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:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Incorporation of the available data on rac in neutrophils, CDC42 in yeast, and rho in fibroblasts suggests a general model for the function of rho-like GTPase (Figure 1). Conversion of an inactive cytoplasmic rho-related p21GDP/GDI complex to active p21. GTP occurs by inhibition of GAP and/or stimulation of exchange factors in response to cell signals. p21.GTP is then able to interact with its target at the plasma membrane. This could result in a conformational change in the target, enabling it to bind cytosolic protein(s). Alternatively, p21.GTP could be actively involved in transporting cytosolic protein(s) to the target. A GAP protein, perhaps intrinsic to the complex, would stimulate GTP hydrolysis allowing p21.GDP to dissociate. Solubilization of p21GDP by interaction with GDI would complete a cycle. What about the nature of the final complex? The rac-regulated NADPH oxidase complex in neutrophils is currently the best understood and most amenable to further biochemical analysis. Two plasma-membrane bound subunits encode the catalytic function necessary for producing superoxide, but the two cytosolic proteins, p47 and p67, are essential for activity. Why the complexity? Production of superoxide is tightly coordinated with phagocytosis, a membrane process driven by rearrangement of cortical actin. This is not unrelated to the membrane ruffling and macropinocytosis that we observe in fibroblasts microinjected with p21rac. It is tempting to speculate, therefore, that in neutrophils rac is involved not only in promoting the assembly of the NADPH oxidase but also in the coordinate reorganization of cortical actin leading to phagocytosis. For CDC42 controlled bud assembly in yeast, the components of the plasma-membrane complex are not so clear. By analogy with rac in neutrophils, it seems likely that CDC42 is involved in promoting the assembly of cytosolic components at the bud site on the plasma membrane. These putative cytosolic proteins have not yet been identified, but BEM1 and ABP1 are two possible candidates. The biochemical basis for the stimulation of adhesion plaques and actin stress fibers by p21rho in fibroblasts is also unclear. However, components of the adhesion plaque such as vinculin and talin are known to be cytosolic when not complexed with integrin receptors, and rho could be involved in regulating their assembly into the adhesion plaque. Several things are still difficult to incorporate into this model. First the target for CDC42, the bud site, although not yet structurally defined requires the activity of another small GTPase, BUD1. Similarly, in activated neutrophils, the NADPH oxidase is found in a complex with rap1, the mammalian homologue of BUD1 (BoKoch et al., 1989). It seems likely, therefore, that the target is not simply a plasma-membrane protein but may be a complex of proteins whose formation is under the control of the rap1/BUD1 GTPase. The other black box in this model is the actin connection: activation of bud assembly by CDC42 is followed by actin polymerization, activation of NADPH oxidase in neutrophils occurs concomitantly with phagocytosis, a cortical actin-dependent process, and p21rho in fibroblasts couples the formation of adhesion plaques to actin stress fibers. One possible link between the GTPase-driven assembly of a plasma-membrane complex and actin polymerization could involve the SH3 domain. Interestingly, both p47 and p67 and yeast ABP1 and BEM1 have SH3 domain. If rho-like GTPases recognize plasma-membrane targets already associated with cortical actin, then this could promote an interaction with a subset of SH3-containing proteins. The result of this would be a GTPase-regulated aggregation of a group of proteins at a single site in the plasma membrane. It is not too difficult to imagine biological processes where such a spatial integration of different biochemical activities would be essential: coupling the assembly of bud components to the formation of actin fibers in yeast; or the activation of NADPH oxidase to phagocytosis in neutrophils; or the assembly of adhesion plaques and the formation of actin stress fibers in fibroblasts are just three examples that have emerged so far. In conclusion, although rho-like GTPases clearly have distinct roles in different mammalian cell types and in yeast, their underlying mechanism of action appears to be strikingly similar. Whether this will remain so when there are some biochemical data to back up these initial observations, time will tell.
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PMID:Ras-related GTPases and the cytoskeleton. 161 Nov 53

Activation of superoxide-producing NADPH oxidase of neutrophils requires the presence of cell membranes, cytosolic components and arachidonate and is markedly enhanced by non-hydrolysable analogues of guanine nucleotides, i.e. guanosine 5'-[gamma-thio]triphosphate and guanosine 5'[beta gamma-imido]triphosphate (p[NH]ppG). Gel filtration and ultrafiltration of the cytosol decreased the basal activity of NADPH oxidase. Activity could be restored by GTP, suggesting participation of the nucleotide in basal activation. Preincubation of neutrophil cytosol with periodate-oxidized p[NH]ppG (ox-p[NH]ppG) followed by gel filtration resulted in a time-dependent enhancement of basal oxidase activity. The presence of GDP or GTP, but not ATP, during the incubation with ox-p[NH]ppG abolished this enhancement. These data are consistent with a stable association of ox-p[NH]ppG with an oxidase-linked cytosolic protein. SDS/PAGE of neutrophil cytosol preincubated with [3H]ox-p[NH]ppG revealed radioactivity in bands migrating as 100, 70, 47, 34 and 22 kDa proteins. Evidence for covalent labelling of the cytosolic protein p47-phox with [3H]ox-p[NH]ppG is presented. Heterogeneity of cytosolic GTP-binding sites and possible participation of protein p47-phox in functional interaction with GTP analogues during cell-free activation are suggested.
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PMID:Involvement of GTP in cell-free activation of neutrophil NADPH oxidase. Studies with GTP analogues. 163 53

