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)

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to generate superoxide, needed for the intracellular killing of microorganisms. This is caused by mutations in any one of the four subunits of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of this enzyme (p67-phox) is missing. We here report on a patient with a mutation in the p67-phox gene that leads to expression of a nonfunctional p67-phox protein. The purified granulocytes of this patient failed to produce superoxide and contained about half of the normal amount of p67-phox. Analysis of the cDNA and genomic DNA of this patient showed that the patient is a compound heterozygote for a triplet nucleotide deletion in the p67-phox gene, predicting an in-frame deletion of lysine 58 in the p67-phox protein and a larger deletion of 11-13 kb in the other allele. Interestingly, the 58Lys deletion in p67-phox disrupts the interaction with p21-rac1, a ras-related protein involved in the activation of the NADPH oxidase. In contrast to normal neutrophils, in which p47-phox and p67-phox translocate to the plasma membrane upon cell activation, the cells of the patient did not show this translocation, indicating that an interaction between p67-phox and p21-rac1 is essential for translocation of these cytosolic proteins and activation of the NADPH oxidase. Moreover, this CGD patient represents the first case of disease caused by a disturbed binding of a ras-related protein to its target protein.
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PMID:Disturbed interaction of p21-rac with mutated p67-phox causes chronic granulomatous disease. 887 95

The human NADPH oxidase is a very intriguing enzyme; although its catalytic unit is retained within cytochrome b558, various additional proteins are required for activity of the NADPH oxidase. In the past few years substantial progress has been made to elucidate the protein-protein interactions and the activation events involved. The following facts have become evident: (1) activation of rac and subsequent interaction with p67-phox is crucial for the interaction of p67-phox with cytochrome b558, and probably with gp91-phox; (2) p47-phox interacts with p22-phox, and phosphorylation of 379Ser of p47-phox is obligatory for this event; (3) p47-phox and p67-phox regulate each other's translocation in a positive sense (see also reference 71). To put it differently: it is vital to gain insight in the intrigues within the phox family and associated characters to fully understand NADPH oxidase activation.
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PMID:Interactions between the components of the human NADPH oxidase: intrigues in the phox family. 890 Feb 89

Flavocytochrome b558, the membrane-spanning component of the NADPH oxidase system of phagocytic cells, is composed of two subunits, p22phox and gp91phox (where phox stands for phagocyte oxidase). The stoichiometry of the subunits has been determined for purified flavocytochrome b556 by: (1) densitometry of Coomassie Blue-stained proteins separated by SDS/PAGE, (2) aromatic absorbance at 280 mm by the subunits after separation by gel filtration under denaturing conditions, (3) crosslinking studies with bis[sulphosuccinimidyl]suberate, where the molecular mass of the cross-linked complex was determined by Western blotting, and (4) radiolabelling of pure flavocytochrome b556 on lysine residues with 125I-labelled Bolton-Hunter reagent (N-succinimidyl-3-(4-hydroxy-5-[125I]iodophenyl)propionate), followed by SDS/PAGE and determination of the radioactivity on each subunit. The ratio of p22phox to gp91phox in the purified flavocytochrome b556 was related back to that in the neutrophil membrane by quantitative Western and dot-blotting to ensure that the stoichiometry was maintained during purification. These measurements showed that the two subunits were present in neutrophil membranes in a molar ratio of 1:1.
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PMID:Stoichiometry of the subunits of flavocytochrome b558 of the NADPH oxidase of phagocytes. 894 64

