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)

Chronic granulomatous disease (CGD) is a congenital disorder in which phagocytes cannot generate superoxide (O2-) and other microbial oxidants due to mutations in any one of four components of the O2(-)-generating complex, NADPH oxidase. We report here a female CGD patient in whom a missense mutation in one of these components, the p22-phox subunit of the neutrophil membrane cytochrome b [where phox indicates phagocyte oxidase (used to designate protein components of the phagocyte NADPH oxidase)] results in a nonfunctional oxidase and failure of neutrophils to produce O2- in response to phorbol 12-myristrate 13-acetate. Cytochrome b in the patient's neutrophils was normal in appearance and abundance as determined by visible spectroscopy and by immunoblots of the gp91 and p22 subunits. However, the neutrophil plasma membranes were devoid of activity in the cell-free oxidase activation system, whereas the cytosol functioned normally. We postulated that the patient was homozygous for a mutation in p22 that results in the synthesis of normal levels of a nonfunctional cytochrome b. A single-base substitution (C----A) was found in the patient's mononuclear cell p22-phox cDNA that predicts a nonconservative Pro----Gln substitution at residue 156. The same mutation was also identified in all clones sequenced from patient genomic DNA, demonstrating homozygosity for the mutant allele. An antipeptide antibody against p22 residues 153-164 was found to bind only to permeabilized neutrophils, indicating that the mutation occurs in a cytoplasmic domain. These studies establish that this domain of p22-phox is cytoplasmic and that mutations in this region can have profound effects on cytochrome b function.
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PMID:Point mutation in the cytoplasmic domain of the neutrophil p22-phox cytochrome b subunit is associated with a nonfunctional NADPH oxidase and chronic granulomatous disease. 176 37

Differentiation of myeloid cells is associated with the gradual acquisition of functional capacity to produce a respiratory burst. In our study HL-60 cells were differentiated to the monocyte phenotype with IFN-gamma or 1,25-dihydroxyvitamin D3, or to the neutrophil phenotype with retinoic acid or DMSO to compare the time-course of expression of membrane and cytosolic oxidase components, and to correlate this with the appearance of a functional oxidase. Over a 6-day period of induction the rank order of the ability of these agents to induce expression of PMA-stimulated superoxide production was: IFN-gamma greater than 1,25(OH)2D3 greater than retinoic acid greater than DMSO. Immunoblot analysis of HL-60 membranes and cytosol was used to assess the amount of specific phagocyte oxidase factors (91 and 22 kDa subunits of membrane cytochrome b558 (gp91 and p22), and 47 and 67 kDa cytosol oxidase factors (p47 and p67)). HL-60 cell membranes or cytosol were tested in a cell-free assay of superoxide production by mixing with normal neutrophil cytosol or membranes, respectively. p47 was first detected at 16 h of differentiation, increasing similarly thereafter with all induction regimens and reaching a maximum by 3 to 4 days. The earliest detection of p67 varied from 2 to 6 days depending on the inducing agent and appeared to be the limiting cytosol component. Small amounts of both subunits of cytochrome b558 were detected in uninduced HL-60 membranes, but were sufficient to support substantial superoxide production when combined with normal neutrophil cytosol. Both cytochrome b558 subunit proteins and membrane oxidase activity increased during differentiation in parallel. We conclude that membrane and cytosol components of the NADPH oxidase complex appear at different times and increase differently during HL-60 differentiation. The production of p67 is the major factor limiting the respiratory burst during HL-60 differentiation.
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PMID:Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression. 217 May 20

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activation involves assembly of membrane-integrated cytochrome b558 comprising gp91(phox) and p22(phox), two specialized cytosolic proteins (p47(phox) and p67(phox)), each containing two Src homology 3 (SH3) domains, and the small G protein Rac. In the present study, we show that the N-terminal SH3 domain of p47(phox) binds to the C-terminal cytoplasmic tail of p22(phox) with high affinity (KD = 0.34 microM). The binding is specific to this domain among several SH3 domains including the C-terminal one of p47(phox) and the two of p67(phox) and requires the Pro156-containing proline-rich sequence but not other putative SH3 domain-binding sites of p22(phox). Replacement of Trp193 by Arg in the N-terminal SH3 domain completely abrogates the association with p22(phox). A mutant p47(phox) with this substitution is incapable of supporting superoxide production under cell-free activation conditions. These findings provide direct evidence that the interaction between the N-terminal SH3 domain of p47(phox) and the proline-rich region of p22(phox) is essential for activation of the NADPH oxidase.
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PMID:Assembly and activation of the phagocyte NADPH oxidase. Specific interaction of the N-terminal Src homology 3 domain of p47phox with p22phox is required for activation of the NADPH oxidase. 870 27

Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.
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PMID:p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. 879 32

A potential candidate for an oxygen-sensing protein in chemoreceptor cells is a heme-linked multicomponent NADPH oxidase, originally described in neutrophils. The postulated function for the oxidase in chemoreceptor cells is to signal changes in oxygen levels (either in the blood or in the airway lumen) via changes in oxygen metabolite production. An alteration in either superoxide (or dismuted hydrogen peroxide) production may affect the gating properties of the O2-sensitive K+ channels. We have previously reported immunohistochemical localization of gp91 glycoprotein component of the oxidase to the plasma membrane of pulmonary neuroepithelial body (NEB) cells. In this study we have investigated the immunocytochemical localization of the other polypeptide components of the oxidase in NEB cells and in the glomus cells of the carotid body. Cultures of dissociated fetal rabbit NEB cells and newborn rat glomus cells were immunostained with specific antibodies recognizing the various polypeptide subunits of the oxidase using indirect immunofluorescence methods. Immunostaining with the anti-oxidase antibodies reveal strong positive reaction in both NEB and glomus cell clusters while other cells were unstained. The positive reaction product was localized to the plasma membrane and/or cytoplasm and no nuclear staining was observed. Live cell labelling studies with anti-p22 antibody showed positive immunofluorescence on the surface of NEB cells, suggesting that this component of the oxidase is also associated with the plasma membrane. In glomus cells, similar strongly positive immunofluorescence signal was observed for p22 and gp91 in paraformaldehyde-fixed cultures, regardless whether they were permeabilized or not. Taken together, our findings of cell surface localization of gp91 and p22 components of the oxidase in chemoreceptive cells suggests that the heme-linked cytochrome b558 component is associated with the plasma membrane. This association allows for direct interaction with the O2-sensitive K+ channel thus forming the molecular complex of membrane bound O2 sensor.
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PMID:Immunocytochemical localization on O2-sensing protein (NADPH oxidase) in chemoreceptor cells. 914 26

The NADPH oxidase cytochrome b558 is a membrane heterodimer comprised of a glycosylated 91-kDa subunit, gp91(phox), and a nonglycosylated 22-kDa subunit, p22(phox). The role of heme in cytochrome b558 biosynthesis was studied using succinyl acetone, an inhibitor of heme synthesis, in PLB-985 myeloid cells undergoing granulocytic differentiation. Succinyl acetone markedly reduced expression of p22(phox) and the mature 91-kDa form of gp91(phox) but not its 65-kDa high mannose precursor, in association with a profound reduction in NADPH oxidase activity. Expression of non-heme-containing cytosolic oxidase components was unaffected. The reduction in cytochrome b558 expression and NADPH oxidase activity was prevented by adding exogenous heme and was reversible upon removal of succinyl acetone. Transgenic expression of gp91(phox) in monkey COS-7 and murine 3T3 cells, both of which lacked endogenous p22(phox) mRNA, demonstrated that p22(phox) was not required for maturation of gp91(phox) carbohydrate to complex oligosaccharides. However, coexpression of transgenic p22(phox) increased the abundance of the mature gp91(phox) glycoprotein. These results suggest that heme incorporation plays an important role in cytochrome b558 assembly and provide further support for the concept that stability of p22(phox) and the mature gp91(phox) subunit is increased by heterodimer formation.
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PMID:Biosynthesis of the phagocyte NADPH oxidase cytochrome b558. Role of heme incorporation and heterodimer formation in maturation and stability of gp91phox and p22phox subunits. 934 Nov 76

