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 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.
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PMID:NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system. 303 Oct 60

We are attempting to identify cytokines that regulate macrophage secretion of reactive oxygen intermediates (ROI) and to analyse the biochemical basis of their effects. In both humans and mice, interferon-gamma (IFN-gamma) appears to be the chief factor secreted by clonally unselected lymphocytes that enhances macrophage oxidative metabolism and antiprotozoal activity. In vivo administration of recombinant IFN-gamma enhances the ROI secretory capacity of monocytes in humans, and the secretion of ROI and killing of protozoa by peritoneal macrophages in mice. A protein secreted by murine tumours and certain non-malignant cells exerts opposing effects. This macrophage deactivation factor (MDF) both blocks the induction of activation by IFN-gamma and reverses pre-existent activation. MDF action is non-toxic and selective, suppressing the secretion of ROI, killing of intracellular protozoa, and expression of Ia antigen, without inhibiting secretion of several other products, or synthesis of protein, ingestion of particles or adherence to culture vessels. The suppressive effect of MDF is reversed over several days after its removal. This reversal is hastened by IFN-gamma. Profound suppression of oxidative metabolism accompanies the differentiation of murine monocytes into Kupffer cells. The capacity of Kupffer cells to secrete ROI and kill intracellular protozoa remains deficient even after exposure to IFN-gamma. Thus, four states of macrophage activation can provisionally be discerned: the transition of mouse peritoneal macrophages from the non-activated to the activated state is accompanied by a ninefold increase in affinity of the superoxide-producing enzyme for NADPH, without a marked increase in cellular Vmax or content of cytochrome b559. The MDF-induced transition of mouse peritoneal macrophages from the activated to the deactivated state is accompanied by both an increase in Km and a decrease in apparent V max of the oxidase. There are no changes in the phorbol myristate acetate receptor number or affinity, glucose transport, NADPH levels, cytochrome b559 content, catalase (EC 1.11.1.6) GSH, GSH peroxidase (EC 1.11.1.9), GSH reductase (EC 1.6.4.2) or myeloperoxidase, consistent with the suppressed ROI secretory capacity and antiprotozoal activity of these cells. The Kupffer cell, whose non-responsiveness to IFN-gamma may mark it as inactivated, appears to lack detectable NADPH oxidase activity, despite the probable presence of cytochrome b559, and in this regard differs from both non-activated and deactivated macrophages.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Secretion of toxic oxygen products by macrophages: regulatory cytokines and their effects on the oxidase. 308 12

Heterocyclic nitrogenous bases, such as pyridine and imidazole which bind to heme-iron in cytochromes, inhibited the respiratory burst in intact neutrophils and NADPH-dependent oxygen consumption in lysed cells. Inhibition was accompanied by a spectral change in reduced cytochrome b558 as judged by low-temperature spectroscopy at 77 K. The position and shape of the alpha-band of the cytochrome were significantly altered upon exposure to pyridine or some other bases. Both inhibition and spectral changes were reversible. The results are consistent with the view that cytochrome b558 is involved in the NADPH oxidase system in neutrophils.
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PMID:Pyridine and imidazole reversibly inhibit the respiratory burst in porcine and human neutrophils: evidence for the involvement of cytochrome b558 in the reaction. 383 71

When amebae were incubated with latex beads, cyanide-insensitive oxygen consumption increased nearly two-fold. This cyanide-insensitive respiration was inhibited by salicylhydroxamate. Furthermore, cell fractionation studies revealed a localization for a portion of the NAD(P)H oxidase activity in phagolysosomes. The presence of low concentrations of divalent metal during fractionation resulted in an increased yield of oxidative activity in the phagolysosome fraction. In addition, the phagolysosome membrane was enriched about two-fold in a b-type cytochrome. These results show that oxidative metabolism in amebae has some striking similarities to the respiratory burst oxidase of neutrophils.
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PMID:Oxidative metabolism associated with phagocytosis in Acanthamoeba castellanii. 392 34

Using nitrogen cavitation and Percoll density gradient centrifugation for subcellular centrifugation of human neutrophils, approximately 90% of the low potential b-cytochrome, unique for phagocytes, as well as 50% of the flavoproteins in normal neutrophils were found in a granule fraction which co-sedimented with the specific granules. Upon stimulation of the intact cells with phorbol myristate acetate, both the b-cytochrome and the flavoprotein translocated from this granule fraction to the fractions which contained the plasma membranes and the NADPH oxidase activity. In neutrophils from two patients with chronic granulomatous disease, both the b-cytochrome and the flavoprotein of the granules were absent, but flavoprotein was present in normal amounts in the membrane and cytosol fractions. Taken together, these findings suggest that the specific granules, or granules co-sedimenting with the specific granules, are important stores for the components of the NADPH oxidase, which is responsible for the respiratory burst. Analysis of the stoichiometry of CO2 generation, H+ secretion and O2 consumption by stimulated neutrophils indicated that the hexose monophosphate shunt is the source of both protons and electrons for the NADPH oxidase activity, as well as of the extra protons secreted during the respiratory burst.
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PMID:The respiratory burst of phagocytosis: biochemistry and subcellular localization. 393 81