NADPH-dependent superoxide generation can be reconstituted in a cell-free system using recombinant cytosolic factors (p47-phox, p67-phox, and Rac) plus flavocytochrome b558. Rac1 and Rac2 are closely related small GTPases, differing primarily in the C-terminal 10 residues where Rac1 but not Rac2 contains a polybasic sequence. In their nonisoprenylated forms, Rac1 was highly effective in reconstituting NADPH oxidase activity (low EC50, high Vmax), whereas Rac2 was only minimally effective (high EC50, low Vmax). In contrast, low concentrations of isoprenylated Rac1 and Rac2 both supported high rates of superoxide generation. Like full length Rac2, truncated forms of both Rac1 and Rac2 in which the C-terminal 10 residues were eliminated were poorly activating, pointing to the C terminus of Rac1 as a determinant of activity. Mutation of single positively charged residues in the C terminus of nonisoprenylated Rac1 markedly reduced its ability to support superoxide generation, affecting both its EC50 and the Vmax. In contrast, mutation or truncation of the C terminus failed to affect the activation of PAK, a Rac-regulated protein kinase. The EC50 for Rac1 increased with increasing salt concentrations, whereas that of Rac2 was independent of salt, implicating the involvement of electrostatic forces for the former. Using flavocytochrome b558 reconstituted into phosphatidylcholine vesicles, the EC50 for Rac1 but not Rac2 decreased (increased binding) when an acidic phospholipid (phosphatidylinositol) was present, supporting a role for the Rac1 polybasic C terminus in binding to the membrane. A model in which Rac must associate simultaneously both with p67-phox and with the membrane to activate the NADPH oxidase can account for the above observations.
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PMID:Membrane association of Rac is required for high activity of the respiratory burst oxidase. 896 31

The superoxide-producing NADPH oxidase consists of membrane-associated cytochrome b558 and cytosolic components, p47-phox and p67-phox. Recently, we have found a novel cytosolic component, p40-phox, which is tightly associated with p67-phox. In this study, we examined the translocation of p40-phox during activation of NADPH oxidase in a cell-free system using the membrane and the purified p47-phox/p67-phox/p40-phox complex. p40-phox was translocated to the membrane by arachidonic acid in a dose-dependent manner. The translocation pattern of p40-phox was similar to those of p47-phox and p67-phox. However, immunoprecipitation assay revealed that p40-phox was dissociated from p47-phox and p67-phox during activation. The translocation of three cytosolic components was not affected by the deletion of GTP-gamma-s from the reaction mixture. Interestingly, a synthetic peptide corresponding to carboxyl-terminus of p40-phox inhibited the activation of NADPH oxidase and translocation of p40-phox, p47-phox, and p67-phox, suggesting that p40-phox might play a role in the activation of NADPH oxidase. These observations suggest that p40-phox is dissociated from p67-phox during activation, and translocates to the membrane by GTP-gamma-s-independent mechanism.
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PMID:Translocation of guinea pig p40-phox during activation of NADPH oxidase. 898 88

Effects of the farnesylcysteine mimetic, farnesylthiosalicylate on the activation of myeloid cells were studied. In dimethyl-sulfoxide-differentiated HL60 cells and in human neutrophils farnesylthiosalicylate (< or = 20 microM) dose-dependently elevated cytosolic Ca2+ concentrations, suggesting phospholipase-C-mediated release of the ion from intracellular stores. In human neutrophils, in addition to the production of inositol trisphosphate, farnesylthiosalicylate induced activation of the NADPH oxidase and translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane. The calcium signal, inositol-trisphosphate production and superoxide generation elicited by farnesylthiosalicylate were partially blocked by treatment of the cells with pertussis toxin, consistent with participation of pertussis-toxin-sensitive and pertussis-toxin-resistant elements. In HL60 cells, farnesylthiosalicylate (< or = 20 microM) did not activate NADPH oxidase but dose-dependently augmented PMA-elicited activity of the enzyme. This effect was resistant to pertussis-toxin treatment. In vitro augmentation of PKC-mediated phosphorylation of histone and cytosolic p47-phox by farnesylthiosalicylate and the finding that downregulation of PKC abrogated potentiation of NADPH oxidase activity by farnesylthiosalicylate were compatible with the involvement of PKC in the response of HL60 cells to farnesylthiosalicylate. It is suggested that the effects of farnesylthiosalicylate on myeloid cells reflect interaction of the analog with prenylcysteine-docking sites on cellular signaling elements.
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PMID:Activation of signaling pathways in HL60 cells and human neutrophils by farnesylthiosalicylate. 902 78

