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 phagocyte NADPH oxidase is a multicomponent membrane-bound electron transport chain that catalyzes the reduction of O2 to superoxide. Cytochrome b558, the terminal electron donor to O2, is an integral membrane heterodimer containing 91- and 22-kDa subunits (gp91-phox and p22-phox, respectively). Synthetic peptides, whose amino acid sequences correspond to a gp91-phox carboxyl-terminal domain, inhibit superoxide production by blocking assembly of the oxidase from membrane and cytosol components. In this study, we examined the amino acid sequence requirements of a series of synthetic truncated gp91-phox peptides for inhibition of human neutrophil NADPH oxidase activation. RGVHFIF, corresponding to gp91-phox residues 559-565, was the minimum sequence capable of inhibiting superoxide generation. Contributions of individual amino acids to overall RGVHFIF inhibitory activity were determined by comparing the abilities of alanine-substituted RGVHFIF peptides to inhibit superoxide production. Substitution of alanine for arginine, valine, isoleucine, or either of the phenylalanines (but not glycine or histidine) within RGVHFIF resulted in loss of inhibitory activity. Synthetic gp91-phox carboxyl-terminal peptides are likely to be competitive inhibitors of the corresponding carboxyl-terminal domain of native gp91-phox by virtue of amino acid identity. We conclude that properties of arginine valine, isoleucine, and phenylalanine side chains within an RGVHFIF-containing domain of gp91-phox contribute significantly to cytochrome b558-mediated activation of the oxidase.
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PMID:Characterization of a phagocyte cytochrome b558 91-kilodalton subunit functional domain: identification of peptide sequence and amino acids essential for activity. 131 44

The subcellular localization of the microbicidal nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and associated b-cytochrome was investigated in human neutrophils. In unperturbed neutrophils 85% of b-cytochrome and the major part of membrane-bound components of the NADPH oxidase co-sedimented with markers for specific granules and gelatinase. Using cytochrome b559 as a marker for membrane-bound components of the NADPH oxidase in quantitative studies we observed that, of the remaining 15%, the vast majority co-sedimented with latent alkaline phosphatase, a marker for a newly identified mobilizable intracellular compartment. Only a small fraction co-localized with the plasma membranes. Azurophil granules contained a protease activity which rapidly inactivated the NADPH oxidase components present in other membranes. Stimulation of the neutrophils with formyl-methionyl-leucyl-phenyl-alanine and leukotriene B4 which caused minimal degranulation of specific granules, resulted in translocation of b-cytochrome to the plasma membrane, concomitant with incorporation of alkaline phosphatase into the plasma membrane.
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PMID:Dual granule localization of the dormant NADPH oxidase and cytochrome b559 in human neutrophils. 254 92

The effect of various soluble stimuli on the superoxide production by guinea pig eosinophils was studied in comparison to neutrophils. Phorbol myristate acetate, A23187, digitonin, NaF, concanavalin A (Con A), and cytochalasin E stimulated eosinophils and neutrophils to release O2-. The O2- production by these active agents, excluding Con A and cytochalasin E, was much greater in eosinophils than in neutrophils. Formyl-Met-Leu-Phe stimulated the O2- production in neutrophils but not in eosinophils. Neither histamine nor Val/Ala-Gly-Ser-Glu stimulated the O2- production in both types of leukocytes. A23187- or Con A-stimulated O2- production was greatly enhanced by cytochalasin B pretreatment in neutrophils but not in eosinophils. Lineweaver-Burk analysis of NADPH oxidase in particulate fractions showed that eosinophils possessed the same Km values as neutrophils and greater Vmax values than neutrophils, suggesting that eosinophils have a similar, but more active, O2- -generating enzyme system than neutrophils.
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PMID:Response of superoxide anion production by guinea pig eosinophils to various soluble stimuli: comparison to neutrophils. 299 68

