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 neutrophil NADPH oxidase activation factors, p47, p67 and the small guanosine-nucleotide-binding regulatory (G) protein Rac1, were expressed in a baculovirus/insect cell system and purified. In coinfection experiments in which Sf9 cells overexpressed concomitantly p47, p67 and Rac1, the latter was not detected in the p47-p67 complex. The propensity of p47 and p67 to associate together was used to purify recombinant p67 from baculovirus-infected Sf9 cells. 20% of the overexpressed Rac1 in infected Sf9 cells was prenylated and was extracted with low doses of detergent from membranes. Elicitation of full oxidase activity on crude neutrophil membranes using a cell-free system required addition of recombinant p47 and p67, but not that of Rac. In contrast, in the case of KCl-washed membranes, addition of Rac, prenylated or unprocessed, together with p47 and p67 was found to enhance oxidase activation up to fivefold. In all experiments, the amount of added arachidonic acid was optimized. In contrast to prenylated Rac, non-prenylated Rac had to be loaded with guanosine 5'-(3-thiotriphosphate) (GTP[S]) to exhibit full activation efficiency. In the cell-free system used, Rac was shown to be the mediator of the GTP[S] effect. The results suggest that the plasma membrane of resting neutrophils contains a sufficient amount of prenylated Rac for efficient oxidase activation. We therefore propose that Rac has a membrane-associated role and helps to dock and position p47 and p67 on the flavocytochrome b component of the oxidase complex.
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PMID:Activation of the O2(-)-generating NADPH oxidase in a semi-recombinant cell-free system. Assessment of the function of Rac in the activation process. 800 73

The production of free oxygen radicals by polymorphonuclear cells (PMNs) was studied in 25 patients after blunt trauma. Superoxide generation significantly increased immediately after trauma and returned to normal soon after the event. Patients were subsequently divided into two groups: those who developed sepsis and those who did not develop infectious complications. Superoxide production by intact PMNs following stimulation by three different stimulants was initially not different in trauma patients who developed sepsis. Follow-up showed an increase in superoxide production when infection complicated the course of trauma patients. Further studies were performed in a cell-free system containing cell membranes and cytosol from patients or healthy controls. No difference in the production of superoxide was found when membranes from trauma patients or controls were mixed with cytosols from controls. When cytosols from patients were mixed with membranes from controls, a significant increase in superoxide production was observed in the group that developed sepsis. Immunoblotting analysis of two protein components of the cytosolic portion of the NADPH oxidase, p47 and p67, were done. The increase in quantity of p47 correlated with the increase in superoxide production during sepsis, and thus may be the major contributor to the high activity.
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PMID:Superoxide production by neutrophils from trauma patients: regulation of NADPH oxidase activity. 802 54

The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome b558, composed of 91-kDa (gp91phox) and 22-kDa (p22phox) subunits, and two cytosolic factors (p47phox and p67phox), each containing two Src homology 3 (SH3) domains. Here we show that the region of the tandem SH3 domains of p47phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22phox and p67phox but fails to interact with a mutant p22phox carrying a Pro-156-->Gln substitution in a proline-rich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.
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PMID:Role of Src homology 3 domains in assembly and activation of the phagocyte NADPH oxidase. 820 90

We have presented evidence that rap1b, a 22 kDa low molecular weight GTP binding protein, becomes associated with the cytoskeleton in thrombin-activated platelets. The initial incorporation is very rapid and occurs as fast as we can measure it. Thus, some rap1b is associated with the cytoskeleton as fast as it is formed. The remainder of the rap1b is incorporated more slowly. This biphasic incorporation of rap1b is similar to the incorporation of GPIIb/IIIa into the cytoskeleton, but no interaction between GPIIb/IIIa and rap1b could be demonstrated. Phosphorylation of rap1b by cAMP-dependent protein kinase did not inhibit its association with the cytoskeleton. We conclude that rap1b is one of an increasing number of proteins that associate with the cytoskeleton during cell activation. The function of rap1b in the cytoskeleton is unclear at this time. However, it is possible to speculate on potential roles. There is growing evidence that low molecular weight G proteins participate in the formation of multi-molecular aggregates. For example, p21rac promotes the assembly of a membrane-associated complex composed of NADPH oxidase, p47, and p67 and this complex is important for activation of NADPH oxidase in neutrophils. Similarly, in yeast, BUD1, a homolog of rap1, forms a complex with BUD5 (a homolog of GDI), BEMI, CDC24, and CDC42 (a homolog of G25K). This multi-protein aggregate may be important in cytoskeletal structure in yeast. In platelets, rad1b, which is membrane associated, may promote the assembly of a complex of proteins during cell activation and may localize this complex to the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Cytoskeletal interactions of Rap1b in platelets. 820 87