The effect of race-specific elicitors on NADPH oxidase was examined in vivo by treating tomato cells with elicitor-containing intercellular fluids prepared from infected tomato leaves inoculated with specific Cladosporium fulvum races. Treatment of Cf-4 or Cf-5 cells with intercellular fluids from incompatible but not from compatible races of C. fulvum increased oxidase activity and the amount of p67-phox, p47-phox, and rac2 in the plasma membrane. Comparison of these three components in the cytosol and plasma membrane indicated that elicitors promoted the translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells carrying the appropriate resistance gene. Protein kinase C activators and inhibitors did not affect enzyme activity or the binding of these three components to the plasma membrane. In contrast, staurosporine, calmodulin antagonists, and EGTA inhibited elicitor-induced oxidase activity and the translocation of the cytosolic components. The assembly process involves a Ca(2+)-dependent protein kinase that catalyzes the phosphorylation of p67-phox and p47-phox, facilitating their translocation to the plasma membrane. Our data suggest that although both plants and animals share common elements in eukaryotic signal transduction, the involvement of different protein kinases mediating the activation of phosphorylation of p67-phox and p47-phox may reflect the unique spatial and temporal distribution of signal transduction pathways in plants.
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PMID:Race-specific elicitors of Cladosporium fulvum promote translocation of cytosolic components of NADPH oxidase to the plasma membrane of tomato cells. 906 55

The superoxide (O2-)-generating NADPH oxidase of phagocytic cells is composed of a membrane-bound flavocytochrome (cytochrome b-559) and three cytosolic components, p47-phox, p67-phox, and the small GTPase rac-1 (or 2). Cytochrome b-559 bears the NADPH binding site and the redox centers (FAD and heme). Electron flow through the redox centers, from NADPH to oxygen, is activated consequent to the assembly of the three cytosolic components with cytochrome b-559. We studied the kinetics of electron flow through the redox centers of NADPH oxidase in a cell-free system, consisting of purified relipidated and reflavinated cytochrome b-559 and recombinant cytosolic components, activated by the anionic amphiphile, lithium dodecyl sulphate. The NADPH oxidase complex assembled in vitro exhibited: (a) a high steady-state electron flow (165 electrons/heme/s); (b) low stationary levels of FAD and heme reduction (about 10%), and (c) a high rate constant of heme oxidation by oxygen (1720 s-1). Surprisingly, the kinetic properties of NADPH oxidase assembled in a semi-recombinant cell-free system, lacking p47-phox (found to generate significant amounts of O2-), were similar to those of the complete system, as shown by a steady-state electron flow of 83 electrons/heme/s, low stationary levels of FAD and heme reduction (10%), and a rate constant of heme oxidation by oxygen of 1455 s-1. The kinetic features of NADPH oxidase assembled in vitro from purified and recombinant components differ considerably from those of solubilized enzyme preparations derived from intact stimulated phagocytes. The fast operation of the cell-free system is best explained by the activation-related facilitation of electron flow at both the FAD-->heme and the heme-->oxygen steps.
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PMID:Electron transfer in the superoxide-generating NADPH oxidase complex reconstituted in vitro. 913 Oct 41

When microorganisms invade the body, they encounter a large asssortment of defense mechanisms. Among these, phagocytes play an important role in the process of killing pathogens. This event is mediated by two important processes, viz. activation of the NADPH oxidase enzyme, which leads to the production of toxic oxygen metabolites, and fusion of intracellular granules with the phagosome (the vesicle that contains the ingested micro-organisms), which causes release of the toxic granule contents into this vesicle. The human NADPH oxidase is a very complex enzyme, in two ways: 1. it exists of at least 6 components: cytochrome b558 (a heterodimer comprised of gp91-phox and p22-phox), p47-phox, p67-phox, p40-phox, rac and Rap1A, and 2. there are multiple signal transduction pathways leading to activation of the NADPH oxidase. The most likely reason for this complexity is the toxicity of the oxygen radicals produced by the active NADPH oxidase; these compounds are not only harmful to the invading pathogens, but also to the surrounding tissues. This latter effect is enforced by the activation of metalloproteases released by neutrophils and by oxidation of protease inhibitors by oxygen metabolites. Therefore, an improper activation of the NADPH oxidase must be prevented at all costs and, when the infection has been cleared, a rapid deactivation mechanism is imperative. In this review, the interaction between the different components of the NADPH oxidase and the activation of these proteins will be discussed.
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PMID:Interactions between the components of the human NADPH oxidase: a review about the intrigues in the phox family. 915 14

The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 x 10(-2) nmol/min/10(6) cells (S.E.M. = +/-0.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox and p47-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.
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PMID:Detection of protein and mRNA of various components of the NADPH oxidase complex in an immortalized human chondrocyte line. 918 52


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