Phagocytosis by rabbit alveolar macrophages (AM) is accompanied by increases in O(2) consumption, glucose oxidation, and H(2)O(2) formation. Two aspects of the interrelations between these metabolic features of phagocytosis have been studied.First, the following evidence indicates that glutathione, glutathione reductase, and peroxidase serve as a cytoplasmic shuttle between H(2)O(2) and NADPH-dependent glucose oxidation: (a) AM contain 5.9 mmumoles of reduced glutathione per 10(6) cells and exhibit glutathione peroxidase and NADPH-specific glutathione reductase activity; (b) oxidized glutathione potentiates NADP stimulation of glucose oxidation; (c) an artificial H(2)O(2)-generating system stimulates glucose oxidation; (d) the cell penetrating thiol inhibitor, N-ethylmaleimide diminishes glucose oxidation. This effect largely depends on inhibition of the glutathione system rather than on inhibition of either H(2)O(2) formation or enzymes directly subserving glucose oxidation.Second, three potential H(2)O(2)-generating oxidases have been sought. No cyanide-insensitive NADH or NADPH oxidase activity could be detected. D-amino acid oxidase activity was 0.48 +/-0.07 U/10(6) cells with D-alanine as substrate.
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PMID:Glutathione-dependent peroxidative metabolism in the alveolar macrophage. 439 62

Endotoxemia, in man, has been associated with an autooxidative reduction in the bioavailability of polymorphonuclear leukocyte receptors. The location and mechanisms of this phenomena have remained unclear; we investigated the effects of lipopolysaccharide (LPS) on intracellular Fc gamma receptor expression. Polymorphonuclear leukocytes (PMN) were incubated with LPS (10 ng/ml), permeabilized with saponin, followed by measurement of CD64, CD32w, and CD16 (Fc gamma RI, II, III) using 125I-monoclonal antibodies directed against these receptors. Exposure of permeabilized PMN to LPS significantly reduced intracellular Fc gamma receptor expression. PMN isolated from patients with chronic granulomatous disease or myeloperoxidase-specific deficiency did not exhibit this effect. Furthermore, specific inhibitors of components of the PMN oxidative burst (NaN3, 10 mM; L-alanine 30 mM) prevented the LPS-induced oxidative reduction in receptor expression. NADPH oxidase inhibition with diphenyleneiodonium also blocked the effect of LPS on intracellular Fc gamma receptor expression. The effects of LPS on intracellular PMN Fc gamma receptors were reproduced with monophosphoryl lipid A but required a 10 times greater concentration than LPS. Preadherence of PMN on fibronectin or arginine-glycine-aspartate-serine (RGDS), but not laminin, prevented the LPS-induced reduction in oxidative receptor expression. The effects of fibronectin/RGDS were blocked by actinomycin D and cycloheximide. Cross-linkage of intracellular Fc gamma receptors prior to exposure to LPS also prevented the LPS-induced oxidative reduction in receptor expression. These results demonstrate that an important pathophysiologic property of LPS is to induce an intracellular oxidative-derived reduction in Fc gamma receptor expression and that the biologically relevant proteins fibronectin and RGDS ameliorate this effect.
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PMID:Regulation of intracellular polymorphonuclear leukocyte Fc receptors by lipopolysaccharide. 806 31

Novel protein kinases that may participate in the signal transduction pathways of neutrophils were sought by a procedure based on the ability of these enzymes to undergo renaturation and catalyze the phosphorylation of a peptide substrate fixed in a gel. We report that neutrophils contain four uncharacterized protein kinases with molecular masses of about 69, 63, 49, and 40 kDa, which are rapidly activated upon stimulation of these cells with the chemoattractant fMet-Leu-Phe. These kinases can catalyze the phosphorylation of a peptide that corresponds to residues 297-331 of the 47-kDa subunit of the NADPH oxidase system (p47-phox). A peptide that corresponds to residues 153-178 of the human myristolyated alanine-rich C kinase substrate (MARCKS) protein was also a substrate for the 69- and 63-kDa kinases. The time course for the activation of these enzymes was similar to the phosphorylation of p47-phox and MARCKS in intact neutrophils. In contrast, stimulation of these cells with 4 beta-phorbol 12-myristate 13-acetate, the calcium ionophore A23187, or the combination of these agonists did not activate these enzymes. Activation of the 63- and 40-kDa protein kinases was blocked by pertussis toxin, calyculin A, and staurosporine. Several other unidentified protein kinases were also active with these peptides but did not exhibit enhanced activity after cell stimulation with this method.
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PMID:Stimulation of neutrophils with a chemoattractant activates several novel protein kinases that can catalyze the phosphorylation of peptides derived from the 47-kDa protein component of the phagocyte oxidase and myristoylated alanine-rich C kinase substrate. 834 15

