Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.3.1 (NADPH oxidase)
 11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

p40(phox) of the phagocyte NADPH oxidase forms a complex with p67(phox) in cytosol, and coincidentally decreases in patients who lack p67(phox). Here we investigated the mode of translocation of p40(phox) to the membrane, its cytoskeletal localization on activation of the NADPH oxidase, and the dependency of its expression relative to that of p67(phox). When human polymorphonuclear leukocytes (PMNs) were stimulated with phorbol myristate acetate (PMA), p40(phox) was translocated to the membrane along with p67(phox), and not was released into the cytosol. Studies with resting PMNs using Triton X-100 revealed the exclusive localization of p67(phox) in the cytoskeletal fraction. Unexpectedly, however, about half of p40(phox), which is deemed to be fully associated with p67(phox), was recovered in the non-cytoskeletal fraction. Unlike p47(phox), the association of p40(phox) with cytoskeleton was not induced by the PMA-stimulation. These results indicate not only that p40(phox) associates with cytoskeleton via a molecule of p67(phox), but also that there are distinct states of p40(phox) that can be manipulated with Triton X-100. Lastly, Western-blot analysis of hematopoietic cells revealed no correlation between p40(phox) and p67(phox) in their protein expressions during cell differentiation, and also that p40(phox) can be stably present alone in cells, unless in the case of mature PMNs. In this regard, definitive proof was obtained with Epstein-Barr virus-transformed B cells of a p67(phox)-deficient patient, in which p40(phox) was normally expressed.
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PMID:Relationships of p40(phox) with p67(phox) in the activation and expression of the human respiratory burst NADPH oxidase. 1105 90

Preparations of plasma membranes isolated from cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells synthesized O2- when incubated with either NADH or NADPH, as measured by an O2--specific assay based on the chemiluminescence of lucigenin. The activities were strongly dependent on the presence of Triton X-100. The Km for NADH was 159 [mu]M; that for NADPH was 19 [mu]M. Neither NADH- nor NADPH-dependent activity was inhibited by azide, an inhibitor of peroxidase, nor by antimycin A, an inhibitor of mitochondrial electron transport; both activities were inhibited by 30 to 100 nM diphenylene iodonium, an inhibitor of the mammalian NADPH oxidase. The NADH- and NADPH-dependent activities could be distinguished by detergent solubilization and ultracentrifugation: the NADH-dependent activity sedimented more easily, whereas the NADPH-dependent activity remained in suspension. One or both of these enzymes may provide the O2- seen when plant cells are exposed to pathogens or pathogen-associated elicitors; however, plasma membranes from rose cells treated with a Phytophthora elicitor had the same activity as control cells.
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PMID:The Superoxide Synthases of Plasma Membrane Preparations from Cultured Rose Cells. 1222 8

Angiotensin II (Ang II) regulates vascular smooth muscle cell (VSMC) function by activating signaling cascades that promote vasoconstriction, growth, and inflammation. Subcellular mechanisms coordinating these processes are unclear. In the present study, we questioned the role of the actin cytoskeleton in Ang II mediated signaling through mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS) in VSMCs. Human VSMCs were studied. Cells were exposed to Ang II (10-7 mol/L) in the absence and presence of cytochalasin B (10-6 mol/L, 60 min), which disrupts the actin cytoskeleton. Phosphorylation of p38MAP kinase, JNK, and ERK1/2 was assessed by immuno blotting. ROS generation was measured using the fluoroprobe chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (4 micromol/L). Interaction between the cytoskeleton and NADPH oxidase was determined by evaluating the presence of p47phox in the Triton X-100 insoluble membrane fraction. Ang II significantly increased phosphorylation of p38MAP kinase, JNK, and ERK1/2 (two- to threefold above control, p < 0.05). Cytochalasin B pretreatment attenuated p38MAP kinase and JNK effects (p < 0.05) without altering ERK1/2 phosphorylation. ROS formation, which was increased in Ang II stimulated cells, was significantly reduced by cytochalasin B (p < 0.01). p47phox, critically involved in NADPH oxidase activation, colocalized with the actin cytoskeleton in Ang II stimulated cells. Our data demonstrate that Ang II mediated ROS formation and activation of p38MAP kinase and JNK, but not ERK1/2, involves the actin cytoskeleton in VSMCs. In addition, Ang II promotes interaction between actin and p47phox. These data indicate that the cytoskeleton is involved in differential MAP kinase signaling and ROS generation by Ang II in VSMCs. Together, these studies suggest that the cytoskeleton may be a central point of crosstalk in growth- and redox-signaling pathways by Ang II, which may be important in the regulation of VSMC function.
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PMID:Role of the actin cytoskeleton in angiotensin II signaling in human vascular smooth muscle cells. 1575 55

Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X-100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)-anchored neutrophil protein, possibly FcgammaRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcgammaRIIIb, the beta2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcgammaRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcgammaRIIIb acts as a membrane adaptor for PR3. FcgammaRIIIb of HMW fractions co-immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo-NHS-biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58-lysed neutrophils were similar in their protein composition to the HMW complexes but not identical.
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PMID:Membrane proteinase 3 and its interactions within microdomains of neutrophil membranes. 1659 72

Some properties of microsomal electron transfer chains, dependent for oxidase activity on addition of NADH or NADPH, duroquinone, and oxygen (L. De Luca et al., 1984, Plant Sci Lett 36: 93-98) are described. Activity is characterized by negatively cooperative kinetics toward reduced pyridine nucleotides, with limiting K(m) of 10 to 50 micromolar at pH 7.0 (increasing at lower pH), as well as toward duroquinone with limiting K(m) of 100 to 400 micromolar regardless of pH. Molecular oxygen is reduced by the enzyme complex with S(0.5) of about 30 micromolar and production of H(2)O and H(2)O(2), without superoxide involvement. The ratio NAD(P)H:O(2) averages 1.35 in the presence of KCN and 1.85 in its absence. The pyridine nucleotide specificity of the dehydrogenases has been investigated by kinetic competition experiments. Some enzyme heterogeneity was established for all preparations. At least two enzymes are detectable in plasma membrane-enriched fractions: a major NAD(P)H dehydrogenase having an acid pH optimum, and an NADPH dehydrogenase active around neutrality. Addition of Triton X-100 strongly enhances the activity over most of the pH scale, but depresses it increasingly at pH values higher than 8.0, to the effect that pH profile shows, under these conditions, a major peak at about pH 5.8 for both NADH and NADPH oxidase. Results with endoplasmic reticulum preparations are similar, except that they suggest the presence of still more activities at and above pH 7. The results are interpreted in terms of different complexes catalyzing electron transfer from NAD(P)H to O(2) without release of intermediates.
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PMID:Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes. 1666 30

The phagocyte oxidase (Phox) protein p40(phox) contains a Phox homology (PX) domain which, when expressed alone, interacts with phosphatidylinositol 3-phosphate (PtdIns (3)P). The functions of the PX domain in p40(phox) localization, association with the cytoskeleton, and superoxide production were examined in transgenic COS-7 cells expressing gp91(phox), p22(phox), p67(phox), and p47(phox) (COS(phox) cells). Full-length p40(phox) exhibited a cytoplasmic localization pattern in resting cells. Upon stimulation with phorbol 12-myristate 13-acetate or fMet-Leu-Phe, p40(phox) translocated to plasma membrane in a p67(phox)- and p47(phox)-dependent manner. Heterologous expression of p40(phox) markedly enhanced superoxide production in phorbol 12-myristate 13-acetate - and fMet-Leu-Phe-stimulated COS(phox) cells. Unexpectedly, mutation of Arg-57 in the PX domain to Gln, which abrogated PtdIns (3)P binding, produced a dominant inhibitory effect on agonist-induced superoxide production and membrane translocation of p47(phox) and p67(phox). The mutant p40(phox) (p40R57Q) displayed increased association with actin and moesin and was found enriched in the Triton X-100-insoluble fraction along with p67(phox) and p47(phox). The enhanced cytoskeleton association of p67(phox) and p47(phox) and the dominant inhibitory effect produced by the p40R57Q were alleviated when a second mutation at Asp-289, which eliminated p40(phox) interaction with p67(phox), was introduced. Likewise, cytochalasin B treatment abolished the dominant inhibitory effect of p40R57Q on superoxide production. These findings suggest a dual regulatory mechanism through the PX domain of p40(phox); its interaction with the actin cytoskeleton may stabilize NADPH oxidase in resting cells, and its binding of PtdIns (3)P potentiates superoxide production upon agonist stimulation. Both functions require the association of p40(phox) with p67(phox).
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PMID:Characterization of a mutation in the Phox homology domain of the NADPH oxidase component p40phox identifies a mechanism for negative regulation of superoxide production. 1769 49

