Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The disruption of the molecular organization of the plasma membrane of leukocytes by phagocytosable particles, or by agents such as surfactants, antibodies, phospholipase C, fatty acids and chemotactic factors, leads to a stimulation of the phagocyte oxidative metabolism. Concanavalin A (Con A) has been used as a tool to study the mechanism of this metabolic regulation. The binding of Con A to the surface of polymorphonuclear leukocytes (PMNL) or macrophages produces a rapid enhancement of oxygen uptake and glucose oxidation through the hexose monophosphate pathway (HMP). This is explained by an activation of the granular NADPH oxidase, the key enzyme in the metabolic stimulation. The effect of Con A is not due to endocytosed lectin, since Con A covalently coupled to large sepharose beads still acts as stimulant. The metabolic changes caused by Con A are reversible. If, after the onset of stimulation, sugars with high affinity for Con A are added to the leukocyte suspension, the activity of granular NADPH oxidase and the rate of respiration and glucose oxidation return to their resting values. The metabolic burst, while partially supressed by treatment of PMNL with iodoacetate, sodium flouride and cytochalasin B, is slightly increased by colchicine. Con A induces a selective release of granular enzymes (beta-glucuronidase, peroxidase, alkaline phosphatase) from PMNL, whereas no leakage of cytoplasmic enzymes is observed. The enzyme release is inhibited by iodoacetate and by drugs known to increase cell levels of cyclic AMP. Based on a current view of the mode of interaction between Con A and cell surfaces, a model of the metabolic disruption of leukocytes is presented.
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PMID:Concanavalin A as a probe for studying the mechanism of metabolic stimulation of leukocytes. 16 45

Activation of the superoxide-generating NADPH oxidase by phorbol ester or zymosan induced a cytoplasmic acidification when liver macrophages were incubated in sodium-free media or in the presence of amiloride. Staurosporine or desensitization of protein kinase C inhibited phorbol ester- and zymosan-induced pH changes and generation of superoxide. The intracellular pH remained unchanged in cells incubated in physiological sodium media. Ionomycin and arachidonic acid did not induce a change in intracellular pH or a generation of superoxide. Fluoride, which has been shown to induce a translocation of protein kinase C in these cells, did not elicit superoxide generation but induced a decrease in intracellular pH. These experiments support (1) a role of the Na+/H+ antiporter in macrophages as a metabolic regulator of intracellular pH upon stimulation of the superoxide-generating NADPH oxidase, and (2) suggest an involvement of protein kinase C in this process.
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PMID:Relationship between intracellular pH changes, activation of protein kinase C and NADPH oxidase in macrophages. 131 15

The superoxide-generating respiratory burst oxidase (NADPH oxidase) from human neutrophils can be activated in a cell-free system consisting of plasma membrane and cytosol by anionic amphiphiles such as sodium dodecyl sulfate and arachidonate (McPhail, L. C., Shirley, P. S., Clayton, C. C., and Snyderman, R. (1985) J. Clin. Invest. 75, 1735-1739; Curnutte, J. T. (1985) J. Clin. Invest. 75, 1740-1743; Bromberg, Y., and Pick, E. (1984) Cell. Immunol. 88, 213-221). Herein, the activity thus obtained is shown to be very labile at 37 degrees C. The rate of inactivation varied inversely with cytosol concentration. The stabilizing factor(s) was destroyed by heat and trypsin, indicating that it is protein in nature. Whereas cytosol from normal cells and from a chronic granulomatous disease patient lacking p67phox stabilized the oxidase activity, that from a chronic granulomatous disease patient lacking p47phox did not. Also, dialdehyde NADPH-treated cytosol showed no stabilizing effect, indicating that p47phox and a putative NADPH-binding component both participate in stabilization. The mechanism of inactivation was further explored by examining the stabilizing effect of agents that can act as chemical cross-linkers. Of several tested, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was the most effective, but others that utilize different chemical mechanisms were also partially effective. EDC extended the half-life at 37 degrees C from 2 to 120 min, protected against the inactivating effects of Triton X-100 and high salt, and did not affect the Km for NADPH. Stabilization required prior activation in the presence of both cytosol and membrane; and EDC treatment of cytosol, membrane, or a mixture of the two prior to the addition of sodium dodecyl sulfate failed to induce stabilization. EDC eliminated the requirement for the continuous presence of cytosol and activator. Dialysis did not cause a loss in activity, whereas control activity was diminished with dialysis and was largely restored with added sodium dodecyl sulfate. In the absence of EDC, the separation of cytosol from the membrane fraction resulted in a significant loss of activity, which was largely restored by the addition of cytosol. However, EDC treatment allowed the isolation of a nearly fully active oxidase in the membrane fraction, the activity of which was not influenced by added cytosol. These results support a model in which the active NADPH oxidase consists of a dissociable complex among membrane and cytosolic components and indicate that the longevity of the activated state requires continuous association of these components.
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PMID:Stabilization of human neutrophil NADPH oxidase activated in a cell-free system by cytosolic proteins and by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. 131 6

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.
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PMID: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. 132 36

