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)

Professional phagocytes, neutrophils, possess a unique membrane-associated NADPH oxidase system, dormant in resting cells, which becomes activated upon exposure to the appropriate stimuli and catalyzes the one-electron reduction of molecular oxygen to superoxide, O2-. Oxidase activation involves the assembly, in the plasma membrane, of membrane-bound and cytosolic constituents of the oxidase system, which are disassembled in the resting state. The oxidase system consists of two plasma membrane-bound components; low-potential cytochrome b558, which is composed of two subunits of 22 kDa and 91 kDa, and a flavoprotein related to the electron transport between NADPH and heme-binding domains of the oxidase. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome b558 (flavocytochrome b558). At least two cytosolic components, 67 kDa protein and a phosphorylated 47 kDa protein, are known to translocate to the plasma membrane, ensuring assembly of an active O2(-)-generating NADPH oxidase system. More recently, the membrane (Raps) and cytosolic (Racs) GTP-binding proteins have been established as essential to oxidase assembly. It is the purpose of this review to focus on recent data concerning the regulatory mechanisms which lead to organization and activation of the neutrophil NADPH oxidase system.
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PMID:Activation factors of neutrophil NADPH oxidase complex. 801 44

Earlier studies have established that mutant strains of Azotobacter vinelandii that do not synthesize ferredoxin I (AvFdI) overexpress another protein designated Protein X (Morgan, T. V., Lundell, P. J., and Burgess, B. K. (1988) J. Biol. Chem. 263, 1370-1375). This protein has now been purified using two-dimensional gel electrophoresis as an assay. The purified protein is a monomer with M(r) approximately 29,000 which degrades slowly to a specific M(r) approximately 22,000 form when stored in solution. The native protein is bright yellow and contains noncovalently attached FAD that is reduced by either dithionite or NADPH without formation of a stable semiquinone. Titration with NADP+/NADPH gives an E0' value of approximately -327 mV versus SHE. Because this E0' is so close to that of the NADP+/NADPH couple it is not clear if Protein X is an NADPH oxidase or an NADP+ reductase in vivo. Comparison of the NH2-terminal sequence and other properties of Protein X with those of other proteins, suggests that it is likely to be related to the Escherichia coli ferredoxin NADP+ reductase (the fpr gene product), and affinity chromatography shows that Protein X binds specifically to AvFdI.
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PMID:Purification and characterization of a NADP+/NADPH-specific flavoprotein that is overexpressed in FdI- strains of Azotobacter vinelandii. 803 7

Neutrophil-membrane-associated NADPH-cytochrome c reductase and cytochrome b558 were separately eluted and highly purified by a combination of ion-exchange Sepharose, N-amino-octylagarose, 2',5'-ADP-Sepharose and heparin-Sepharose column chromatographies. The purified cytochrome c reductase with an apparent molecular mass of 68 kDa contained FMN and FAD (FMN/FAD approx. 1). Cytochrome b558 prepared in the presence of phospholipids and FAD showed marked O2-.-producing activity (Vmax., 8.53 mumol of O2-./min per mg of cytochrome; Km for NADPH 58.8 microM) in a cell-free assay system consisting of cytosol, arachidonate and GTP[S]. However, when it was obtained without FAD added to the purification process, it had negligible FAD and little or no O2-.-forming activity in the reconstituted system. The NADPH oxidase activity was not markedly stimulated on incubation of the purified reductase with either flavinated or flavin-depleted cytochrome b558 in the cell-free system, suggesting that the reductase is not likely to be involved in neutrophil O2-. generation. The purified reductase cross-reacted with polyclonal antibodies against both hepatic NADPH-cytochrome P-450 reductase and a synthetic peptide, ILVGPGTGIAPFRSF, which indicates residues 529-543 located in the glycine-rich NADPH-binding domain of the P-450 reductase, but cytochrome b558 did not produce any immunoreactive bands to these antibodies. These antibodies also produced a positive reaction with a 76 kDa protein from dimethyl sulphoxide-induced HL-60-cell microsomes. After solubilization of the microsomal membranes, the 76 kDa protein was readily converted into a partially proteolysed form (68 kDa) even in the presence of antiproteases. In addition, the microsomal fraction shows a CO difference spectrum with a peak at about 454 nm and a trough at 476 nm in the presence of dithionite, indicating the presence of a cytochrome P-450-like haemoprotein.
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PMID:NADPH-cytochrome c reductase from human neutrophil membranes: purification, characterization and localization. 811 Jan 98

