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)

A novel peroxidase that catalyses the dimerization of ferulic acid or caffeic acid via oxidative coupling and formation of beta beta'-linkage to the lignan-type compounds 8,8'-bis(caffeic acid) or 8,8'-bis(ferulic acid) respectively was purified from the leaves of Bupleurum salicifolium. The enzyme, for which the name caffeate peroxidase is proposed, was purified 2700-fold. It is a glycoprotein and has an Mr of 38,000 as determined by gel filtration and SDS/PAGE. The Km values for ferulic acid and caffeic acid were 0.24 mM and for H2O2 0.04 mM with caffeic acid and 0.48 mM with ferulic acid. The purified peroxidase does not exhibit activity on other phenylpropanoids tested and has no detectable phenol oxidase or NADPH oxidase activity. The caffeate peroxidase could be involved in the biosynthesis of lignans.
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PMID:Purification of a new peroxidase catalysing the formation of lignan-type compounds. 184 25

The presumed NADPH dehydrogenase function of the heterodimeric cytochrome b558 in the neutrophil oxidase complex has been investigated by combined photoaffinity labeling and immunoblot analysis of membrane proteins from bovine neutrophils. The photoaffinity probe was a radiolabeled analog of NADPH, [4-[N-(4-azido-2-nitrophenyl)[3H]amino]butyryl]NADPH ([3H]azido-NADPH), and the antibodies were directed against the C-terminal regions of the two subunits of cytochrome b558. Plasma membrane vesicles obtained by differential centrifugation of bovine neutrophil homogenates were routinely used as a source of NADPH oxidase. They were permeabilized by sodium deoxycholate to facilitate the access of NADPH or its azido analog to the totality of the specific binding sites. In the absence of light, azido-NADPH behaved as a competitive inhibitor of NADPH oxidase with a Ki of 6 microM, and was able to bind to high-affinity specific binding sites with a Kd of 5-6 microM, indicating a higher affinity of the oxidase for the photoprobe than for the substrate NADPH (KM = 30-40 microM). Upon photolabeling, the oxidase was fully inactivated. Following resolution of the membrane proteins by SDS-PAGE, a predominant photolabeled protein band of 80-100 kDa was revealed, which coincided with the large subunit (beta) of cytochrome b558 identified by immunoblot in a parallel gel. The enzymatic deglycosylation of photolabeled neutrophil membranes shifted the masses of both the photolabeled band and the immunoreactive beta subunit from 80-100 to 55-65 kDa in accordance with the glycoprotein nature of the beta subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Critical assessment of the presence of an NADPH binding site on neutrophil cytochrome b558 by photoaffinity and immunochemical labeling. 836 34

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

Plasma membranes of neutrophil cells contain the redox component of the O2(-)-generating NADPH oxidase complex, namely a heterodimeric flavocytochrome b consisting of an alpha subunit of 22 kDa and a beta subunit of 85-105 kDa of a glycoprotein nature. The NADPH oxidase is dormant in resting neutrophils. When neutrophils are exposed to a variety of particulate or soluble stimuli, the oxidase becomes activated, due to the assembly on the membrane-bound flavocytochrome b of three cytosolic factors, p47phox, p67phox and Rac 2 (or Rac 1). The effect of phenylarsine oxide (PAO), which reacts specifically with vicinal and neighbouring thiol groups in proteins, was assayed on the NADPH oxidase activity of bovine neutrophils, elicited after activation of the oxidase in a cell-free system consisting of plasma membranes and cytosol from resting neutrophils, GTP[S], ATP and arachidonic acid; the effect of PAO on the oxidase activation itself was measured independently. PAO preferentially inhibited oxidase activation rather than the elicited oxidase activity, and inhibition resulted from binding of PAO to the membrane component of the cell-free system. To determine the PAO-binding protein responsible for the loss of oxidase activation, we used photoaffinity labeling with a tritiated azido derivative of PAO, 4-[N-(4-azido-2-nitrophenyl)amino-[3H]acetamido]phenylarsine oxide, ([3H]azido-PAO). Photoirradiation of plasma membranes from resting neutrophils in the presence of [3H]azido-PAO resulted in the prominent labeling of a protein of 85-105 kDa whose migration on SDS/PAGE coincided with that of the beta subunit of flavocytochrome b as identified by immunoreaction. Upon deglycosylation, the photolabeled band at 85-105 kDa was shifted to 50-60 kDa as was the immunodetected beta subunit. Similar results were obtained with isolated flavocytochrome b in liposomes. Photoaffinity labeling of the beta subunit of the membrane-bound flavocytochrome b or the isolated flavocytochrome b in liposomes resulted in abolition of oxidase activation in the reconstituted cell-free system. Incorporation of [3H]azido-PAO into flavocytochrome b was negligible when photoaffinity labeling was performed on neutrophil membranes that had been previously activated. The results suggest that the beta subunit of flavocytochrome contains two target sites for PAO which are accessible in resting neutrophils, but not in activated neutrophils.
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PMID:Phenylarsine oxide as an inhibitor of the activation of the neutrophil NADPH oxidase--identification of the beta subunit of the flavocytochrome b component of the NADPH oxidase as a target site for phenylarsine oxide by photoaffinity labeling and photoinactivation. 949 37