In neutrophils, receptor-mediated activation of the respiratory burst requires ATP, possibly for phosphotransferase reactions. The oxidative response is only partially inhibited by blockers of protein kinase C, suggesting the involvement of other kinases. Recent evidence has demonstrated activation of tyrosine phosphorylation in chemoattractant-stimulated cells. This effect is likely mediated by G proteins because it is obliterated by pretreatment with pertussis toxin. In this report we have attempted to correlate the respiratory burst and phosphotyrosine accumulation induced by activation of G proteins, accomplished by treatment of electroporated cells with nonhydrolyzable analogues of GTP. In cells stimulated with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) both responses displayed similar time course and concentration dependence. The guanine nucleotide selectivity sequence and the divalent cation requirements were also similar for both responses. These similarities suggest a relationship between tyrosine phosphorylation and the activation of the NADPH oxidase. GTP gamma S-induced phosphotyrosine accumulation was found to be inhibited by pretreatment of the cells with phorbol esters, underlining the existence of regulatory interactions between different signal transduction pathways in neutrophils.
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PMID:Tyrosine phosphorylation and oxygen consumption induced by G proteins in neutrophils. 170 84

When a particulate NADPH oxidase prepared from phorbol ester-activated human neutrophils was treated with pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP), the superoxide anion-producing activity was inhibited according to affinity labeling kinetics. NADPH afforded a protection against inactivation which was competitive with respect to PLP-AMP; 2',5'-ADP and 2'-phospho-5' diphosphoadenosine (ATP ribose) appeared to be as potent as NADPH as protecting agents. NADP+ and ATP were less effective, while ADP and GTP-gamma-S did not protect significantly. These results suggest that PLP-AMP can be used, in conjunction with tritiated cyanoborohydride, to identify the elusive NADPH-dependent flavoprotein which is part of the electron transfer chain of NADPH oxidase.
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PMID:Inactivation of NADPH oxidase from human neutrophils by affinity labeling with pyridoxal 5'-diphospho-5'-adenosine. 176 75

Activation of the membrane-associated NADPH oxidase in intact human neutrophils requires a receptor-associated heterotrimeric GTP-binding protein that is sensitive to pertussis toxin. Activation of this NADPH oxidase by arachidonate in a cell-free system requires an additional downstream pertussis toxin-insensitive G protein (Gabig, T. G., English, D., Akard, L. P., and Schell, M. J. (1987) (J. Biol. Chem. 262, 1685-1690) that is located in the cytosolic fraction of unstimulated cells (Gabig, T. G., Eklund, E. A., Potter, G. B., and Dykes, J. R. (1990) J. Immunol. 145, 945-951). In the present study, immunodepletion of G proteins from the cytosolic fraction of unstimulated neutrophils resulted in a loss of the ability to activate NADPH oxidase in the membrane fraction. The activity in immunodepleted cytosol was fully reconstituted by a partially purified fraction from neutrophil cytosol that contained a 21-kDa GTP-binding protein. Purified human recombinant Krev-1 p21 also completely reconstituted immunodepleted cytosol whereas recombinant human H-ras p21 or yeast RAS GTP-binding proteins had no reconstitutive activity. Rabbit antisera raised against a synthetic peptide corresponding to the effector region of Krev-1 (amino acids 31-43) completely inhibited cell-free NADPH oxidase activation, and this inhibition was blocked by the synthetic 31-43 peptide. An inhibitory monoclonal antibody specific for ras p21 amino acids 60-77 (Y13-259) had no effect on cell-free NADPH oxidase activation. Activation of the NADPH oxidase in intact neutrophils by stimulation with phorbol myristate acetate caused a marked increase in the amount of membrane-associated antigen recognized by 151 antiserum on Western blot. Thus a G protein in the cytosol of unstimulated neutrophils antigenically and functionally related to Krev-1 may be the downstream effector G protein for NADPH oxidase activation. This system represents a unique model to study molecular interactions of a ras-like G protein.
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PMID:Resolution of a low molecular weight G protein in neutrophil cytosol required for NADPH oxidase activation and reconstitution by recombinant Krev-1 protein. 190 90

