Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: KEGG:D02011 (FAD)
 5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytochrome b558 is the only membrane component of the phagocyte O2(-)-producing NADPH oxidase. The O2- production by the oxidase reconstituted in vitro with the crude membrane fraction is enhanced several-fold by addition of FAD, whereas that with the partially purified cytochrome is completely dependent on exogenous FAD, suggesting that FAD acts through the membrane component, cytochrome b558. The alignments of the amino acid sequence of the large subunit of the cytochrome (gp91-phox) with those of previously characterized flavoproteins reveal that the middle and C-terminal portions of gp91-phox are likely to be FAD- and NADPH-binding domains, respectively. Cytochrome b558, thus, appears to be a flavoprotein with an NADPH-binding site, of the NADPH oxidase.
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PMID:Cytochrome b558, a component of the phagocyte NADPH oxidase, is a flavoprotein. 132 65

Succinate:menaquinone-7 oxidoreductase (complex II) of the Gram-positive bacterium Bacillus subtilis consists of equimolar amounts of three polypeptides; a 65-kDa FAD-containing polypeptide, a 28-kDa iron-sulfur cluster containing polypeptide, and a 23-kDa membrane-spanning cytochrome b558 polypeptide. The enzyme complex was overproduced 2-3-fold in membranes of B. subtilis cells containing the sdhCAB operon on a low copy number plasmid and was purified in the presence of detergent. The cytochrome b558 subunit alone was similarly overexpressed in a complex II deficient mutant and partially purified. Isolated complex II catalyzed the reduction of various quinones and also quinol oxidation. Both activities were efficiently albeit not completely blocked by 2-n-heptyl-4-hydroxyquinoline N-oxide. Chemical analysis demonstrated two protoheme IX per complex II. One heme component was found to have an Em,7.4 of +65 mV and an EPR gmax signal at 3.68, to be fully reducible by succinate, and showed a symmetrical alpha-band absorption peak at 555 nm at 77 K. The other heme component was found to have an Em,7.4 of -95 mV and an EPR gmax signal at 3.42, was not reducible by succinate under steady-state conditions, and showed in the reduced state an apparent split alpha-band absorption peak with maxima at 553 and 558 nm at 77 K. Potentiometric titrations of partially purified cytochrome b558 subunit demonstrated that the isolated cytochrome b558 also contains two hemes. Some of the properties, i.e., the alpha-band light absorption peak at 77 K, the line shapes of the EPR gmax signals, and reactivity with carbon monoxide were observed to be different in B. subtilis cytochrome b558 isolated and in complex II. This suggests that the bound flavoprotein and iron-sulfur protein subunits protect or affect the heme environment in the assembled complex.
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PMID:Two hemes in Bacillus subtilis succinate:menaquinone oxidoreductase (complex II). 132 13

Cytochrome b558 of pig blood neutrophils was purified from the membranes of resting cells to examine its ability to reconstitute superoxide (O2-)-forming NADPH oxidase activity in a cell-free assay system containing cytosol and fatty acid. The membrane-associated cytochrome b558 was solubilized with a detergent, n-heptyl beta-thioglucoside, and purified by DEAE-Sepharose, heparin-Sepharose, and Mono Q column chromatography. The final preparation of cytochrome containing 11.5 nmol of protoheme/mg of protein gave bands of the large and small subunits on immunoblotted gel. The cell-free system with the purified cytochrome alone as a membrane component showed little O2(-)-generating activity in the absence of exogenous FAD. However, the system showed high O2(-)-generating activity of 31.8 mol/s/mol of cytochrome b558 (52.5% of the original O2(-)-generating activity of the solubilized membranes) in the presence of a nitro blue tetrazolium (NBT) reductase fraction that was separated from the cytochrome b fraction by heparin-Sepharose chromatography. Heat treatment of the NBT reductase fraction resulted in loss of the O2(-)-generating activity in the reconstituted system. The O2(-)-forming activity of the reconstituted system was markedly decreased by removal of FAD from the NBT reductase fraction and was restored by readdition of FAD to the FAD-depleted reductase. The reconstituted system containing purified cytochrome b558 plus the NBT reductase showed approximately 100 times higher O2(-)-generating activity than a system containing rabbit liver NADPH-cytochrome P-450 reductase instead. These results suggest that both the FAD-dependent NBT reductase and cytochrome b558 are required as membrane redox components for O2(-)-forming NADPH oxidase activity. The present data are discussed in comparison with previously reported results on reconstituted systems containing added free FAD.
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PMID:Reconstitution of superoxide-forming NADPH oxidase activity with cytochrome b558 purified from porcine neutrophils. Requirement of a membrane-bound flavin enzyme for reconstitution of activity. 132 33

