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)

Superoxide generation by phagocytic cells requires assembly of the enzymatic complex NADPH oxidase, consisting of membranous and cytosolic components which translocate to the plasma membrane upon cell activation. Two highly homologous cytosolic small GTP-binding proteins, rac1 and rac2, have recently been implicated in the activation of NADPH oxidase. We report that, in resting neutrophils, the amount of rac detectable in the plasma membrane-enriched fraction accounted for 1.5% of the cytosolic protein. When the O2- generation was induced by stimulating neutrophils with PMA, fMLP or opsonized zymosan rac proteins were not recruited at the plasma membrane as analysed by immunoblot or immunofluorescence assays. We conclude that rac proteins do not assemble with the NADPH oxidase complex in the plasma membrane, and we propose that they might instead transduce translocation signals to the other cytosolic components.
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PMID:The small GTP-binding protein rac is not recruited to the plasma membrane upon NADPH oxidase activation in human neutrophils. 811 79

Phagocytes produce superoxide by the assembly of a multicomponent complex that utilizes NADPH for the reduction of molecular oxygen (NADPH oxidase). The components participating in the assembly are a membrane-bound flavocytochrome and three cytosolic proteins, one of which was shown to be a dimer of the small GTP-binding protein (G protein) Rac1 p21 or Rac2 p21 with GDP dissociation inhibitor for Rho (Rho GDI). We determined the identity and quantity of the nucleotide bound to Rac1 p21 by high performance anion exchange chromatography of extracts prepared from highly purified Rac1 p21-Rho GDI, isolated from guinea pig macrophage cytosol. Rac1 p21 contained only GDP at a ratio of close to 1 mol of GDP per mol of G protein. The GDP-bound form of Rac1 p21 complexed to Rho GDI functioned as a potent activator of NADPH oxidase in a cell-free system that contained no free GTP or ATP. We propose that the GDP-bound form of Rac1 p21 might be the physiological activator of NADPH oxidase in macrophages, following its dissociation from Rho GDI, and that nucleotide exchange or conversion to GTP is not necessarily involved.
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PMID:The GDP-bound form of the small G protein Rac1 p21 is a potent activator of the superoxide-forming NADPH oxidase of macrophages. 812 10

Activation of the NADPH oxidase of phagocytes involves the small GTP-binding protein p21rac. In this paper we report that neutrophil cytosol contains predominantly p21rac2 rather than p21rac1, and that the P21rac2 is almost entirely complexed with rhoGDI (GDP dissociation inhibitor) to form a heterodimer with a molecular mass of 45-50 kDa. Activation of superoxide production by phorbol 12-myristate 13-acetate or formylmethionyl-leucyl-phenylalanine in whole cells, and by SDS in the cell-free assay, led to the dissociation of some of the p21rac2 from rhoGDI and its movement to the plasma membrane together with p47phox and p67phox. The appearance of these proteins at the plasma membrane was related to the dose of the agonist and to the rate of superoxide generation. The nucleotide bound to p21rac2 in this complex following isolation was almost exclusively GDP, with less than 2% GTP, and the complex was active in the cell-free assay. Although the rac/GDI complex could activate the NADPH oxidase in the absence of exogenous GTP, the rate of superoxide production was increased 3-fold by the addition of GTP and was almost completely inhibited by GDP. Our findings confirm that rhoGDI serves as GDP dissociation inhibitor and that the release of p21rac2 from this inhibitor is an important step in activation of the NADPH oxidase.
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PMID:Activation of NADPH oxidase involves the dissociation of p21rac from its inhibitory GDP/GTP exchange protein (rhoGDI) followed by its translocation to the plasma membrane. 814 70

Recent progress in understanding the regulation of the phagocyte NADPH oxidase by the Rac GTP-binding protein(s) provides the first detailed glimpse into the mechanisms of leukocyte regulation by a small GTP-binding protein. Studies over the past year indicate that the activity of NADPH oxidase can be modulated by regulation of the GTP- versus GDP-bound state of Rac. Additional proteins of the Ras superfamily are likely to be involved in a variety of normal leukocyte functions.
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PMID:The role of small GTP-binding proteins in leukocyte function. 817 86

The highly regulated enzyme HMG-CoA reductase generates mevalonate, the precursor of a complex series of isoprenoids that posttranslationally modify (isoprenylate) certain proteins (e.g., the low-molecular-weight GTP-binding proteins) or that are incorporated into cholesterol and other end products. We recently reported that isoprenoids are required for NADPH oxidase activity in granulocytes via LMW GTP-binding protein isoprenylation. In this study, we evaluated the effects of isoprenoid depletion on the expression of proinflammatory genes in human monocytic THP-1 cells. We selected conditions under which pretreatment for 24 h with isoprenoid synthesis inhibitors (HMG-CoA reductase inhibitor lovastatin or compactin at 10 microM) did not compromise cell viability but markedly suppressed H2O2 generation. Under these conditions interleukin-8 (IL-8) production was attenuated (by 50-90%) in response to lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, and phorbol myristate acetate. Coincubation of reductase inhibitor-treated cells with mevalonate prevented the attenuation of IL-8 production by reductase inhibitors. The effects of isoprenoid depletion on cytokine production were selective: IL-1 beta generation was not inhibited but the production of IL-6 and IL-8 was concomitantly suppressed. IL-8 induction was suppressed at least in part through attenuation of the increase in mRNA in stimulated cells. We conclude that isoprenoid generation through the mevalonate pathway is a requirement for IL-8 induction by activated monocytic cells in vitro. Isoprenylation inhibitors have the potential to alter monocyte proinflammatory function.
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PMID:Role of the mevalonate pathway of isoprenoid synthesis in IL-8 generation by activated monocytic cells. 819 1

