyl isocyanide (close to 100% binding at 10mM); butyl isocyanide also inhibited the oxidase activity of stimulated whole neutrophils (22.5% inhibition at 10mM). Photoreduced FMN stimulated O2 uptake by the oxidase. The results support a scheme of electron transport within the oxidase complex involving NADPH, FAD, cytochrome b-245 and O2 in that sequence.     

Certain lymphoid cells contain the membrane-associated component of the phagocyte-specific NADPH oxidase

Anionic amphiphiles such as long chain unsaturated fatty acids and SDS were shown to activate the superoxide (O2-) producing NADPH oxidase in a cell-free system derived from sonically disrupted phagocytes (macrophages and granulocytes). O2- production required the cooperation of a membrane associated component sedimenting at 48,000 X g (pi) and a cytosolic factor (sigma). The purpose of the present investigation was to find out whether components pi and sigma were also present in non-phagocytic cells that do not produce O2- when stimulated. It was found that the 48,000 X g pellets of guinea pig lymph node and thymus cell sonicates contained significant amounts of component pi, as shown by their ability to support SDS-elicited NADPH-dependent O2- production when supplemented with macrophage cytosol. Lymph node and thymus pi could be extracted from the membrane by 30 mM octyl glucoside, just as its macrophage-derived equivalent. Combining lymph node and thymus 48,000 X g pellet with autologous cytosol did not yield an active enzyme preparation. Also, cytosol from lymph node and thymus cells could not cooperate with macrophage 48,000 X g pellet, indicating that component sigma was lacking in lymphoid cells. Neither pi nor sigma could be detected in guinea pig kidney, the mouse myeloma cell line MOPC 315 and the canine cell line Cf2Th. The 48,000 X g pellet of all nonphagocytic cells examined contained a b-cytochrome that resembled, by its spectral characteristics, the cytochrome b559 thought to be characteristic of phagocytes. In macrophages, cytochrome b559 represented 80% of b-cytochrome content of the 48,000 X g pellet, whereas in non-phagocytic cells, the equivalent material represented only 50 to 60%. There was no correlation between the presence and quantity of the cytochrome b559-like chromophore in the 48,000 X g pellet of a particular cell type and its ability to cooperate with macrophage cytosol in SDS-elicited O2- production.     

Rat macrophage treatment with lipopolysaccharide leads to a reduction in respiratory burst product secretion and a decrease in NADPH oxidase affinity

The effect of LPS on the respiratory burst in resident rat peritoneal macrophages has been examined. Rat macrophages secreted high levels of both O2- and H2O2 in response to triggering with phorbol esters, opsonized zymosan, and immune complexes. After culture in vitro with LPS these macrophages exhibited a marked diminution in their capacity to secrete high levels of respiratory burst products. The LPS-mediated loss of secretory activity was apparent after 2 hr of exposure to LPS and was inhibitable by polymyxin B in a dose-dependent fashion. The effect was not selective for any triggering agent type as inhibition of secretory activity occurred after triggering with PMA, zymosan and immune complexes. PGE2 added at levels secreted by the macrophages in response to LPS also inhibited respiratory burst product secretion. In addition, indomethacin prevented the LPS-mediated inhibition of secretion. Because the inhibition of secretion was common to all triggering agents tested, this suggested that the basis for the impaired secretion was at a level other than the receptor for the triggering agent. Both LPS and PGE2 treatment of the macrophages increased the Km of the oxidase for NADPH (1.7- to 2.3-fold) without affecting significantly the Vmax of the enzyme. These data suggest that stimulation of rat peritoneal macrophages by LPS results in an impaired ability to secrete respiratory burst products as a result of a PGE2-mediated decrease in NADPH oxidase affinity and that this alteration is independent of alterations in tumoricidal activity.     