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.
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PMID:Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid. 215 45

The NADPH-binding component of the neutrophil superoxide-generating oxidase was studied in the particulate oxidase fractions obtained from the neutrophils of normal and chronic-granulomatous-disease (CGD) patients. The molecular mass of the NADPH-binding component of the stimulated human neutrophils, which was labelled with the 2',3'-dialdehyde derivative of NADPH and sodium cyanoboro[3H]hydride, was 66 kDa. The 66 kDa component was also labelled in monocytes, but not in red blood cells, platelets and lymphocytes. The particulate oxidase fractions obtained from the patients with CGD had a diminished amount of FAD, whether they contained cytochrome b558 or not. The fractions labelled with the NADPH analogue showed that CGD patients had the NADPH-binding component in the neutrophils. The molecular mass of the component was identical with that of the normal neutrophils. The patients are thought to have an intact NADPH-binding domain of the oxidase in the neutrophils in spite of a diminished amount of FAD in the particulate fractions. The component of the oxidase in the resting neutrophils was also labelled with the analogue. The molecular mass of the component in the resting neutrophils was identical with that of the stimulated neutrophils, and the component was not phosphorylated during the activation process. These results indicate that the NADPH-binding component of the oxidase, which is specific to phagocytes, is present in the resting neutrophils and that the component does not change with respect to molecular mass during the activation process.
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PMID:NADPH-binding component of the superoxide-generating oxidase in unstimulated neutrophils and the neutrophils from the patients with chronic granulomatous disease. 363 31

Cytochrome b558 isolated from human neutrophils was inactive and contained no detectable FAD. However, high NADPH oxidase activity was seen upon reconstitution of the cytochrome with either native FAD or 8-mercapto-FAD in the presence of phospholipids (phosphatidylcholine/phosphatidylethanolamine/phosphatidylinositol/ sphingomyelin/cholesterol, 4:2:1:3:3 (w/w)). Their cell-free superoxide-generating activities were 40.5 and 35.5 mol/s/mol of heme, respectively, which corresponded to 70 and 61% of the original activity of the plasma membranes. Both flavins co-eluted with heme and protein on gel exclusion chromatography. The respective specific flavin content was 6.45 and 7.93 nmol/mg of protein and corresponded to a flavin:heme molar ratio of 0.41 and 0.51 consistent with a 2:1 ratio of heme to flavin. Mixing of 8-mercapto-FAD with flavin-depleted cytochrome b558 caused a red-shift of the flavin absorption maximum from 520 nm to around 560 nm, as has been seen when a variety of other apoflavoprotein dehydrogenases bind this analog. The 8-mercapto-FAD reconstituted into the cytochrome reacted readily with either iodoacetamide (k = 38.8 M-1.min-1) or iodoacetic acid (k = 12.1 M-1.min-1) to give a fluorescence spectrum characteristic of a 8-mercaptoflavin derivative, 8-SCH2CONH2 FAD or 8-SCH2COOH FAD. These results indicate that position 8 of FAD bound to the protein is freely accessible to solvent. These studies support the idea that cytochrome b558 is a flavocytochrome.
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PMID:Reconstitution of flavin-depleted neutrophil flavocytochrome b558 with 8-mercapto-FAD and characterization of the flavin-reconstituted enzyme. 760 14