NADPH is a system in phagocytic cells that generates O2- and hydrogen peroxide in the endocytic vacuole, both of which are important for killing of the engulfed microbe. Dysfunction of this oxidase results in the syndrome of chronic granulomatous disease, characterized by a profound predisposition to bacterial and fungal infections. A flavocytochrome b is the site of most of the mutations causing this syndrome. The FAD and NADPH binding sites have been located on the beta subunit of this molecule, the C-terminal half of which showed weak sequence similarity to other reductases, including the ferredoxin-NADP reductase (FNR) of known structure. This enabled us to build a model of the nucleotide binding domains of the flavocytochrome using this structure as a template. The model was built initially using a novel automatic modeling method based on distance-matrix projection and then refined using energy minimization with appropriate side-chain torsional constraints. The resulting model rationalized much of the observed sequence conservation and identified a large insertion as a potential regulatory domain. It confirms the inclusion of the neutrophil flavocytochrome b-245 (Cb-245) as a member of the FNR family of reductases and strongly supports its function as the proximal electron transporting component of the NADPH oxidase.
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PMID:A structural model for the nucleotide binding domains of the flavocytochrome b-245 beta-chain. 825 42

Nicotinic acid hydroxylase from Clostridium barkeri contains selenium in an unidentified form that is dissociated as a low molecular weight compound upon denaturation of the enzyme. Other cofactors of this enzyme are molybdopterin, FAD, and iron-sulfur clusters. In the current study, we show that the enzyme, as isolated, exhibits a stable Mo(V) electron paramagnetic resonance (EPR) signal ("resting" signal) and that this signal is correlated with the selenium content and nicotinate hydroxylase activity of the enzyme. Substitution of 77Se for normal selenium isotope abundance results in splitting of the Mo(V) EPR signal of the native protein without affecting the iron signals of the FeS clusters. The Mo(V) EPR signal and nicotinic acid hydroxylase activity of enzyme isolated from cells grown in selenium-deficient medium are barely detectable. In contrast, the EPR signals of the FeS clusters, the electronic absorption spectrum, the NADPH oxidase activity, and the chromatographic behavior are changed little and are typical of active selenium-containing enzyme. An EPR signal indicative of the presence of molybdenum in the selenium-deficient enzyme also is exhibited. From these results, we conclude that a dissociable selenium moiety is coordinated directly with molybdenum in the molybdopterin cofactor and, moreover, this selenium is essential for nicotinic acid hydroxylase activity.
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PMID:Nicotinic acid hydroxylase from Clostridium barkeri: electron paramagnetic resonance studies show that selenium is coordinated with molybdenum in the catalytically active selenium-dependent enzyme. 827 71

Purified cytochrome b559 relipidated with either a mixture of phosphatidylcholine and phosphatidic acid or with phosphatidylcholine only exhibits high and low superoxide (O2-) producing ability, respectively, in the absence of cytosolic activators [Koshkin, V. and Pick, E. (1993) FEBS Lett. 327, 57-62]. This system was used as a model for the study of the mechanism of NADPH oxidase activation. It is shown that, depending on the composition of the phospholipid environment, cytochrome b599 binds FAD with high or low affinity, this being accompanied by changes in flavin absorbance and fluorescence. High affinity binding of FAD to cytochrome b559 relipidated with phosphatidylcholine combined with phosphatidic acid is associated with an enhanced NADPH-driven O2- producing capacity. A kinetic study of O2- production by cytochrome b559 reflavinated under stoichiometric FAD binding conditions revealed an FAD/heme ratio of 1:2. A further kinetic study of O2- production by high- and low-activity relipidated and reflavinated cytochrome b559, at varying substrate concentrations, and the determination of steady-state difference spectra of such preparations, reduced by NADPH, indicated that O2- production is activated by facilitation of electron transfer from NADPH to FAD rather than by an enhancement of NADPH binding.
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PMID:Superoxide production by cytochrome b559. Mechanism of cytosol-independent activation. 830 96