The NADPH oxidase of guinea pig polymorphonuclear leucocytes

Summary NADPH oxidase from stimulated guinea pig granulocytes was extracted with deoxycholate. The solubilized enzyme was stable in 20% glycerol. Solubilized enzyme was free of myeloperoxidase activity. The properties of the deoxycholate solubilized enzyme indicated that it is a high molecular weight complex with a flavoprotein, calmodulin and cytochrome b possibly forming part of the complex. Maximum activity was between pH 7.0 and 7.5. The K_m value was 15.8 µM for NADPH and 434 µM for NADH indicating that NADPH is the preferential substrate.     

Release of arachidonate and reduction of oxygen. Independent metabolic bursts of the mouse peritoneal macrophage

Diverse particulate and soluble stimuli trigger two metabolic bursts in mouse peritoneal macrophages important in the inflammatory and/or cytotoxic actions of the cells: release, oxygenation, and further metabolism of arachidonic acid from endogenous phospholipids and reduction of molecular oxygen to reactive intermediates. We tested the hypothesis that the release of arachidonic acid or formation of its metabolites are obligatory intermediate steps in triggering the NADPH oxidase that reduces O2 to O-2. With phorbol diesters as stimuli, the following inhibitors of phospholipase A2 and lipoxygenase suppressed release of H2O2 at nontoxic concentrations (microM range): p-bromophenacyl bromide, quinacrine, eicosatetraenoic acid, nordihydroguaiaretic acid, and phenidone. Indomethacin and acetylsalicylic acid were ineffective. However, the suppressive effect of the first five agents on H2O2 release could be attributed to their suppression of macrophage glucose uptake at the same concentrations, a previously unrecognized effect of these compounds. Further, concanavalin A, wheat germ agglutinin, and thrombin each stimulated abundant arachidonate release without H2O2 release. Finally, noncytolytic concentrations of cycloheximide and/or emetine suppressed arachidonate release without affecting H2O2 secretion triggered either by phorbol esters or zymosan. Release and metabolism of arachidonic acid and secretion of reactive oxygen intermediates appear to be two frequently coincident but mutually independent metabolic pathways in the mouse peritoneal macrophage.     

Effect of chelating agents and superoxide on human neutrophil NAD(P)H oxidation

NAD(P)H oxidation is frequently measured to assay the activity of the neutrophil O-2-generating oxidase. It was found that 10(-4) M ethylene glycol bis (beta-aminoethyl ether)-N-N'-tetraacetic acid (EGTA) increased NAD(P)H oxidation by the 27,000 g granule fraction of resting and stimulated human neutrophils without altering net O-2 production. The commonly used chelating agents EDTA and diethylene triamine pentaacetic acid had similar effects. The addition of superoxide dismutase eliminated the effect of the chelating agents and thus demonstrated that the stimulated reaction was dependent upon O-2. KCN and bathophenanthroline disulfonate, an iron-chelating agent, prevented O-2-dependent NADPH oxidation by neutrophil granule fractions in the presence of EGTA. In contrast, bathocuproine disulfonate, a copper-chelating agent, mimicked the EGTA effect. The effects of both bathophenanthroline disulfonate and bathocuproine disulfonate were completely abolished when the agents were saturated with iron and copper, respectively. All the chelating agents studied, except bathophenonthroline disulfonate, also promoted O-2-dependent NADPH oxidation in a system wherein O-2 was generated by xanthine oxidase. Thus, commonly used chelating agents, by interacting with available iron and copper, may alter the apparent stoichiometry of the neutrophil O-2-generating oxidase and artifactually increase NADPH oxidation in other systems where O-2 is present.     