Botulinum D toxin has been shown to ADP-ribosylate 22-kD proteins in neutrophilic leukocytes, but the function of these GTP-binding proteins remains unknown. In analogy to small GTP-binding proteins like SEC4 to YPT1, it has been suggested that botulinum D toxin substrates might be involved in secretory process of myeloid cells. Three main findings lead to the opposite conclusion. First of all, in human neutrophils, botulinum D toxin does not modify the release of azurophilic and specific granules induced by a chemoattractant (a formylpeptide) or a phorbol ester. Second, botulinum D toxin ADP-ribosylates 24 to 26-kD proteins that are only present in plasma membranes of human neutrophils. The membrane location of these substrates differs largely from that of the GTP-binding proteins involved in exocytosis and located in granules. Finally, since the same quantity of the toxin substrates is present in neutrophils as in their precursors, HL60 cells (which are devoid of specific granules and characterized by immature azurophilic granules and NADPH oxidase), it is unlikely that endogenous botulinum D toxin substrates are directly involved in the secretory responses of neutrophils.
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PMID:Effect of botulinum D toxin on human neutrophilic leukocytes and localization of its substrates. 196 13

Superoxide production by neutrophil NADPH oxidase activated in a cell-free system consisting of plasma membranes, cytosol and arachidonate is enhanced by nonhydrolyzable analogs of GTP and reduced by GDP. To characterize the interaction of guanine nucleotides with the system, dialdehyde analogs of GTP and GDP (oGTP and oGDP) were employed. oGDP or oGTP caused an irreversible and dose dependent inactivation of NADPH oxidase-supporting cytosolic activity. Cytosol was fractionated on S and Q Sepharose ion exchange columns into three fractions, combinations of which synergistically supported activation of NADPH oxidase. Two fractions shown by immunoblotting to contain the oxidase-linked p47 and p67 proteins were inactivated by oGDP. Labeling with [alpha-32P]-oGTP lead to incorporation of the label into several proteins.
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PMID:Dialdehyde-GDP blocks activity of cytosolic components of neutrophil NADPH oxidase. 204 36

The dormant O2(-)-generating oxidase in plasma membranes from unstimulated neutrophils becomes activated in the presence of arachidonate and a multicomponent cytosolic fraction. This process is stimulated by nonhydrolyzable GTP analogues and may involve a pertussis toxin insensitive GTP-binding protein. Our studies were designed to characterize the putative GTP-binding protein, localizing it to either membrane or cytosolic fraction in this system. Exposure of the isolated membrane fraction to guanosine-5'-(3-O-thio)triphosphate (GTP gamma S), with or without arachidonate, had no effect on subsequent NADPH oxidase activation by the cytosolic fraction. Preexposure of the cytosolic fraction to GTP gamma S alone did not enhance activation of the membrane oxidase. However, preexposure of the cytosol to GTP gamma S then arachidonate caused a four-fold enhancement of its ability to activate the membrane oxidase. This enhancement was evident after removal of unbound GTP gamma S and arachidonate, and was not augmented by additional GTP gamma S during membrane activation. A reconstitution assay was developed for cytosolic component(s) responsible for the GTP gamma S effect. Cytosol preincubated with GTP gamma 35S then arachidonate was fractionated by anion exchange chromatography. A single peak of protein-bound GTP gamma 35S was recovered that had reconstitutive activity. Cytosol preincubated with GTP gamma 35S alone was similarly fractionated and the same peak of protein-bound GTP gamma 35S was observed. However, this peak had no reconstitutive activity. We conclude that the GTP-binding protein regulating this cellfree system is located in the cytosolic fraction. The GTP gamma S-liganded form of this protein may be activated or stabilized by arachidonate.
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PMID:A neutrophil GTP-binding protein that regulates cell free NADPH oxidase activation is located in the cytosolic fraction. 211 50

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.
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PMID:Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate. 215 19

The superoxide generating NADPH oxidase was studied in an SDS-activated cell-free system. This system requires the participation of both membranal and cytosolic components. Cytosol derived from elicited peritoneal guinea pig macrophages was fractionated by several nucleotide affinity chromatography procedures. Various such fractionations led to the separation of two distinct factors, both of which are necessary for the activation and/or activity of the superoxide-forming NADPH oxidase. One factor (sigma 2), bound to octyl, 2',5'-ADP-, 5'-ATP-, 5'-GTP-agarose and carboxymethyl-Sepharose but did not bind to hexyl, 5'-AMP-, 5'-ADP- and 5'-GDP-agarose. The other factor (sigma 1) did not bind to any of the above matrices. Subsequent elution of sigma 2 from 2',5'-ADP-agarose was effected by ATP, GTP and NADPH but not by NADH. Elution from GTP-agarose was by ATP and GTP but not by NADPH. Elution from ATP-agarose was by ATP, GTP and also, albeit weakly, by NADPH. The above results suggest that sigma 2 contains a site which recognizes the phosphate group at the ribose 2' position in adenosine, and a site that recognizes purine nucleotide triphosphates.
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PMID:Nucleotide binding properties of cytosolic components required for expression of activity of the superoxide generating NADPH oxidase. 215 58


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