The effects of anti-allergic drugs with or without H1-receptor antagonism on the NADPH oxidase (EC 1.6.99.6) from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Three anti-allergic drugs with H1-receptor antagonism, azelastine, ketotifen and oxatomide, were found to inhibit the superoxide generation of human neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of these drugs required for 50% inhibition of the oxidase (IC50) were: azelastine--0.7 microM in the whole-cell system and 0.5 microM in the cell-free system; ketotifen--60 microM in the whole-cell system and 6.8 microM in the cell-free system; and oxatomide--25 microM in the whole-cell system and 9.7 microM in the cell-free system. In addition, in the cell-free system, these drugs did not change the Km values for the NADPH of the oxidase. However, these drugs did not inhibit the superoxide generation of NADPH oxidase after its activation in whole-cell and cell-free systems, suggesting that these drugs do not have superoxide-scavenger actions. Concentrations of less than 200 microM of anti-allergic drugs without H1-receptor antagonism, tranilast, repirinast and ibudilast, did not inhibit neutrophil NADPH oxidase in whole-cell and cell-free systems. The IC50 of hydrocortisone in the cell-free system was 60 microM. These results suggest that anti-allergic drugs with H1-receptor antagonism inhibit activation of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in cell-free systems and that they have much stronger anti-inflammatory action than hydrocortisone.
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PMID:Effects of anti-allergic drugs on human neutrophil superoxide-generating NADPH oxidase. 137 6

Fluorescence intensified/enhanced microscopy has been used to study the metabolic activation of living human neutrophils in time-lapse sequences. The autofluorescence associated with NAD(P)H's emission band was studied within individual quiescent and stimulated cells. Excitation of NAD(P)H-associated autofluorescence was provided by a high-intensity Hg-vapor lamp. The background-subtracted autofluorescence signals were computer enhanced. In some cases the ratio image of NAD(P)H-associated autofluorescence to tetramethyl-rhodamine methyl ester (TRME) fluorescence, which was found to be uniformly distributed within neutrophils, was calculated to normalize autofluorescence intensities for cell thickness. Activation of the NADPH oxidase by phorbol myristate acetate, F-, N-formyl-methionyl-leucyl-phenylalanine (FMLP), or tumor necrosis factor (TNF) dramatically reduced autofluorescence levels. Membrane solubilization with sodium dodecyl sulfate eliminated autofluorescence. Thus, control experiments indicated that most or all of the detectable NAD(P)H-associated autofluorescence was due to NAD(P)H, consistent with previous non-microscopic studies. To understand the metabolic events surrounding the internalization and oxidative destruction of targets, we have imaged the NAD(P)H-associated autofluorescence of neutrophils and the Soret band of antibody coated target erythrocytes during cell-mediated cytotoxicity. Absorption contrast microscopy of the erythrocyte's Soret band is an especially sensitive indicator of the entry of reactive oxygen metabolites into this target's cytosol. Thus, it is possible to spectroscopically dissect and image the substrate (NADPH) and product (O2-) reactions of the NADPH oxidase in living unlabeled neutrophils. During real-time experiments at 37 degrees C, the level of NAD(P)H-associated autofluorescence surrounding phagosomes greatly increases before the disappearance of the target's Soret band. NAD(P)H-associated autofluorescence in the vicinity of phagocytosed erythrocytes is greatly diminished after target oxidation. This suggests that NAD(P)H is translocated to the vicinity of phagosomes prior to the oxidation of targets. The apparent cytosolic redistribution of NAD(P)H was confirmed by ratio imaging microscopy to control for cell thickness. We suggest that NADPH including its sources and/or carriers accumulate near phagosomes prior to target oxidation and that local NADPH molecules are consumed during target oxidation.
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PMID:Imaging neutrophil activation: analysis of the translocation and utilization of NAD(P)H-associated autofluorescence during antibody-dependent target oxidation. 161 16

A putative reservoir of functional plasma membrane proteins, the secretory vesicle identified by latent alkaline phosphatase and tetranectin, has previously been demonstrated based on indirect evidence (Borregaard, N., Miller, L. J., and Springer, T. A. (1987) Science 237, 1204-1206; Borregaard, N., Christensen, L., Bjerrum, O. W., Birgens, H. S., and Clemmesen, I. (1990) J. Clin. Invest. 85, 408-416). Difficulties in separating plasma membranes from this entity by density gradient centrifugation has prohibited discriminative dynamic and quantitative studies of secretory vesicles and plasma membranes. By combining density centrifugation with free flow electrophoresis we overcame this obstacle. Freshly prepared unperturbed human neutrophils were subjected to nitrogen cavitation followed by density centrifugation on Percoll gradients. Light membrane fractions containing plasma membranes and secretory vesicles were applied to high voltage free flow electrophoresis on an Elphor VaP 22. Plasma membrane vesicles, identified by HLA class I antigen mixed enzyme-linked immunosorbent assay (Bjerrum, O. W., and Borregaard, N. (1990) Scand. J. Immunol. 31, 305-313) and 125I applied to cells before cavitation, were clearly separated from secretory vesicles. Electron microscopy revealed a morphology typical of plasma membranes in the former fraction and a population of vesicles with markedly different appearance in the latter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles demonstrated distinct differences in protein patterns between the two fractions. Superoxide generating capacity induced by sodium dodecyl sulfate and cytosol, an entity traditionally ascribed to the plasma membrane, was largely confined to fractions containing secretory vesicles. Thus, the majority of membrane-bound NADPH oxidase components of light membranes of human neutrophils colocalize with secretory vesicles.
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PMID:Separation of human neutrophil plasma membrane from intracellular vesicles containing alkaline phosphatase and NADPH oxidase activity by free flow electrophoresis. 163 31