2,6-Dichlorophenolindophenol-reducing activity of phagocytosis-associated NADPH oxidase

The 2,6-dichlorophenolindophenol (DCIP)-reducing activity of the phagocytosis-associated NADPH oxidase was investigated using homogenates and a membrane fraction (F2) of elicited guinea pig peritoneal macrophages stimulated by phorbol myristate acetate. Essentially all of the stimulation-specific DCIP reduction under aerobic conditions could be inhibited when high concentrations of superoxide dismutase (SOD), about 10 times those usually used to inhibit the superoxide (O-2)-mediated cytochrome c reduction, were used. SOD inhibited the DCIP reduction by chemically generated O2- in the same manner as the stimulation-specific DCIP reduction by the macrophage F2, and the concentration of SOD necessary for 50% inhibition was about 10 times that for the reduction of cytochrome c. Under anaerobic conditions, however, the NADPH oxidase could reduce DCIP, though the rate was slow because we could not use a sufficiently high DCIP concentration. The observations indicate that the NADPH oxidase preferentially reduces oxygen under aerobic conditions, though the oxidase can reduce DCIP in the anaerobic state.     

Oxidant-mediated inhibition of ligand uptake by the macrophage mannose receptor

We have investigated the effect of oxidants on ligand recognition and internalization by the macrophage mannose receptor. Rat bone marrow macrophages were treated with increasing concentrations of H2O2 for 30 min at 37 degrees C. Fifty percent inhibition of ligand uptake was observed at 250 microM, with only 10% of control uptake remaining following exposure to 1 mM H2O2 for 30 min. Electron micrographic analysis of macrophages following H2O2 treatment showed no morphological alterations compared to untreated cells. Ligand uptake was also inhibited by the following H2O2 generating systems: menadione, xanthine/xanthine oxidase, glucose/glucose oxidase, and phorbol 12-myristate 13-acetate-stimulated polymorphonuclear leukocytes. Inhibition could be blocked by catalase plus or minus superoxide dismutase. Treatment of macrophages at 4 degrees C with H2O2 had no effect on ligand binding, whereas treatment with H2O2 at 37 degrees C reduced binding to 15% of control levels and decreased the number of surface receptors to one-third of control cells. H2O2 treatment inhibited ligand degradation by macrophages, but did not prevent ligand movement from the surface to the interior of the cell. In addition, ligand delivery to lysosomes was blocked by oxidant treatment. These results suggest that treatment of macrophages with reagent H2O2 or H2O2-generating systems inhibits the normal ligand delivery and receptor recycling process involving the mannose receptor. Potential mechanisms might include receptor oxidation, alterations in ATP levels, or membrane lipid peroxidation.     

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 in alveolar macrophages of diabetic rats

Bactericidal ability of alveolar macrophages is depressed in rats with diabetes mellitus. To define the mechanism of this abnormality, we measured the parameters of respiratory burst in alveolar macrophages, peripheral blood monocytes, and neutrophils of rats 8 wk after the induction of diabetes by streptozocin. Superoxide anion (O2-.) generation during basal conditions and after stimulation with phorbol myristate acetate (PMA) was measured as superoxide dismutase-inhibitable cytochrome c reduction. NADPH, the principal substrate for NADPH-oxidase-dependent O2-. generation, was measured in the alveolar macrophages and quick-frozen lungs by the enzyme-cycling method. O2-. generation after PMA was significantly lower in the alveolar macrophages of diabetics than in the controls (14.4 +/- 2.0 nmol.10(6) cells-1.20 min-1 vs. 26.2 +/- 1.9, P less than 0.05). Conversely the peripheral blood monocytes of diabetics demonstrated an enhanced O2-. production after PMA stimulation. There was no significant difference in the neutrophil O2-.-generation between the groups. The alveolar macrophage NADPH (control 0.44 +/- 0.15 nmol/10(6) cells vs. diabetic 0.21 +/- 0.04, P less than 0.05) and lung tissue NADPH levels (control 81.4 +/- 16.3 nmol/g dry wt vs. diabetic 35.8 +/- 20.5, P less than 0.05) were significantly lower in the diabetics than in the controls. These data indicate that the O2-.-generating capacity of alveolar macrophages is markedly depressed in diabetes, whereas their precursors, monocytes, are primed to generate O2-. with PMA stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Purification and properties of an O2-.-generating oxidase from bovine polymorphonuclear neutrophils