In murine ventricular myocytes, activation of mechanosensitive ion channels (MSCs) includes activation of non-selective cation currents and deactivation of inwardly rectifying potassium currents. Using pharmacological inhibitors and knockout models, we analyzed signaling steps that are critical to transduce the mechanical signal (stretch) into electrophysiological events (MSC). We provide evidence for an activation of NAD(P)H oxidase and NOS3 in response to stretch putatively via the angiotensin II receptor type 1. The involvement of superoxide and nitric oxide was verified by the block of MSC using specific scavengers (tiron and PTIO, respectively). Superoxide and nitric oxide are known to combine very rapidly to form peroxynitrite. Accordingly, MSC were blocked by the peroxynitrite scavenger uric acid and could be mimicked by application of exogenous peroxynitrite. Peroxynitrite formation may activate phospholipases generating amphipaths that modulates channel function via changing the curvature of the surrounding lipid bilayer. This conclusion is supported by our findings that MSC were suppressed by inhibitors of phospholipases but could be mimicked by exogenous phospholipases or by amphipaths (oleic acid, Triton X-100).
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PMID:Modulation of cardiac mechanosensitive ion channels involves superoxide, nitric oxide and peroxynitrite. 1863 30

As recently demonstrated, angiotensin II (Ang II) induces an increase in myocardial distensibility. Although endothelin-1 and the endocardial endothelium (EE) also modulate myocardial diastolic properties, their interaction with Ang II at this level has not yet been investigated. Increasing concentrations of Ang II (from 10(-8) to 10(-5) M) were studied in rabbit right papillary muscles in the following conditions: (1) baseline; (2) after selective removal of EE with Triton X-100; and (3) with intact EE in presence of a non-selective endothelin receptor antagonist (PD-145065), a selective endothelin type A receptor antagonist (BQ-123), an inhibitor of nitric oxide synthesis (N(G)-nitro-L-arginine (L-NA) or an inhibitor of the NAD(P)H oxidase (apocynin). At baseline, Ang II induced a concentration-dependent positive inotropic effect and an increase in passive muscle length (L) up to 1.020 +/- 0.004 L/L(max). After restoring muscle length to maximal physiological length (L(max)), passive tension decreased by 46.1 +/- 4.0%. When the EE was removed, the effect on myocardial distensibility was abolished. With intact EE in presence of PD-145065, BQ-123 or L-NA, the effects of Ang II on myocardial distensibility were attenuated, with a maximal increase in passive muscle length of 1.0087 +/- 0.0012, 1.0068 +/- 0.0022 and 1.0066 +/- 0.0020 L/L(max) and a decrease in resting tension of 22.6 +/- 3.6, 16.1 +/- 6.0 and 20.4 +/- 5.6%, respectively. In the presence of apocynin, the effect on myocardial distensibility was abolished. In conclusion, the Ang II-dependent acute increase in myocardial distensibility is abolished by the selective removal of the EE and attenuated in the presence of endothelin-1 receptor antagonists, an inhibitor of nitric oxide synthesis or an inhibitor of NAD(P)H oxidase.
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PMID:Angiotensin II-induced increase in myocardial distensibility and its modulation by the endocardial endothelium in the rabbit heart. 1934 35

OsRac1, a member of the Rac/Rop GTPase family, plays important roles as a molecular switch in rice innate immunity, and the active form of OsRac1 functions in the plasma membrane (PM). To study the precise localization of OsRac1 in the PM and its possible association with other signaling components, we performed proteomic analysis of DRMs (detergent-resistant membranes) isolated from rice suspension-cultured cells transformed with myc-tagged constitutively active (CA) OsRac1. DRMs are regions of the PM that are insoluble after Triton X-100 treatment under cold conditions and are thought to be involved in various signaling processes in animal, yeast and plant cells. We identified 192 proteins in DRMs that included receptor-like kinases (RLKs) such as Xa21, nucleotide-binding leucine-rich repeat (NB-LRR)-type disease resistance proteins, a glycosylphosphatidylinositol (GPI)-anchored protein, syntaxin, NADPH oxidase, a WD-40 repeat family protein and various GTP-binding proteins. Many of these proteins have been previously identified in the DRMs isolated from other plant species, and animal and yeast cells, validating the methods used in our study. To examine the possible association of DRMs and OsRac1-mediated innate immunity, we used rice suspension-cultured cells transformed with myc-tagged wild-type (WT) OsRac1 and found that OsRac1 and RACK1A, an effector of OsRac1, shifted to the DRMs after chitin elicitor treatment. These results suggest that OsRac1-mediated innate immunity is associated with DRMs in the PM.
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PMID:Proteome analysis of detergent-resistant membranes (DRMs) associated with OsRac1-mediated innate immunity in rice. 1950 82


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