This work tests the hypothesis that chronic alcohol intoxication suppresses the microbicidal activity of Kupffer cells by modulating the expression of cell surface receptors associated with respiratory burst and the release of potent microbicidal agents [i.e., reactive oxygen species (ROS)]. Because alcohol is also a potential risk factor in human immunodeficiency virus-1 (HIV-1) infection, this study examines the effect of HIV-1 glycoprotein 120 (gp120)-induced ROS release by isolated Kupffer cells. After 16 weeks of ethanol feeding, Kupffer cells from male Sprague-Dawley rats were isolated and assayed for HIV-1 gp120-induced superoxide release. Fluorescein isothiocyanate (FITC)-HIV-1 gp120 binding, NADPH oxidase, and protein kinase C activity in Kupffer cells were measured. Results show that HIV-1 gp120 induced the release of superoxide anion in a dose-dependent manner in normal rats. Mannosylated-bovine serum albumin inhibited FITC-HIV-1 gp120-mediated superoxide release in normal Kupffer cells by 85%. Moreover, 83 +/- 6% of Kupffer cells were FITC-HIV 1 gp120-positive, whereas <30% were CD4-positive. In alcohol-fed rats, HIV-1 gp120-induced ROS release was reduced by 70% and FITC-HIV-1 gp120 binding (in terms of fluorescence intensity per 10[6] Kupffer cells) by 44% in Kupffer cells, without any change in percent positive cells for this ligand. Concomitantly, HIV-1 gp120-induced translocation of NADPH oxidase to the plasma membranes of Kupffer cells in alcohol-fed rats was suppressed by 60%. In contrast, alcohol consumption significantly increased total protein kinase C activity and phorbol ester-induced superoxide release by Kupffer cells. These studies demonstrate that Kupffer cells are likely targets of HIV-1 whose binding sites on macrophages could also include mannose-specific receptors. These observations further suggest that suppression of HIV-1 gp120-mediated ROS production in chronic alcoholics is due to altered cell surface receptor expression for gp120, and defective postreceptor signaling mechanisms, which in turn could lead to attenuated microbicidal activity of hepatic macrophages.
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PMID:Chronic alcohol intoxication attenuates human immunodeficiency virus-1 glycoprotein 120-induced superoxide anion release by isolated Kupffer cells. 958 56

Chronic granulomatous disease (CGD) is a hereditary disorder of host defense due to absent or decreased activity of phagocyte NADPH oxidase. The X-linked form of the disease derives from defects in the CYBB gene, which encodes the 91-kD glycoprotein component (termed "gp91-phox") of the oxidase. We have identified the mutations in the CYBB gene responsible for X-linked CGD in 131 consecutive independent kindreds. Screening by SSCP analysis identified mutations in 124 of the kindreds, and sequencing of all exons and intron boundary regions revealed the other seven mutations. We detected 103 different specific mutations; no single mutation appeared in more than seven independent kindreds. The types of mutations included large and small deletions (11%), frameshifts (24%), nonsense mutations (23%), missense mutations (23%), splice-region mutations (17%), and regulatory-region mutations (2%). The distribution of mutations within the CYBB gene exhibited great heterogeneity, with no apparent mutational hot spots. Evaluation of 87 available mothers revealed X-linked carrier status in all but 10. The heterogeneity of mutations and the lack of any predominant genotype indicate that the disease represents many different mutational events, without a founder effect, as is expected for a disorder with a previously lethal phenotype.
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PMID:X-Linked chronic granulomatous disease: mutations in the CYBB gene encoding the gp91-phox component of respiratory-burst oxidase. 958 2