A membrane-associated O2-.-generating oxidase has been purified from activated bovine polymorphonuclear neutrophils (PMN). The oxidase was extracted with Triton X-100 from a PMN membrane fraction largely devoid of lysosomal granules. The Triton extract was purified by a series of steps, including ion-exchange chromatography on DE-52 cellulose, gel filtration on Sephadex G-200, and isoelectric focusing. The O2-.-generating oxidase activity was assayed as a superoxide dismutase inhibitable cytochrome c reductase. The activity of the purified enzyme was strictly dependent on NADPH as electron donor. The purification factor with respect to the phorbol myristate acetate activated PMN was 75, and the recovery was about 6%. The reactivity of the purified oxidase was increased by 3-4-fold after incubation with asolectin. The minimum molecular weight of the oxidase, deduced from migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 65 000 +/- 3000. The optimum pH of the oxidase was 7.5, its KM,NADPH was congruent to 30 microM, and its isoelectric point was at pH 5.0. The enzyme was inhibited by low concentrations of mersalyl (half-inhibition congruent to 10 microM) and Cibacron Blue (half-inhibition less than 10 microM). It was insensitive to 1 mM cyanide. Rapid loss of activity occurred at 0-2 degrees C, concomitantly with a decrease in sensitivity to superoxide dismutase: both activity and sensitivity to superoxide dismutase could be restored by addition of asolectin. The purified oxidase contained no spectrophotometrically detectable cytochrome b, and enzymatic assay failed to detect FAD in oxidase preparations subjected to heat treatment or trypsin digestion.     

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.     

Solubilization and Separation of a Plant Plasma Membrane NADPH-O2- Synthase from Other NAD(P)H Oxidoreductases

Solubilization and ion-exchange chromatography of plasma membrane proteins obtained from bean (Phaseolus vulgaris L.) seedlings resulted in a single NAD(P)H-O2--synthase protein peak. This enzyme showed a high preference toward NADPH as a substrate (reaction rate, 27.4 nmol O2- produced min-1 mg-1 protein), whereas NADH reactions ranged from 0 to maximally 15% of the NADPH reactions. The protein functions as an oxidase and it was clearly resolved from NAD(P)H dehydrogenases identified with commonly used strong oxidants (ferricyanide, cytochrome c, DCIP, and oxaloacetate). The involvement of peroxidases in O2- production is excluded on the basis of potassium-cyanide insensitivity and NADPH specificity. The NADPH oxidase is only moderately stimulated by flavins (1.5-fold with 25 [mu]M flavine adenine dinucleotide and 2.5-fold with 25 [mu]M flavin mononucleotide) and inhibited by 100 [mu]M p-chloromercuribenzenesulfonic acid, 200 [mu]M diphenyleneiodonium, 10 mM quinacrine, 40 mM pyridine, and 20 mM imidazole. The presence of flavins was demonstrated in the O2-synthase fraction, but no b-type cytochromes were detected. The effect of these inhibitors and the detection of flavins and cytochromes in the plant O2- synthase make it possible to compare this enzyme with the NADPH O2- synthase of animal neutrophil cells.     

Electrophoretic isolation of a membrane-bound NADPH oxidase from guinea-pig polymorphonuclear leukocytes

Electrophoretic isolation of a membrane-bound NADPH oxidase of guinea-pig polymorphonuclear leukocytes was attempted with the O2- -generating membranes of cells unstimulated or stimulated with C3b-zymosan or sodium dodecyl sulfate, and also with the phagosomes isolated from the phorbol myristate acetate-coated latex particle-phagocytosing cells. When these vesicles were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of Triton X-100 and then assayed for NADPH-Nitroblue tetrazolium reducing activity, the activity was detected by the appearance of a single, blue band of the reduced dye on the gel, independent of the source of vesicles. In addition, the enzyme was able to generate O2- and its activity was significantly augmented with the homologous liver microsomal cytochrome b5. Its activity was heat-labile and inactivated by N-ethylmaleimide and p-chloromercuribenzene sulfonate. The enzyme, with an apparent molecular weight of 150 000, in the phagosomes was easily susceptible to limited proteolysis by trypsin and formed an active fragment with a molecular weight of 70 000, accompanying the loss of O2- -generating activity of the vesicles.     