This study evaluates the role of inositol phosphates as possible mediators of the activation of phospholipase A2 and NADPH oxidase in cultured rat liver macrophages. Inositol phosphate formation was achieved by zymosan, immune complexes, latex particles and calcium ionophore while the release of arachidonic acid and the formation of prostaglandin E2 was also elicited by phorbol ester and NaF, but not by latex particles; generation of superoxide was obtained by zymosan and phorbol ester only. The kinetics of the formation of inositol phosphates revealed that within the first few minutes after zymosan addition inositol trisphosphate was formed, followed by inositol bisphosphate and inositol monophosphate. Pre-treatment of the cells with dexamethasone or removal of extracellular calcium led to an inhibition of the zymosan-induced formation of inositol phosphates and prostaglandin E2 but had no effect on the generation of superoxide; inhibition of the Na+/H+ exchanger by removal of extracellular sodium ions led to a decrease of the zymosan-induced synthesis of prostaglandin E2, but did not affect the formation of inositol phosphates and superoxide. Pre-treatment of the cells with phorbol ester decreased the zymosan-induced synthesis of prostaglandin E2 and superoxide, but even enhanced the zymosan-induced formation of inositol phosphates. These data indicate that in cultured rat liver macrophages the formation of prostaglandins and superoxide cannot be correlated to an activation of phospholipase C.
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PMID:Activation of phospholipase C is not correlated to the formation of prostaglandins and superoxide in cultured rat liver macrophages. 164 76

Effects of non-steroidal anti-inflammatory drugs (NSAID: amfenac sodium, diclofenac sodium, indomethacin and ketoprofen) on the generation of superoxide anion (O2-) by isolated rat polymorphonuclear leukocytes (PMN) were studied spectrophotometrically using cytochrome c. The effects of these drugs were also studied on O2- production by the xanthine-xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-NADPH oxidase systems. Amfenac sodium, at 0.1 mM, inhibited significantly O2- generation in rat PMN induced by opsonized zymosan. At 0.5 mM, diclofenac sodium and indomethacin inhibited the O2- generation in rat PMN. All of the above drugs slightly inhibited O2- production by the xanthine-xanthine oxidase system. On the other hand, O2- production by the NADPH-NADPH oxidase system was significantly inhibited by the addition of amfenac sodium, ketoprofen or indomethacin. These results suggest that non-steroidal anti-inflammatory drugs do not work as an O2- scavenger and block O2- production by the NADPH-NADPH oxidase system of rat PMN. It is concluded that amfenac sodium and the other drugs are able to inhibit granulocyte O2- production by blocking the activation of NADPH-oxidase.
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PMID:Inhibitory effects of non-steroidal anti-inflammatory drugs on superoxide generation. 165 19

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes in a cell-free system by anionic amphiphiles requires the participation of both membrane and cytosolic components. We reported that ammonium sulfate fractionation (Pick, E., Kroizman, T., and Abo, A. (1989) J. Immunol. 143, 4180-4187) and affinity chromatography on 2',5'-ADP-agarose (Shaag, D., and Pick, E. (1990) Biochim. Biophys. Acta 1037, 405-412) permit separation of cytosol in two fractions (sigma 1 and sigma 2) that support O2- production by solubilized membrane synergistically. We now describe the purification of sigma 1 to near homogeneity and demonstrate that it represents a cytosolic component distinct from p47-phox and p67-phox, that are both found in fraction sigma 2. Sigma 1 was absolutely required for the full expression of amphiphile-activated NADPH-oxidase activity. This requirement was evident whether sigma 1 was added to cell-free systems composed of: (a) solubilized membrane and a sigma 2-enriched cytosolic fraction, or (b) purified cytochrome b559, incorporated in liposomes, and purified sigma 2. Sigma 1 was purified by a sequence comprising ammonium sulfate fractionation, hydrophobic chromatography on phenyl-Superose, absorption with CM-Sepharose, anion exchange chromatography on DEAE-Sepharose, and gel filtration on Superose 12. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of sigma 1 of maximal purity, under both reducing and nonreducing conditions, demonstrated the presence of two proteins, of 24 and 22 kDa. On gel filtration, sigma 1 was eluted as a symmetrical peak of 46 kDa that by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed the presence of both 24- and 22-kDa bands. We suggest that, in its native form, sigma 1 might represent a complex of the 24- and 22-kDa proteins. The specific roles of each molecule in NADPH oxidase function remain to be determined.
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PMID:Purification and characterization of a third cytosolic component of the superoxide-generating NADPH oxidase of macrophages. 166 Aug 77


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