We have reported a deficiency of a 91-kDa glycoprotein component of the phagocyte NADPH oxidase (gp91(phox)) in neutrophils, monocytes, and B lymphocytes of a patient with X chromosome-linked chronic granulomatous disease. Sequence analysis of his gp91(phox) gene revealed a single-base mutation (C --> T) at position -53. Electrophoresis mobility-shift assays showed that both PU.1 and hematopoietic-associated factor 1 (HAF-1) bound to the inverted PU.1 consensus sequence centered at position -53 of the gp91(phox) promoter, and the mutation at position -53 strongly inhibited the binding of both factors. It was also indicated that a mutation at position -50 strongly inhibited PU.1 binding but hardly inhibited HAF-1 binding, and a mutation at position -56 had an opposite binding specificity for these factors. In transient expression assay using HEL cells, which express PU.1 and HAF-1, the mutations at positions -53 and -50 significantly reduced the gp91(phox) promoter activity; however, the mutation at position -56 did not affect the promoter activity. In transient cotransfection study, PU.1 dramatically activated the gp91(phox) promoter in Jurkat T cells, which originally contained HAF-1 but not PU.1. In addition, the single-base mutation (C --> T) at position -52 that was identified in a patient with chronic granulomatous disease inhibited the binding of PU.1 to the promoter. We therefore conclude that PU.1 is an essential activator for the expression of gp91(phox) gene in human neutrophils, monocytes, and B lymphocytes.
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PMID:PU.1 as an essential activator for the expression of gp91(phox) gene in human peripheral neutrophils, monocytes, and B lymphocytes. 960 Sep 21

Lactoferrin (LF) is an iron-binding glycoprotein present in various secretions including milk and the specific granules of neutrophils. The main biological properties of this protein are thought to concern the regulation of iron absorption, antimicrobial activity and modulation of neutrophil activity. Copper bound LF (Cu-LF) inhibited the stimulation-dependent reduction of cytochrome c (Cyt. c) in guinea pig peritoneal neutrophils (GPMN) but were without effect on NADPH oxidase activity of the respiratory burst. However, Cu-LF stimulated the stimulation-dependent production of hydrogen peroxide as seen with superoxide dismutase (SOD). Similar but weaker inhibition of Cyt. c reduction than that shown by Cu-LF was observed with manganese-LF (Mn-LF) but not with ferrous-LF (Fe-LF) or apo-LF (Apo-LF). The inhibitory activity was concentration-dependent and the ID50s of Cu-LF and of Mn-LF were 0.1 and 5 microM, respectively. Reactive oxygen species (ROS) detected by luminol chemiluminescence (LCL) of stimulated-GPMN were partially inhibited by Cu-LF. Changes in LCL of stimulated GPMN induced by Cu-LF were similar to those of superoxide dismutase (SOD). Thus, it is concluded that low concentrations of Cu-LF had SOD-like activity and high concentrations of Cu-LF inhibited the stimulation-dependent generation of ROS.
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PMID:Superoxide dismutase-like activity of metal substituted lactoferrin derivatives. 980 32

Protein-A, 42KD cell wall glycoprotein of S. aureus Cowan I enhance mononuclear and polymorphonuclear cell counts in vivo and possesses antitoxic, antitumor, properties. In order to explain the mechanism of its function, the respiratory burst phenomenon in cell and cell free system was studied using lucigenin-dependent chemiluminescence technique. A dose dependent increase in protein A-mediated generation of superoxide radical was observed in resting and PMA stimulated neutrophils. Superoxide dismutase (SOD) was used to confirm the production of superoxide radicals (O2-). To understand the mechanism of protein-A induced O2- generation; NADPH oxidase activity was measured in cell free system using NADPH as a substrate. A significant increase in NADPH oxidase activity was observed in the membrane and post-nuclear supernatant fraction of activated human neutrophils. Cytosolic fraction showed slight enzyme activation. Protein A (SpA)-induced NADPH oxidase activation in the membrane fraction was observed even in the absence of the substrate NADPH. These data indicate that protein A attenuate the NADPH oxidase system to produce O2- radicals.
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PMID:Protein-A activates membrane bound multicomponent enzyme complex, NADPH oxidase in human neutrophils. 1058 4


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