Studies on the mechanism of NADPH oxidation by the granule fraction isolated from human resting polymorphonuclear blood cells.

Various factor affecting NADPH-oxidation by resting human leucocyte granules (LG) at acid pH, have been investigated. It was found that: 1) oxidation of NADPH by LG was increasingly inhibited by increased cyanide concentrations in the medium and was abolished by 4 mM cyanide. 2) with or without cyanide in the incubation medium, LG omitted, Mn++ in the presence of NADPH induced superoxide anion (O- WITH 2) production, as evidenced by oxygen consumption and H2O2 production, which were abolished (in the absence of cyanide) by cytochrome C (a potent O- with 2 scavenger). 3) Both NADPH oxidation in the presence of 2 mM cyanide (cyanide-resistant) and in its absence (cyanide-sensitive) by LG occurred only in the presence of Mn++, and both were inhibited by superoxide dismutase. 4) Cyanide-resistant NADPH oxidation by LG generated H2O2, was inhibited by H2O2 and was not modified by "active" catalase. The ratio of cyanide-resistant NADPH oxidation/O2 uptake was 1 up to 1.25 mM NADPH, and increased above this concentration. 5) Cyanide-sensitive NADPH oxidation was inhibited by catalase and increased upon addition of H2O2. The ratio of cyanide-sensitive NADPH oxidation/O2 uptake was 2. It was concluded that after initiation by O - with 2, produced independently of LG, two sequential types of LG dependent NADPH oxidations occur. First, an O - with 2-dependent protein mediated NADPH oxidation (cyanide-resistant) which generates H2O2 and O - with 2 occurs. Second, NADPH peroxidation (cyanide-sensitive) which utilizes H2O2 takes place.     

The respiratory burst oxidase of neutrophils. Separation of an FAD enzyme and its characterization

Pig blood neutrophils were briefly activated by various fatty acids and then fractionated into membrane vesicles with different NADPH oxidase activities. Treatment of these membranes with a detergent, octyl glucoside, resulted in a high yield of solubilized oxidase, which was subjected to isoelectric focusing on gels (pI 4.0-8.0). 1) A distinct band staining with NADPH-nitroblue tetrazolium focused at pI 5.0. The enzyme (pI 5.0) showed high specificity for NADPH and similar characteristics to the oxidase involved in the respiratory burst. 2) The enzyme was extracted from gel slices and analyzed. When measured promptly after its extraction, its NADPH oxidase activity was high, but there was apparent superoxide dismutase-insensitive cytochrome c reduction, probably due to direct electron transfer to the heme protein. However, it could produce superoxide anion (O2-) under some micelle conditions. 3) Therefore, the formation of the enzyme-substrate complex of yeast cytochrome c peroxidase was employed for the detection of H2O2. A fresh extract of stimulated cells catalyzed equimolar NADPH oxidation and H2O2 production of 306 and 300 nmol min-1 (mg protein)-1, respectively. The Km value of the enzyme for NADPH was 30 +/- 13 (S.D.) microM. The recovery of the extract (pI 5.0) was 19% of the total activity. 4) The enzyme extract contained 1.1-1.9 nmol of FAD/mg of protein, giving a turnover number of 300-600 min-1 in terms of O2- generation/FAD. No heme protein was found in the enzyme. The enzyme was mainly of 67-kDa molecular mass.