Superoxide is produced by a NADPH oxidase of phagocytic cells and contributes to their microbicidal activities. The oxidase is activated when receptors in the neutrophil plasma membrane bind to the target microbe. These receptors recognise antibodies and complement fragments which coat the target cell. The oxidase electron transport chain, located in the plasma membrane, comprises a low potential cytochrome b heterodimer (gp 91-phox and p22-phox) associated with FAD. It is non-functional until at least three proteins, p67-phox, p47-phox and p21rac (and possibly others), move from the cytosol to dock on the cytochrome b. The docking involves the interaction of SH3 domains on p47-phox or p67-phox with a proline-rich sequence on the small subunit of the cytochrome b. These SH3 domains may become exposed following phosphorylation of p47-phox by protein kinase C or, in model systems, by addition of arachidonic acid to reconstitution mixtures. Following the docking process the electron-transporting component is able to transfer electrons from NADPH to oxygen. This electrogenic event is charge-compensated by the opening of a proton channel. Components of the oxidase are expressed in non-phagocytes, where their function is uncertain but could be related to some signal function of superoxide.
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PMID:The regulation of superoxide production by the NADPH oxidase of neutrophils and other mammalian cells. 784 Jul 72

A photoactivable derivative of FAD, 4-[N-(4-azido-2-nitrophenyl)amino]butyryl-FAD (NAP4-FAD), was synthesized in a tritiated form with tritium placed in the NAP4 moiety of the photoprobe. [3H]NAP4-FAD was used to photolabel the putative flavin binding site of the O2(-)-generating NADPH oxidase located in the plasma membrane of bovine neutrophils. Effective photolabeling required partial deflavination of membranes, which was achieved by mild treatment with ammonium sulfate added to 50% saturation and 0.05% Triton X-100 for 30 min at 2-4 degrees C. Under these conditions, 40-50% of the oxidase activity was lost, but it could be fully recovered by the addition of nanomolar amounts of FAD (KM = 10-20 nM). Added FAD could be substituted by [3H]NAP4-FAD in photolabeling experiments. In the dark, [3H]NNAP4-FAD bound reversibly with high affinity to deflavinated neutrophil plasma membranes (Kd = 50 nM), did not transport electrons, and efficiently inhibited the FAD-dependent restoration of oxidase activity (Ki = 60 nM). Upon photoirradiation of neutrophil plasma membranes in the presence of [3H]NAP4-FAD, the nitrene derivative formed bound covalently to a 80-120 kDa protein that was identified as the beta-subunit of cytochrome b558 by immunodetection and enzymatic deglycosylation. The amount of [3H]NAP4-FAD covalently incorporated into the beta-subunit of cytochrome b558 was 80-90% of the amount of photoprobe specifically bound to neutrophil plasma membranes. A linear relationship between the extent of specific photolabeling by [3H]NAP4-FAD and the percentage of NADPH oxidase inactivation was observed for percentages of inactivation of up to 70-80%, extrapolating to 0.5 mol of covalently bound [3H]NAP4-FAD per mol of heme b558.
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PMID:Photoaffinity labeling and photoinactivation of the O2(-)-generating oxidase of neutrophils by an azido derivative of FAD. 784 36

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

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 possible flavoprotein related to the electron transport between NADPH and cytochrome b558. Recent reports have indicated that FAD-binding sites of the oxidase are contained in cytochrome 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. It is the purpose of this review to focus on recent data concerning electron transfer mechanisms of the activated neutrophil NADPH-oxidase complex and molecular pathology of chronic granulomatous disease.
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PMID:Mechanisms for the activation/electron transfer of neutrophil NADPH-oxidase complex and molecular pathology of chronic granulomatous disease. 803 32


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