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: KEGG:D02011 (FAD)
5,530 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The superoxide (O-2)-generating NADPH oxidase of phagocytes is a multicomponent complex consisting of a membrane-associated flavocytochrome (cytochrome b559), bearing the NADPH binding site and two redox centers (FAD and heme) and three cytosolic activating components: p47(phox), p67(phox), and the small GTPase Rac (1 or 2). The canonical view is that the induction of O-2 generation involves the stimulus-dependent assembly of all three cytosolic components with cytochrome b559, a process mimicked in vitro by a cell-free system activated by anionic amphiphiles. We studied the requirement for individual cytosolic components in the activation of NADPH oxidase in a cell-free system consisting of purified and relipidated cytochrome b559, recombinant p47(phox), p67(phox), and Rac1, and the amphiphile, lithium dodecyl sulfate. We found that pronounced activation of NADPH oxidase can be achieved by exposing cytochrome b559 to p67(phox) and Rac1, in the total absence of p47(phox) (turnover = 60 mol O-2/s/mol cytochrome b559). However, maximal activation (turnover = 153 mol O-2/s/mol cytochrome b559) could only be obtained in the presence of p47(phox). O-2 production, in the absence of p47(phox), was dependent on: high molar ratios of p67(phox) and Rac1 to cytochrome b559, Rac1 being in the GTP-bound form, cytochrome b559 being saturated with FAD, and an optimal concentration of amphiphile. Single cytosolic components or combinations of two cytosolic components, other than p67(phox) and Rac1, were incapable of activation. We conclude that p67(phox) and Rac1 are the only cytosolic components directly involved in the induction of electron transport in cytochrome b559. p47(phox) appears to facilitate or stabilize the interaction of p67(phox) and, possibly, Rac1 with cytochrome b559, and is required for optimal generation of O-2 under physiological conditions.
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PMID:The cytosolic component p47(phox) is not a sine qua non participant in the activation of NADPH oxidase but is required for optimal superoxide production. 893 91

Defective NADPH oxidase components prevent superoxide (O-2) generation, causing chronic granulomatous disease (CGD). X-linked CGD patients have mutations in the gene encoding the gp91(phox) subunit of cytochrome b558 and usually lack gp91(phox) protein completely (X91(0)). gp91(phox) is considered to be a flavocytochrome that contains binding sites for NADPH, FAD, as well as heme. We here report a rare X-linked CGD patient whose neutrophils entirely failed to produce O-2, but presented a diminished expression of gp91(phox) containing about one-third of the heme present in normal individuals by Soret absorption. Translocation of cytosolic factors p67(phox) and p47(phox) was normal. However, the FAD content in his neutrophil membranes was as low as that of X91(0) patients, suggesting complete depletion of FAD in his gp91(phox). This was in agreement with the finding that a single base substitution (C1024 to T) changed His-338 to Tyr in gp91(phox) in a predicted FAD-binding domain of the flavocytochrome model. The loss of FAD could not be corrected even after addition of reagent FAD or a FAD-rich dehydrogenase fraction isolated from normal neutrophils to the patient's membranes, in a reconstitution in vitro with normal cytosol. These results indicate that His-338 is a very critical residue for FAD incorporation into the NADPH oxidase system. This is the first such mutation found in CGD.
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PMID:Mutation at histidine 338 of gp91(phox) depletes FAD and affects expression of cytochrome b558 of the human NADPH oxidase. 977 99

An activation domain in p67(phox) (residues within 199-210) is essential for cytochrome b(558)-dependent activation of NADPH superoxide (O2(-.)) generation in a cell-free system (Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663-16668). To determine the steady state reduction flavin in the presence of highly absorbing hemes, 8-nor-8-S-thioacetamido-FAD ("thioacetamido-FAD") was reconstituted into the flavocytochrome, and the fluorescence of its oxidized form was monitored. Thioacetamido-FAD-reconstituted cytochrome showed lower activity (7% versus 100%) and increased steady state flavin reduction (28 versus <5%) compared with the enzyme reconstituted with native FAD. Omission of p67(phox) decreased the percent steady state reduction of the flavin to 4%, but omission of p47(phox) had little effect. The activation domain on p67(phox) was critical for regulating flavin reduction, since mutations in this region that decreased O2(-.) generation also decreased the steady state reduction of flavin. Thus, the activation domain on p67(phox) regulates the reductive half-reaction for FAD. This reaction is comprised of the binding of NADPH followed by hydride transfer to the flavin. Kinetic deuterium isotope effects along with K(m) values permitted calculation of the K(d) for NADPH. (R)-NADPD but not (S)-NADPD showed kinetic deuterium isotope effects on V and V/K of about 1.9 and 1.5, respectively, demonstrating stereospecificity for the R hydride transfer. The calculated K(d) for NADPH was 40 microM in the presence of wild type p67(phox) and was approximately 55 microM using the weakly activating p67(phox)(V205A). Thus, the activation domain of p67(phox) regulates the reduction of FAD but has only a small effect on NADPH binding, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.
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PMID:The p67(phox) activation domain regulates electron flow from NADPH to flavin in flavocytochrome b(558). 1043 66