Phagocytic white blood cells contain a multicomponent oxidase that generates microbicidal products by catalyzing electron transfer from NADPH to molecular oxygen. Activation of this oxidase requires interactions of a unique membrane flavocytochrome with the cytosolic proteins p47phox, p67phox, and p21Rac. This flavocytochrome, designated cytochrome b558, is a heteromer comprising a 22-kDa alpha-subunit (p22phox) and a glycosylated approximately 91-kDa beta-subunit (gp91phox). Cytochrome b558 was expressed in Sf9 insect cells coinfected with recombinant baculoviruses carrying cDNAs for p22phox and gp91phox. Membranes of these cells contained a b-type cytochrome with a dithionite-reduced minus oxidized difference spectrum similar to that of neutrophil cytochrome b558. The recombinant cytochrome b558 beta-subunit was heterogeneously N-glycosylated as demonstrated by its susceptibility to cleavage with endoglycosidases F and H. In contrast to the neutrophil cytochrome b558, a portion of the N-linked oligosaccharide was of the high mannose type. Recombinant cytochrome b558 supported superoxide production in a cell-free assay containing recombinant p47phox, p67phox, and p21Rac. The enzymatic turnover of the partially purified recombinant cytochrome b558 and neutrophil cytochrome b558 were similar (approximately 100-160 mol of superoxide generated/s/mol of cytochrome heme, range of two experiments) and the native and recombinant cytochromes showed similar requirements for NADPH and exogenous FAD. These studies represent the first reconstitution of the NADPH oxidase solely from recombinant proteins and define a model system to explore the structure and function of cytochrome b558.
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PMID:Production of recombinant cytochrome b558 allows reconstitution of the phagocyte NADPH oxidase solely from recombinant proteins. 831 88

Purified cytochrome b559 from guinea pig macrophages was relipidated with several phospholipid mixtures. Relipidated cytochrome b559 was found capable of NADPH-dependent superoxide (O2-) production in the absence of the cytosolic components of the NADPH oxidase complex. The rate of O2- generation by cytochrome b559 varied with the type of phospholipid utilized for relipidation, was absolutely dependent on exogenous FAD, and was enhanced by a critical concentration of anionic amphiphile. It is demonstrated that exogenous FAD acts by binding to cytochrome b559. These results provide firm experimental evidence for the proposal that cytochrome b559 comprises the complete electron transporting apparatus of the O2- forming NADPH oxidase and that the cytosolic components function merely as activators.
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PMID:Generation of superoxide by purified and relipidated cytochrome b559 in the absence of cytosolic activators. 839 46

Superoxide is produced by phagocytic cells at rates sufficient to have cytocidal effects. A wide variety of receptor-dependent and -independent agonists triggers this respiratory burst, including immunoglobin aggregates, complement fragments, and leukotriene B4. Lower rates of O2-. production are triggered by addition of specific cytokines into B-lymphocytes, endothelial cells, fibroblasts, and kidney mesangial cells; low concentration of radicals may act as signals for proliferation or other changes. The NADPH oxidase of phagocytes, characterized by the presence of FAD and a low potential cytochrome b, is organized to transfer electrons electrogenically across the plasma membrane from NADPH to O2. A proton channel permits movement of compensating H+.
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PMID:The mechanism of the production of superoxide by phagocytes. 839 50

Diphenyleneiodonium (DPI) and its analogues have been previously shown to react via a radical mechanism whereby an electron is abstracted from a nucleophile to form a radical, which then adds back to the nucleophile to form covalent adducts [Banks (1966) Chem. Rev. 66, 243-266]. We propose that the inhibition of neutrophil NADPH oxidase by DPI occurs via a similar mechanism. A reduced redox centre in the oxidase could serve as electron donor to DPI, and inhibition would occur after direct phenylation of the redox cofactor, or of adjacent amino acid groups by the DPI radical. In the absence of an activatory stimulus, human neutrophil NADPH-oxidase was not inhibited by DPI. The Ki for time-dependent inhibition by DPI of human neutrophil membrane NADPH oxidase was found to be 5.6 microM. Inhibitory potency of DPI was shown to be directly related to rate of enzyme turnover, indicating the need for a reduced redox centre. Adducts were formed between photoreduced flavin (FAD or FMN) and inhibitor (DPI or diphenyliodonium). These were separated by h.p.l.c. and characterized by absorbance spectroscopy, 1H-n.m.r. and fast-atom-bombardment m.s. and found to have properties consistent with substituted 4a,5-dihydroflavins. After incubation of pig neutrophil membranes with DPI, the quantity of recoverable intact flavin was greatly diminished when NADPH was present to initiate oxidase turnover, indicating that the flavin may be the site of DPI activation. These results may provide a common mechanism of action for iodonium compounds as inhibitors of other flavoenzymes.
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PMID:Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase. 843 98


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