Phospholipase A2 (PLA2) inhibitors suppressed simultaneously, in a dose-dependent manner, the activation of NADPH oxidase and the release of 3H-labelled arachidonic acid ([3H]AA) stimulated by either phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ) in human neutrophils. In spite of total inhibition of superoxide production in the presence of the PLA2 inhibitors, 10 microM bromophenacyl bromide (BPB) or 20 microM quinacrine, a maximal phosphorylation of p47 and translocation of p47 and p67 to the neutrophil membranes induced by PMA or OZ was observed. Addition of 10 microM free AA, which by itself did not stimulate superoxide generation, restored oxidase activity in neutrophils treated with PLA2 inhibitors. These findings indicate that phosphorylation and translocation of the cytosolic factors to the membranes are not sufficient for generating superoxide; a functional PLA2 is also needed to stimulate the oxidase activity. The inhibition of PLA2 activity did not prevent the phosphorylation of p47, suggesting that the location of PLA2 is downstream of and does not activate protein kinase C.
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PMID:The requirement for phospholipase A2 for activation of the assembled NADPH oxidase in human neutrophils. 828 Jan 2

Stimulated superoxide generation was 2-fold higher in neutrophils from 20 rats with common bile-duct ligation (CBDL) compared to that of 20 sham-operated control rats. In order to study the mechanism of the higher NADPH oxidase activity in CBDL rats, the kinetic parameters of NADPH oxidase were analyzed. The Vmax of the NADPH oxidase in CBDL rat neutrophils was significantly higher than that of control rat neutrophils (10.2 and 5.3 nmol/min, respectively). The membrane and cytosol fractions of the oxidase were studied in a cell-free system. Neutrophil cytosol from CBDL rats added to neutrophil membranes from either CBDL or control rats produced 22.4 +/- 1.6 and 21.0 +/- 1.4 nmol/10(6) cells per 10 min, respectively. When neutrophil cytosol from control rats was mixed with neutrophil membranes from control or CBDL rats the generation of superoxide was 10.6 +/- 1.4 and 10.0 +/- 1.5 nmol/10(6) cells per 10 min, respectively. These results suggest that the cytosol components of the oxidase regulate its activity. By immunoblot analysis it was shown that the amount of the cytosolic factor p47 in neutrophils of CBDL rats is higher than that present in an equal number of neutrophils from control rats.
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PMID:Elevated NADPH-oxidase activity in neutrophils from bile-duct-ligated rats: changes in the kinetic parameters and in the oxidase cytosolic factor p47. 830 98

Erythropoietin (Epo)-producing hepatoma cells (HepG2) reveal, in addition to the cytochromes of the respiratory chain, a photometrically measurable haem signal with absorbance maxima at 559 nm and 427 nm, suggesting the presence of a b-type cytochrome. This activity exhibited a low midpoint potential, CO-binding spectra and reduction which was insensitive to both cyanide and antimycin. This haem possessed a 22 kDa subunit and might be part of an electron transfer chain similar to the NADPH oxidase, since the NADPH oxidase cytosolic activating factor (p47) could be identified by Western blot analysis. H2O2, which was detected inside the cells by confocal microscopy, might therefore be produced by the suggested electron transfer chain. This cyanide- and antimycin-insensitive but hypoxia-sensitive cytochrome b would be an attractive candidate for controlled Epo production in response to pO2.
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PMID:Photometric characteristics of haem proteins in erythropoietin-producing hepatoma cells (HepG2). 838 44