Chronic granulomatous disease (CGD) is a group of inherited disorders in which phagocytes are unable to generate superoxide (O(2)(-)) due to genetic defects in any 1 of 4 essential NADPH oxidase components. Mutations in the X-linked gene for gp91(phox), the large subunit of the flavocytochrome b(558) heterodimer, account for the majority of CGD. An X-CGD patient in which a splice junction mutation results in an in-frame deletion of 30 nucleotides encoding amino acids 488 to 497 of gp91(phox) (delta488-497 gp91(phox)) has previously been reported. In this study, we generated myeloid PLB-985 cells expressing the mutant triangle delta488-497 gp91(phox) to further characterize its functional properties. These cells mimicked the phenotype of the patient's neutrophils with normal expression of a nonfunctional delta488-497 gp91(phox) flavocytochrome. Translocation of p47(phox) and p67(phox) to delta488-497 gp91(phox) PLB-985 plasma membranes was not affected, as determined both in activated intact cells and in the cell-free system. Furthermore, a synthetic peptide corresponding to residues 488-497 of gp91(phox) was relatively ineffective in inhibiting O(2)(-) production in the cell-free oxidase assay (IC50, approximately 500 micromol/L), suggesting that residues 488-497 of gp91(phox) are not directly involved in oxidase assembly. Mutant delta488-497 gp91(phox) flavocytochrome failed to support iodonitrotetrazolium (INT) reduction, showing a disruption of electron transfer from NADPH to the FAD center of gp91(phox). However, the FAD binding capacity of the mutant flavocytochrome was normal, as measured by equilibrium dialysis. Taken together, these results suggest that the delta488-497 deletion in gp91(phox) disrupts electron transfer to FAD, either due to a defect in NADPH binding or to impaired delivery of electrons from NADPH.
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PMID:Functional analysis of NADPH oxidase in granulocytic cells expressing a delta488-497 gp91(phox) deletion mutant. 1049 23

Hydrogen peroxide is the final electron acceptor for the biosynthesis of thyroid hormone catalyzed by thyroperoxidase at the apical surface of thyrocytes. Pig and human thyroid plasma membrane contain a Ca(2+)-dependent NAD(P)H oxidase that generates H(2)O(2) by transferring electrons from NAD(P)H to molecular oxygen. We purified from pig thyroid plasma membrane a flavoprotein which constitutes the main, if not the sole, component of the thyroid NAD(P)H oxidase. Microsequences permitted the cloning of porcine and human full-length cDNAs encoding, respectively, 1207- and 1210-amino acid proteins with a predicted molecular mass of 138 kDa (p138(Tox)). Human and porcine p138(Tox) have 86.7% identity. The strongest similarity was to a predicted polypeptide encoded by a Caenorhabditis cDNA and with rbohA, a protein involved in the Arabidopsis NADPH oxidase. p138(Tox) shows also similarity to the p65(Mox) and to the gp91(Phox) in their C-terminal region and have consensus sequences for FAD- and NADPH-binding sites. Compared with gp91(Phox), p138(Tox) shows an extended N-terminal containing two EF-hand motifs that may account for its calcium-dependent activity, whereas three of four sequences implicated in the interaction of gp91(Phox) with the p47(Phox) cytosolic factor are absent in p138(Tox). The expression of porcine p138(Tox) mRNA analyzed by Northern blot is specific of thyroid tissue and induced by cyclic AMP showing that p138(Tox) is a differentiation marker of thyrocytes. The gene of human p138(Tox) has been localized on chromosome 15q15.
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PMID:Purification of a novel flavoprotein involved in the thyroid NADPH oxidase. Cloning of the porcine and human cdnas. 1060 Dec 91

Diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, has been utilized to demonstrate the importance of histidine residues in the functioning of proteins. In previous studies of the NADPH oxidase, histidine residues have been determined to be important in the ability of gp91(phox) to function as an H(+) pathway and in the binding of haem and FAD. We have investigated the ability of DEPC to inhibit H(+) flux and superoxide generation by human neutrophils. Proton flux through the NADPH oxidase-associated H(+) channel was inhibited by DEPC only if applied simultaneously with an activator of the channel. This suggested that the site modified by DEPC is not accessible in the closed channel. Superoxide generation by the NADPH oxidase was also inhibited by DEPC when applied after or simultaneously with the activator. Translocation of the NADPH oxidase cytosolic components, p67(phox) and p47(phox), to the membrane was unaffected by DEPC. In a cell-free system, DEPC-treated membranes failed to support superoxide generation or the reduction of Iodonitrotetrazolium Violet and showed a loss of the characteristic cytochrome b(558) spectrum. Superoxide generation by DEPC-treated cytosol was inhibited slightly. Therefore it can be concluded that there are two sites within the NADPH oxidase that interact with DEPC, one in the H(+) pathway, only accessible in the activated oxidase, and a second accessible prior to activation of the NADPH oxidase. The latter non-proton pathway DEPC site is located within the membrane components of the NADPH oxidase and is associated with the binding of haem in the enzyme complex.
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PMID:Inhibition of the neutrophil NADPH oxidase and associated H+ channel by diethyl pyrocarbonate (DEPC), a histidine-modifying agent: evidence for at least two target sites. 1151 29

Activation of the phagocyte NADPH oxidase occurs via assembly of cytosolic p47(phox), p67(phox), and Rac with the membrane-bound flavocytochrome b(558). Recently, we have found that p67(phox)-(1-210) (p67N) fused with p47(phox)-(1-286) (p47N) or with Rac efficiently stabilizes the oxidase in a cell-free reconstitution system. In an attempt to further stabilize the oxidase, we herein used a constitutively active Rac, RacQ61L, and examined its effect on the oxidase stability. The half-life (t(1/2)) of the activity reconstituted with wild-type Rac was 12 min at 37 degrees C, which was extended 6-fold by RacQ61L. Also, the stability of the oxidase without p47(phox) increased 8-fold using RacQ61L. RacQ61L had a higher affinity for the complex than wild-type Rac and increased the affinity of p67N for the complex. Far-western blotting showed an enhanced binding between RacQ61L and p67N. The oxidase was stabilized by nanomolar FAD, and RacQ61L lowered the FAD concentration required. The combination of RacQ61L and a fusion protein consisting of p67N and p47N produced an extremely stable enzyme (t(1/2) = 184 min at 37 degrees C). The effectiveness of RacQ61L and fusion proteins on stabilization was in the following order: p67N-Rac < p67N + RacQ61L < or = p67N-RacQ61L << p67N-p47N + RacQ61L. These results indicate that a tightly bound ternary complex of p67(phox), Rac, and p47(phox) is very effective in maintaining the oxidase and confirm that the longevity of the activated state requires continuous association of these components. This simple and efficient method of stabilization may provide a useful tool to elucidate the nature of the activated oxidase.
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PMID:Remarkable stabilization of neutrophil NADPH oxidase using RacQ61L and a p67phox-p47phox fusion protein. 1251 53

Truncated forms of gp91(phox) were expressed in E. coli in which the N-terminal hydrophobic transmembrane region was replaced with a portion of the highly soluble bacterial protein thioredoxin (TRX). TRX-gp91(phox) (306-569), which contains the putative FAD and NADPH binding sites, showed NADPH-dependent NBT (nitroblue tetrazolium) reductase activity, whereas TRX-gp91(phox) (304-423) and TRX-gp91(phox) (424-569) were inactive. Activity saturated at about a 1:1 molar ratio of FAD to TRX-gp91(phox) (306- 569), and showed the same Km for NADPH as that for superoxide generating activity by the intact enzyme. Activity was not inhibited by superoxide dismutase, indicating that it was not mediated by superoxide, but was blocked by an inhibitor of the respiratory burst oxidase, diphenylene iodonium (DPI). In the presence of Rac1, the cytosolic regulatory protein p67(phox) stimulated the NBT reductase activity, but p47(phox) had no effect. Truncated p67(phox) containing the activation domain (residues 199- 210) stimulated activity approximately 2-fold, whereas forms mutated or lacking this region failed to stimulate the activity. Our data indicate that: 1) TRX-gp91(phox) (306-569) contains the binding sites for both pyridine and flavin nucleotides; 2) this flavoprotein domain shows NBT reductase activity; and 3) the flavin-binding domain of gp91(phox) is the target of regulation by the activation domain of p67(phox).
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PMID:Expression and characterization of the flavoprotein domain of gp91phox. 1461 16

An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system [10] To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored. Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect. A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction. The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558). The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction. Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.
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PMID:Activation domain in P67phox regulates the steady state reduction of FAD in gp91phox. 1461 17


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