The effects of antibiotics and other commonly used medications on the human polymorphonuclear neutrophil leukocytes' (PMNs) nicotinamide-adenine dinucleotide phosphate-reduced (NADPH) oxidase activity have been investigated in vitro. Five antibiotics (penicillin G sodium, cefamandole nafate, metronidazole hydrochloride, clindamycin phosphate, and tobramycin sulfate, and a triple combination of penicillin G sodium-metronidazole hydrochloride-tobramycin sulfate) and two sedatives (morphine sulfate and diazepam) were incubated with normal human PMNs at therapeutic, infratherapeutic, and supratherapeutic drug levels. The superoxide dismutase-inhibitable, NADPH-dependent reduction of cytochrome C in the PMNs was studied after stimulation with formyl-methionyl-leucine-phenylalanine. Tobramycin sulfate and the triple combination of penicillin G sodium-metronidazole hydrochloride-tobramycin sulfate significantly reduced the NADPH oxidase activity at all dosages studied. Clindamycin phosphate, morphine sulfate, and diazepam also showed significant reduction at therapeutic and supratherapeutic concentrations. Penicillin G sodium, cefamandole nafate, and metronidazole hydrochloride did not cause a decrease in enzyme activity at any levels tested. We conclude that NADPH oxidase activity can be adversely affected by the circulating levels of common antibiotics and sedatives.
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PMID:Effect of antibiotics and sedatives on normal neutrophil nicotinamide-adenine dinucleotide phosphate-reduced oxidase activity. 394 1

NADPH oxidase activity was solubilized by detergent treatment of subcellular particles obtained from guinea-pig peritoneal macrophages stimulated with phorbol myristate acetate. Gel filtration of the material containing the NADPH oxidase activity gave two peaks of proteins, one of which eluted with the void and the other with the included volume of an AcA 22 column. The material eluted in the void volume contained more than 50% of the NADPH oxidase activity and less than 10% of the NAD(P)H cytochrome c reductase activity. A b-type cytochrome with peaks of absorption at 558, 528 and 426 nm was also enriched in the fraction which contained the NADPH oxidase activity. The distribution of flavoproteins as revealed by the measurement of FAD was different from that of NADPH oxidase and cytochrome b, and followed the elution profile of NADH cytochrome c reductase. Studies in subcellular particles showed that the b cytochromes of mitochondria and endoplasmic reticulum reduced by selective biochemical means accounted for only a minor part of the total b-type cytochromes and that the new cytochrome b previously described in neutrophils is the major chromophore also in macrophages. Oxidation-reduction midpoint potential of the partially purified cytochrome b was shown to be -247 mV. Association of cytochrome b with the NADPH oxidase activity and its very low Em7.0 makes it a suitable candidate to be part of the superoxide-generating system also in macrophages.
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PMID:Partial purification of the superoxide-generating system of macrophages. Possible association of the NADPH oxidase activity with a low-potential (-247 mV) cytochrome b. 406 52

A comparative analysis was made of the effectiveness of three methods for the reconstitution of microsomal electron-transfer chains, namely, self-assembly, incorporation of electron carriers into liposomes (non-specific template) and incorporation into ;ghosts' of microsomal vesicles (specific template). It was shown that when the ;ghosts' of the microsomal vesicles were used as a specific template extra cytochrome b(5) and NADH-specific flavoprotein were incorporated into them, but cytochrome P-450 and NADPH-specific flavoprotein were not incorporated into the membrane. As a result of the self-assembly and incorporation into liposomes all the electron carriers were present in the reconstituted membrane. Cytochrome P-450 reactivation took place and the inactive form, cytochrome P-420, was converted into the active form, cytochrome P-450. Of the four enzyme hydroxylation systems studied, i.e. NADPH- and NADH-dependent p-hydroxylation of aniline, and NADPH- and NADH-dependent N-demethylation of dimethylaniline, only the NADH-dependent demethylation of dimethylaniline (60% of the initial value) and NADH-dependent p-hydroxylation of aniline (30% of the initial value) were reconstituted by self-assembly. NADPH oxidase and NADH oxidase activities were only properly reconstituted by self-assembly and incorporation into liposomes. In contrast, the NADPH-specific system of peroxidation of unsaturated fatty acids was reconstituted by specific template-binding.
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PMID:The reconstitution of microsomal redox chains. A comparitive analysis of the effectiveness of membrane self-assembly and template binding of electron carriers. 415 29

A patient with an X-linked genetic disease resembling chronic granulomatous disease (CGD) but differing in several aspects from previously studied cases is described. The oxidase enzyme of the patient's granulocytes was normally activated, but had reduced activity as shown by an increased Michaelis constant and decreased maximum velocity of NADPH-dependent superoxide production. Cytochrome-b was undetectable in dithionite difference spectra. This CGD-like disease further implicates cytochrome-b as an important component of the microbicidal NADPH oxidase system and provides insight into its role in the enzyme complex.
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PMID:Chronic granulomatous disease due to granulocytes with abnormal NADPH oxidase activity and deficient cytochrome-b. 629 35

Human neutrophils were fractionated by nitrogen cavitation and Percoll density centrifugation, and the subcellular localization of FAD-flavoprotein, b-cytochrome, NADH-cytochrome b5 reductase, and NADPH-dependent cytochrome c reductase were determined in normal cells, cells from two patients with chronic granulomatous disease (CGD), and normal cells that had been stimulated with phorbol myristate acetate. In normal cells, a FAD-flavoprotein is found in a 1:2 molar ratio, with cytochrome b in the fractions containing the specific granules. Triton X-114 phase distribution indicates that the b-cytochrome but not the b-cytochrome-associated flavoprotein is an integral membrane protein. 80% of this flavoprotein, as well as all the b-cytochrome, was absent in these fractions from 2 CGD patients, although these patients had normal quantities of FAD in the fractions containing plasma membranes and cytosol. During stimulation the b-cytochrome-associated flavoprotein of the granules translocates with the b-cytochrome to the plasma membrane where NADPH oxidase is localized. Definition of the role of these NADPH oxidase constituents may provide a molecular description of the normal neutrophil respiratory burst and the molecular defect(s) in CGD.
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PMID:Subcellular localization of the human neutrophil NADPH oxidase. b-Cytochrome and associated flavoprotein. 670 48


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