Superoxide anion (O2-) plays a key role in the endogenous suppression of endothelium-derived nitric oxide (NO) bioactivity and has been implicated in the development of hypertension. In previous studies, we found that O2- is produced predominantly in the adventitia of isolated rabbit aorta and acts as a barrier to NO. In the present studies, we characterize the enzyme responsible for O2- production in the adventitia and show that this enzyme is a constitutively active NADPH oxidase with similar composition as the phagocyte NADPH oxidase. Constitutive O2--generating activity was localized to aortic adventitial fibroblasts and was enhanced by the potent vasoconstrictor angiotensin II. Immunohistochemistry of aortic sections demonstrated the presence of p22(phox), gp91(phox), p47(phox), and p67(phox) localized exclusively in rabbit aortic adventitia, coincident with the site of staining for O2- production. Furthermore, immunodepletion of p67(phox) from adventitial fibroblast particulates resulted in the loss of NADPH oxidase activity, which could be restored by the addition of recombinant p67(phox). Further study into the regulation of this adventitial source of O2- is important in elucidating the mechanisms regulating the bioactivity of NO and may contribute to our understanding of the pathogenesis of hypertension.
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PMID:Localization of a constitutively active, phagocyte-like NADPH oxidase in rabbit aortic adventitia: enhancement by angiotensin II. 940 39

Increasing experimental evidence suggests that non-phagocytic cells express a potent superoxide (O2-.)-producing NADH oxidase that might be related to the phagocytic NADPH oxidase. Here we show that the cytokine tumour necrosis factor alpha (TNF-alpha) activates, in a time- and dose-dependent manner, a O2-.-producing NADH oxidase in cultured rat aortic smooth-muscle cells. Dose-response experiments for NADH showed an upward shift of the curve for TNF-alpha-treated cells, suggesting that TNF-alpha increased the amount of available enzyme. Using the anti-sense transfection technique, we further demonstrate that the molecular identity of this oxidase includes p22(phox) (the alpha subunit of cytochrome b558 and part of the electron transfer component of the phagocytic NADPH oxidase), which we recently cloned from a rat vascular smooth-muscle cell cDNA library. In addition, prolonged treatment with TNF-alpha increased p22phox mRNA expression without affecting p22phox mRNA stability, and only when transcriptional activity was intact. These findings identify a p22phox-containing NADH oxidase as a source for cytokine-induced free radical production in vascular smooth-muscle cells and clarify some of the mechanisms involved in the regulation of vascular oxidase activity.
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PMID:Tumour necrosis factor alpha activates a p22phox-based NADH oxidase in vascular smooth muscle. 944 95

Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b558 comprising gp91(phox) and p22(phox), and three cytosolic proteins: p47(phox) and p67(phox), each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47(phox), retaining the N-terminal and the two SH3 domains, and the N terminus of p67(phox). When either truncated p47(phox) or p67(phox) is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47(phox) and p67(phox) are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47(phox), but not the full-length one, can bind to p22(phox), a binding required for the oxidase activation. The N-terminal SH3 domain of p47(phox) is responsible for the binding not only to p22(phox), but also to the p47(phox) C terminus. Thus the SH3 domain is accessible in the active p47(phox), but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions.
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PMID:Anionic amphiphile-independent activation of the phagocyte NADPH oxidase in a cell-free system by p47phox and p67phox, both in C terminally truncated forms. Implication for regulatory Src homology 3 domain-mediated interactions. 946 21

The phagocyte NADPH oxidase flavocytochrome b558 is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91(phox), and a nonglycosylated subunit, p22(phox). It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O2), resulting in the generation of superoxide ion (O2-). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91(phox), p22(phox), or both polypeptides to examine the relative role of each flavocytochrome b558 subunit in heme binding and O2- formation. A similar membrane localization was observed when gp91(phox) and p22(phox) were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91(phox) or both gp91(phox) and p22(phox) showed a b-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome b558. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22(phox). Furthermore, redox titration studies suggested that two heme groups were contained in gp91(phox) expressed in COS7 membranes, with midpoint potentials of -264 and -233 mV that were very similar to those obtained for neutrophil flavocytochrome b558. These results provide strong support for the hypothesis that gp91(phox) is the sole heme binding subunit of flavocytochrome b558. However, coexpression of gp91(phox) and p22(phox) in COS7 membranes was required to support O2- production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b558.
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PMID:Gp91(phox) is the heme binding subunit of the superoxide-generating NADPH oxidase. 965 28


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