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)

Recent evidence suggests that a number of non-phagocytic cell types may contain a superoxide generating NADPH oxidase. Studies to data on cultured human fibroblasts have primarily concerned the identification of cytochrome b558, whilst expression of other NADPH oxidase components have not been addressed. In this study we have investigated the expression of NADPH oxidase with particular reference to the cytosolic factors p47-phox and p67-phox. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed that human fibroblasts express mRNA for p47-phox, p67-phox and p22-phox. Expression of the gp91-phox transcript was not detected, indicating that human fibroblasts may possess an NADPH oxidase isoenzyme. Western blot analysis of human fibroblast cytosol, using an anti-p47-phox antibody (JW-1), identified a 47 kDa protein. Cell-free reconstitution assays showed that fibroblast cytosol could initiate superoxide generation when mixed with either human fibroblast membranes (0.16 nmol superoxide/min/microgram membrane protein), or resting human neutrophil membranes (0.20 nmol superoxide/min/microgram membrane protein). These data indicate that the expression of p47-phox and p67-phox by human fibroblasts may contribute to the cells' generation of superoxide.
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PMID:The functional expression of p47-phox and p67-phox may contribute to the generation of superoxide by an NADPH oxidase-like system in human fibroblasts. 798 96

Phagocytes are able to generate reactive oxygen species by an activatable NADPH oxidase system. We investigated the inhibition of NADPH oxidase activation by a methoxy-substituted catechol, apocynin. Oxygen uptake by neutrophils incubated with 300 microM apocynin was completely inhibited at 7 min after addition of serum-treated zymosan (STZ), with a lagtime of inhibition of 2 to 3 min. The lagtime of effect of apocynin in neutrophils relatively deficient of myeloperoxidase was about 50% longer when compared with normal cells. Inhibition of the STZ-induced respiratory burst by apocynin was also observed in human eosinophils but not in human alveolar macrophages. Immunoblots of neutrophil membranes, isolated at 2 and 7 min after STZ stimulation of neutrophils, demonstrated translocation of the cytosolic oxidase components p47-phox and p67-phox to the membrane fraction. Translocation at 7 min after STZ stimulation was markedly reduced when the neutrophils had been incubated with 300 microM apocynin, but translocation was normal after 2 min of stimulation. These properties suggest that apocynin is an intracellular inhibitor of the assembly of NADPH oxidase in neutrophils and eosinophils and that apocynin requires conversion by peroxidases to exert its inhibitory effect. The capacity of neutrophils for intracellular killing of Staphylococcus aureus was not affected by apocynin. The potential therapeutic value of apocynin was demonstrated in vitro by its ability to protect secretory leukocyte proteinase inhibitor from oxidative inactivation by neutrophils.
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PMID:Characteristics of the inhibition of NADPH oxidase activation in neutrophils by apocynin, a methoxy-substituted catechol. 801 41

The phagocyte superoxide-generating NADPH oxidase, a multicomponent, membrane-bound electron transport chain, consists of cytochrome b558, p47-phox, p67-phox, and p21rac1 or p21rac2. The mechanisms of oxidase assembly are poorly understood. In previous studies using a cell-free NADPH oxidase system, we showed that preincubation of neutrophil membrane with neutrophil cytosol containing p47-phox, but not p67-phox, led to formation of a long-lived NADPH oxidase intermediate. This suggested that p47-phox interacted with cytochrome b558 in the early stages of oxidase assembly while p67-phox participated in a later stage. Peptides containing the sequence RGVHFIF (corresponding to amino acids 559-565 of the 91-kDa subunit of cytochrome b558) inhibit NADPH oxidase activity by blocking the early interaction between p47-phox and cytochrome b558. In the present study, we examined whether p21rac facilitated the interaction between p47-phox and cytochrome b558. We preincubated pure recombinant p47-phox with neutrophil membrane containing cytochrome b558 in the cell-free system. Superoxide-generating activity was subsequently reconstituted by adding pure rp67-phox and partially purified p21rac. RGVHFIF inhibited superoxide production if added to the cell-free system during preincubation of rp47-phox with membrane. RGVHFIF was markedly less inhibitory if added to the cell-free system after membrane was preincubated with pure rp47-phox. In contrast to p47-phox, preincubation of membrane with either p21rac or rp67-phox conferred no protection from inhibition of superoxide-generating activity by RGVHFIF added after preincubation.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:p21rac does not participate in the early interaction between p47-phox and cytochrome b558 that leads to phagocyte NADPH oxidase activation in vitro. 811 10