The O2- generating NADPH oxidase of human Epstein-Barr virus immortalized B lymphocytes (EBV-B lymphocytes) and the NADPH oxidase of human neutrophils were compared. The capacity of the oxidase of EBV-B lymphocytes to generate O2- is 100-fold less than that of neutrophils. Like the oxidase of neutrophils, the oxidase of EBV-B lymphocytes is decreased or abolished in chronic granulomatous disease (CGD). Activation of neutrophil oxidase in an heterologous cell-free system, using human neutrophil membranes and EBV-B lymphocyte cytosol from healthy and CGD patients, combined with immunoblotting investigations of the cytosolic activating factors p47 and p67 involved in O2- production, suggests that neutrophils and EBV-B lymphocytes possess similar complements of cytosolic factors p47 and p67. Cytochrome b -245, the major membrane redox component of the O2- generating oxidase, is only slightly expressed in the membrane of EBV-B lymphocytes. A sensitive and specific immunocytochemical method for detection of the two subunits of cytochrome b -245 is described; it shows that both subunits are virtually absent in EBV-B lymphocytes from CGD patients deficient in the large subunit.
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PMID:The O2- generating oxidase of B lymphocytes: Epstein-Barr virus-immortalized B lymphocytes as a tool for the identification of defective components of the oxidase in chronic granulomatous disease. 839 41

The NADPH oxidase of phagocytic cells is a multimeric enzyme complex activated during phagocytosis. It catalyzes the production of the superoxide anion, precursor of many toxic oxygen metabolites involved in the defense against microorganisms. The enzyme becomes active after assembly on a membrane bound flavocytochrome b of cytosolic factors p47 phox, p67 phox and p40 phox and of low molecular mass GTP binding proteins. This paper reviews recent results concerning the role of two small G proteins, Rac and Rap 1A in oxidase activation. Native prenylated small G proteins are either in the form of a complex in which the GDP bound G protein is associated with a guanine nucleotide dissociation inhibitor, GDI, or in an active GTP bound form able to trigger the activity of its effector. Rac and Rho share a common GDI. As chemotaxis, under Rho control, and oxidase activation, under Rac control, show mutually exclusive signalling pathways, we propose a model where the GDI would switch from one pathway to the other by sequestering either Rac or Rho.
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PMID:Small G proteins and the neutrophil NADPH oxidase. 858 75

The latent NADPH oxidase activity of purified cytochrome b(558) from rabbit peritoneal neutrophils was expressed in a cell-free system consisting of either gel-filtrated cytosol from resting neutrophils, or a mixture of the three cytosolic activation factors, namely p47, p67 and the G protein Rac1. The cell-free system was supplemented with arachidonic acid and GTPgammaS. With gel-filtrated cytosol, the oxidase activity was relatively high (22 moles O(2)(-)/s/mole heme b in the absence of added FAD), and enhanced by less than one fourth upon addition of FAD. In contrast, with the purified cytosolic activation factors the rate of O(2)(-) production was low (8 moles O(2)(-)/s/mole heme b), and enhanced more than two-fold by a saturating concentration of FAD. The specificity of FAD was demonstrated by the lack of effect of FMN. FAD was determined together with heme b and the oxidase activity in eluates from a Sepharcryl column at the last step of the purification of cytochrome b(558). In the eluted fraction that contained both the maximal inducible oxidase activity and the highest amount of heme b, the molar amount of FAD was 20 times less than that of heme b. It is concluded that cytochrome b(558) is an NADPH-dependent flavocytochrome oxido-reductase (NADPH oxidase) in which one part of FAD is firmly bound and another, loosely attached. On the other hand, there may exist a parallel pathway of electron transfer from NADPH via distinct FAD dehydrogenase(s) to the heme b component of the NADPH oxidase.
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PMID:Assessment of the flavoprotein nature of the redox core of neutrophil NADPH oxidase. 864 81


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