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages. 822 83

Members of the Rho family of GTP-binding proteins are localized in the cytosol of cells by complexation with a protein known as (Rho)GDI. We show by sucrose gradient equilibrium sedimentation analysis that all of the Rac protein present in human neutrophil cytosol exists as a complex with (Rho)GDI under non-activating conditions. This interaction can be disrupted in the presence of various lipids which have been shown to have biological activity in a variety of systems, including NADPH oxidase activation. Particularly effective were arachidonic acid, phosphatidic acid, and phosphatidylinositols. These lipids were active at concentrations from 0.5-50 microM and were capable of disrupting complexation of (Rho)GDI with both GDP- and GTP-bound forms of Rac, although the latter were more sensitive to lipid. These data suggest that certain lipids generated in chemoattractant-stimulated neutrophils may play a role in modulating the activity of Rac and thus NADPH oxidase activity.
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PMID:Biologically active lipids are regulators of Rac.GDI complexation. 825 41

To investigate a possible role of phospholipase A2 (PLA2) in the respiratory burst in bovine eosinophilic and neutrophilic leukocytes dependent on GTP-binding protein (G-protein), we permeabilized these cells with Staphylococcus aureus alpha-toxin and induced NADPH oxidase activity with the non-hydrolysable GTP analogue GTP[S] or the aluminium tetrafluoro complex AlF4-. Under same experimental conditions, cells responded with different onset times. The onset time for eosinophils was 50-200 s, for neutrophils it was only a few seconds. GTP[S] stimulated in neutrophils only 5% of the respiratory burst compared to eosinophils, whereas AlF4(-)-induced comparable responses (neutrophils 120% of eosinophils). GDP inhibited these responses with an IC50 value of 2.4 mM. Arachidonic acid showed, with the exception of AlF4- stimulated neutrophils, on both stimuli and cell types an enhancing effect (150%) that reached its maximum at 0.1-1 microM. The PLA2 inhibitor 4-bromophenacylbromide reduced the GTP[S]- and AlF4(-)-induced response almost completely (10 microM) and the inhibition was not significantly different for eosinophils and neutrophils (IC50 1-3 microM). If the respiratory burst was reduced with 4-bromophenacylbromide to 1-4% of the original value, 10% of the basal NADPH oxidase activity could be restored by addition of only 20-100 nM arachidonic acid. In addition, the PLA2 activator adriamycin enhanced the response in a dose-dependent manner and in the same order as arachidonic acid did. The results presented above suggest that the respiratory burst may be regulated by different low-molecular-mass and/or heterotrimeric G-proteins and an active role for arachidonic acid or its metabolites in the activation and the maintenance of the direct G-protein-stimulated respiratory burst in bovine eosinophils and neutrophils.
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PMID:Nanomolar arachidonic acid influences the respiratory burst in eosinophils and neutrophils induced by GTP-binding protein. A comparative study of the respiratory burst in bovine eosinophils and neutrophils. 826 58

The mechanisms used by phagocytic leukocytes in the process of bacterial killing are regulated by GTP-binding proteins of the Ras superfamily. In particular, the formation of toxic oxygen metabolites via the NADPH oxidase requires the action of both Rac and Rap1A proteins. Rac2 forms a third cytosolic component of the human neutrophil NADPH oxidase. Rac2 is active in its GTP-bound form, and requires post-translational processing (isoprenylation) in order to interact with regulatory proteins which stimulate the exchange of GTP for GDP. In the resting neutrophil, Rac is localized to the cytosol in the form of a complex with a GDP dissociation inhibitor (GDI) protein. Upon cell activation, this complex is disrupted to enable Rac to translocate to the active oxidase at the plasma membrane. The Rac-GDI complex may be regulated by the release of specific lipids known to be generated during phagocyte activation.
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PMID:Regulation of phagocyte function by low molecular weight GTP-binding proteins. 828 94

Activation of the NADPH oxidase of phagocytic cells requires the action of Rac2 or Rac1, members of the Ras superfamily of GTP-binding proteins. Rac proteins are active when in the GTP-bound form and can be regulated by a variety of proteins that modulate the exchange of GDP for GTP and/or GTP hydrolysis. The p190 Rac GTPase Activating Protein (GAP) inhibits human neutrophil NADPH oxidase activity in a cell-free assay system with a K1 of approximately 100 nM. Inhibition by p190 was prevented by GTP gamma S, a nonhydrolyzable analogue of GTP. Similar inhibition was seen with a second protein exhibiting Rac GAP activity, CDC42Hs GAP. The effect of p190 on superoxide (O2-) formation was reversed by the addition of a constitutively GTP-bound Rac2 mutant or Rac1-GTP gamma S but not by RhoA-GTP gamma S. Addition of p190 to an activated oxidase produced no inhibitory effect, suggesting either that p190 no longer has access to Rac in the assembled oxidase or that Rac-GTP is not required for activity once O2- generation has been initiated. These data confirm the role of Rac in NADPH oxidase regulation and support the view that it is the GTP form of Rac that is necessary for oxidase activation. Finally, they raise the possibility that NADPH oxidase may be regulated by the action of GAPs for Rac proteins.
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PMID:Regulation of NADPH oxidase activity by Rac GTPase activating protein(s). 830 40


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