We have investigated the relationship between the expression of the p47-phox and p67-phox cytosolic components of the NADPH oxidase and priming of the macrophage respiratory burst. Western blot analysis revealed that murine bone marrow-derived macrophages (BMM) contain immunoreactive proteins detected by antisera raised against recombinant human p47-phox and p67-phox. Priming BMM by exposure to tumor necrosis factor alpha (TNF-alpha) or lipopolysaccharide (LPS) increased the levels of p47-phox and p67-phox. Colony-stimulating factor 1 (CSF-1), which we previously found to have a negative effect on the priming of murine macrophages, had no effect on the level of p47-phox but down-regulated that of p67-phox. Our results suggest that the regulatory effects of LPS, TNF-alpha, and CSF-1 on the respiratory burst of BMM may be due to modulation of the expression of the p47-phox and p67-phox cytosolic components of the NADPH oxidase.
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PMID:Expression of p47-phox and p67-phox proteins in murine bone marrow-derived macrophages: enhancement by lipopolysaccharide and tumor necrosis factor alpha but not colony stimulating factor 1. 814 24

The superoxide-forming NADPH oxidase of human phagocytes is composed of membrane-bound and cytosolic proteins which, upon cell activation, assemble on the plasma membrane to form the active enzyme. Patients suffering from chronic granulomatous disease (CGD) are defective in one of the following components: p47-phox and p67-phox, residing in the cytosol of resting phagocytes, and gp91-phox and p22-phox, constituting the membrane-bound cytochrome b558. In an X-linked CGD patient we identified a novel missense mutation predicting an Asp-->Gly substitution at residue 500 of gp91-phox, associated with normal amounts of nonfunctional cytochrome b558 in the patient's neutrophils. In PMA-stimulated neutrophils and in a cell-free translocation assay with neutrophil membranes and cytosol, the association of the cytosolic proteins p47-phox and p67-phox with the membrane fraction of the patient was strongly disturbed. Furthermore, a synthetic peptide mimicking domain 491-504 of gp91-phox inhibited NADPH oxidase activity in the cell-free assay (IC50 about 10 microM), and the translocation of p47-phox and p67-phox in the cell-free translocation assay. We conclude that residue 500 of gp91-phox resides in a region critical for stable binding of p47-phox and p67-phox.
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PMID:A point mutation in gp91-phox of cytochrome b558 of the human NADPH oxidase leading to defective translocation of the cytosolic proteins p47-phox and p67-phox. 818 43

Src homology 3 (SH3) domains direct cellular localization and signal transduction through specific protein-protein interactions with proline-rich target sequences. The two SH3 domain in p67-phox, a cytosolic component of the phagocyte NADPH oxidase system, may mediate interactions within the oxidase complex and direct its translocation to membranes. The requirement for SH3 domains in p67-phox was studied both in cell-free and whole cell oxidase assay systems. The amino-terminal domain of p67-phox (amino acids 1-246) that lacks both SH3 domains was active in vitro. Various forms of p67-phox lacking one or both SH3 domains were produced in whole cells using episomal expression vectors to stably transfect p67-phox-deficient Epstein-Barr virus-B cells derived from chronic granulomatous disease patients. Complete restoration of NADPH oxidase activity was achieved with full-length p67-phox cDNA expression. Deletion of either SH3 domain resulted in dramatic reductions of NADPH oxidase activity relative to corrected transfected cells, which correlated with decreases in membrane binding. Deletion of both SH3 domains completely abolished p67-phox membrane binding and oxidase activity. Thus, in contrast to oxidase reconstitution in a cell-free system, we observed a requirement for both SH3 motifs for restoration of oxidase activity and binding of p67-phox to membranes.
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PMID:Role of p67-phox SH3 domains in assembly of the NADPH oxidase system. 820 39