To further define the role played by protein kinase C (PKC) in the activation of the neutrophil NADPH oxidase, we have utilized a pseudosubstrate of PKC which was myristoylated at the N terminus. In electropermeabilized neutrophils, the myristoylated pseudosubstrate Phe-Ala-Arg-Lys-Gly-Ala-Leu-Arg-Gln (myr-psi PKC) inhibited PMA-induced protein phosphorylations and activation of the NADPH oxidase, induced either by PMA or by the receptor agonist formyl-methionyl-leucyl-phenylalanine. Both the pseudosubstrate lacking the N-terminal myristate (psi PKC) and a myristoylated control peptide (Phe-Ala-Glu-Asp-Gly-Ala-Leu-Glu-Gln, myr-CP) were without effect on these responses. The myristoylated pseudosubstrate was also tested in a cell-free system, in which NADPH oxidase activation can be achieved by addition of SDS and guanosine 5'-3-O-(thio)triphosphate in a staurosporine-insensitive manner. Myr-psi PKC, but not psi PKC or myr-CP, proved to be a potent inhibitor of NADPH oxidase activity in the cell-free system, indicating that the inhibition observed in permeabilized neutrophils may have been caused by an effect other than PKC inhibition. In the presence of myr-psi PKC, translocation in the cell-free system of the cytosolic oxidase components p47-phox and p67-phox to the plasma membrane was inhibited. From these results we conclude that myristoylation profoundly increases the ability of pseudosubstrates of PKC to inhibit not only PKC-mediated phosphorylations, but also NADPH oxidase activation. The latter effect, however, is most probably not related to PKC inhibition but may indicate a critical role of the membrane surface charge in the translocation of the cytosolic oxidase components p47-phox and p67-phox.
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PMID:Inhibition of neutrophil NADPH oxidase assembly by a myristoylated pseudosubstrate of protein kinase C. 836 Jan 54

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222-->Phe mutant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222-->Ala mutant, [A222]p-hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, [A222]p-hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p-hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H.A., Mattevi, A., Oblomova, G., Kalk, K.H., Hol, W.G.J., van der Bolt, F.J.T. & van Berkel, W.J.H. (1994) Biochemistry 33, 10161-10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin. [A222]p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towards the productive "in' position. [A222]p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the "out' conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222-->Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring.
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PMID:Flavin motion in p-hydroxybenzoate hydroxylase. Substrate and effector specificity of the Tyr22-->Ala mutant. 864 2

A plasma membrane iron reductase, required for cellular iron acquisition by Saccharomyces cerevisiae, and the human phagocytic NADPH oxidase, implicated in cellular defense, contain low potential plasma membrane b cytochromes that share elements of structure and function. Four critical histidine residues in the FRE1 protein of the iron reductase were identified by site-directed mutagenesis. Individual mutation of each histidine to alanine eliminated the entire heme spectrum without affecting expression of the apoprotein, documenting the specificity of the requirement for the histidine residues. These critical residues are predicted to coordinate a bis-heme structure between transmembrane domains of the FRE1 protein. The histidine residues are conserved in the related gp91(phox) protein of the NADPH oxidase of human granulocytes, predicting the sites of heme coordination in that protein complex. Similarly spaced histidine residues have also been implicated in heme binding by organelle b cytochromes with little overall sequence similarity to the plasma membrane b cytochromes. This bis-heme motif may play a role in transmembrane electron transport by distinct families of polytopic b cytochromes.
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PMID:Intramembrane bis-heme motif for transmembrane electron transport conserved in a yeast iron reductase and the human NADPH oxidase. 894 93

The leukocyte NADPH oxidase is a multi-subunit enzyme that catalyzes the reduction of oxygen to O2- at the expense of a reduced pyridine nucleotide. We have used site-directed mutagenesis to examine the functional role of the four cysteines in p47PHOX, one of the subunits of the oxidase. For these experiments, mutant proteins in which a single cysteine was replaced with alanine were expressed in p47PHOX-deficient Epstein-Barr virus-transformed B lymphoblasts, and O2- production by these transfected cells was measured. The activity of the mutant C98A was similar to that of wild type, but the maximum rate of O2- production by C196A was significantly larger than seen with wild type. The other two mutants (i.e., C111A and C378A) differed from wild type not only in maximum O2- production, but also in the time required for activation, which was considerably delayed with both of these mutants. The similarity in the time courses of oxidase activation with the C111A and C378A mutants, and the finding that C378A occurs in the sequence CSE, raises the possibility that these cysteines may be involved in redox regulation of oxidase activity.
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PMID:The leukocyte NADPH oxidase subunit p47PHOX: the role of the cysteine residues. 946 17


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