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages. 822 83

The superoxide-generating NADPH oxidase of neutrophils can be activated in a cell-free system consisting of cell membranes, cytosol and an activating detergent (e.g. arachidonate or SDS). It has previously been reported [Aviram and Sharabani (1989) Biochem. Biophys. Res. Commun. 161, 712-719] that a mixture of phosphoinositides (PPIs), as well as the individual inositol lipids, interfere with the activation process. In the present study it is shown that exposure of the cytosol to PPI results in a progressive (t1/2 = 30 s) loss of its oxidase-supporting activity and that Mg2+ ions eliminate this inactivation. Neomycin, previously described as an inhibitor of cell-free activation, counteracted the effect of PPI and vice versa. Fractionation experiments implicated the p67-phox cytosolic component of the oxidase in the association with PPI. PPI blocked activity of recombinant p67-phox also and quenched the fluorescence intensity of its tryptophan residues. It is suggested that PPIs may mediate the interaction of the oxidase with the cytoskeleton and/or with the membrane.
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PMID:The interaction of cytosolic components of neutrophil NADPH oxidase with phosphoinositides. 824 Feb 58

The NADPH oxidase generates superoxide in phagocytic cells. It is important for immunity and its deficiency leads to chronic granulomatous disease (CGD). It consists of a membrane-bound flavocytochrome b that lies dormant until activated by the translocation to the plasma membrane of cytosolic proteins, p47phox (phox for phagocyte oxidase), p67phox and p21rac, a small GTP-binding protein. We show here that a novel component, p40phox, forms an activation complex with p47phox and p67phox with which it translocates to the membrane to associate with the flavocytochrome b. cDNA cloning and amino acid analysis revealed that p40phox has an src homology 3 (SH3) domain and a large region of sequence similarity with the N-terminus of p47phox. The primary association of p40phox appears to be with p67phox, and it is present in reduced amounts in patients with CGD lacking p67phox.
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PMID:p40phox, a third cytosolic component of the activation complex of the NADPH oxidase to contain src homology 3 domains. 828 52

Chronic granulomatous disease (CGD) is characterized by the failure of phagocytic leukocytes to kill certain bacteria and fungi. This is caused by deficiencies in one of the components of NADPH oxidase, the enzyme in phagocytic leukocytes that generates superoxide. In a rare, autosomal recessive form of CGD, a 67-kD cytosolic component of NADPH oxidase (p67-phox) is missing. Until now, mutations in the gene coding for this protein have not been identified. We now report on a 10-year-old girl with lymph node and liver abscesses who was recognized as an A67(0) CGD patient by lack of NADPH oxidase activity in her granulocytes, a cytosolic defect in a cell-free oxidase system, and lack of immunoreactive material with an antiserum against the p67-phox protein. mRNA for this protein was present in normal amounts in her monocytes. This p67-phox mRNA was reverse-transcribed, and the coding region was amplified by polymerase chain reaction in six overlapping fragments and was sequenced. The patient appeared to be homozygous for a G-233-->A mutation, resulting in a nonconservative amino acid change (78Gly-->Glu). This mutation was also found in the genomic DNA of this patient but not in that of 38 normal donors. Both parents and a sister proved to be carriers of the disease, as deduced from the mutation in only one allele. The carrier state was also manifested by intermediate superoxide production by their intact granulocytes and in the cell-free system.
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PMID:Autosomal recessive chronic granulomatous disease with absence of the 67-kD cytosolic NADPH oxidase component: identification of mutation and detection of carriers. 828 49


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