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)

The effects of anti-allergic drugs with or without H1-receptor antagonism on the NADPH oxidase from human neutrophils in both whole-cell and fully soluble (cell-free) systems were investigated. Three anti-allergic drugs with H1-receptor antagonism, azelastine, ketotifen and oxatomide, were found to inhibit the superoxide generation of human neutrophils exposed to phorbol myristate acetate in a whole-cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. The concentrations of three drugs required for 50% inhibition of the oxidase (IC50) were as follows: azelastin--0.7 microM in the whole-cell system and 0.5 microM in the cell-free system, ketotifen--60 microM in the whole-cell system and 6.8 microM in the cell-free system, and oxatomide--25 microM in the whole-cell system and 9.7 microM in the cell-free system. In addition, in the cell-free system, these drugs did not change the Km values for the NADPH of the oxidase. However, micromoles of tranilast, an anti-allergic drug without H1-receptor antagonism, did not inhibit neutrophil NADPH oxidase in the whole-cell and cell-free systems. The IC50 of hydrocortisone in the cell-free system was 60 microM. These results suggest that anti-allergic drugs with H1-receptor antagonism inhibit reconstitution of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in cell-free systems and that they have a strong anti-inflammatory action. Anti-allergic drugs are not the drugs of first choice for asthma, but these drugs, especially basic anti-allergic drugs, should be used more frequently for the treatment of chronic asthma, infectious-typed asthma and mixed-typed asthma closely associated with acute and chronic inflammation of the airways as well as atopic asthma.
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PMID:[Biochemical study on anti-inflammatory action of anti-allergic drugs--with regard to NADPH oxidase]. 168 45

The superoxide (O2-) forming NADPH oxidase complex of resting phagocytes can be activated in a cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). For O2- production to occur, the participation of both membrane-associated and cytosol-derived components is required. The purpose of this investigation was to isolate and characterize the membrane component of NADPH oxidase. For this purpose, guinea pig macrophage membranes were extracted with 1 M NaCl, solubilized by 40 mM octyl glucoside, and subjected to a purification sequence consisting of absorption with DEAE-Sepharose, affinity chromatography on heparin-agarose, and chromatography on hydroxylapatite. At each purification step, fractions were assayed for their ability to support SDS-elicited, cytosol-dependent O2- production, following incorporation in liposomes of phosphatidylcholine. We found that membrane oxidase activity copurified strictly with cytochrome b559. Peak hydroxylapatite fractions exhibited specific O2(-)-forming activity in the range of 81-115 mumol of O2-/mg protein/min and a specific cytochrome b559 content of 7-14 nmol of cytochrome b559/mg protein. SDS-polyacrylamide gel electrophoresis analysis of the peak oxidase activity fractions, derived by hydroxylapatite chromatography, revealed essentially two bands that were identified as the beta (54-60 kDa) and alpha (21/22 kDa) subunits of guinea pig cytochrome b559. The relation of the two polypeptides to cytochrome b559 was established by correlation with a spectral signal characteristic of cytochrome b559, immunoblotting with antibodies against defined human cytochrome b559 beta and alpha chain peptides, cross-linking studies, and deglycosylation experiments. Hydroxylapatite-purified membrane oxidase preparations did not contain FAD and were free of cytochrome c reductase activity. Purified membrane oxidase preparations were also capable of cooperating with purified cytosolic components in SDS-elicited cell-free O2- production. We conclude that the membrane-associated component of the O2- generating NADPH oxidase is identical to cytochrome b559.
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PMID:The membrane-associated component of the amphiphile-activated, cytosol-dependent superoxide-forming NADPH oxidase of macrophages is identical to cytochrome b559. 184 35

We studied the effect of bilirubin on the NADPH-dependent superoxide production induced by sodium dodecyl sulfate in a cell-free system consisting of the membrane and cytosolic fractions of pig neutrophils. Preincubation of the cytosolic fraction with bilirubin before the addition of sodium dodecyl sulfate resulted in the time- and dose-dependent inhibition of the superoxide production while the preincubation of the membrane fraction with the tetrapyrrole did not result in the inhibition. When the pigment was added after the initiation of the reaction, the ongoing production was not affected by the addition. Other tetrapyrroles, such as hemin, protoporphyrin and biliverdin, also inhibited the production. The results indicate that bilirubin inhibits the activation process of the superoxide producing NADPH oxidase by decreasing the potency of the cytosolic fraction and its inhibitory effect seems to be due to the hydrophobic nature of the tetrapyrrole.
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PMID:Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system. 184 4

Phagocytic leukocytes contain an activatable NADPH:O2 oxidoreductase. Components of this enzyme system include cytochrome b558, and three soluble oxidase components (SOC I, SOC II, and SOC III) found in the cytosol of resting cells. Previously, we found that SOC II copurifies with, and is probably identical to, a 47-kDa substrate of protein kinase C. In the present study we investigated the change in location of several of these oxidase components after activation of intact neutrophils with phorbol myristate acetate (PMA) and separation of subcellular fraction on sucrose density gradients. On Western blots with fractions of resting cells, the alpha subunit of cytochrome b558 was detected with a monoclonal antibody as a doublet of Mr 22,000 and 24,000 in the specific granules and as a single band of Mr 24,000 in the plasma membrane. PMA induced an increase of cytochrome b558 in the plasma membrane, including the Mr 22,000 band. PMA also induced translocation of the 47-kDa protein from the cytosol to the membrane fraction, as revealed by in vitro phosphorylation experiments. When NADPH oxidase activity was determined in a cell-free system in the presence of sodium dodecyl sulfate and GTP with plasma membranes from resting cells, cytosol from PMA-treated cells was deficient compared with cytosol from resting cells. This deficiency could be partially restored by the addition of SOC I. Concomitantly, SOC I activity appeared in the plasma membranes of PMA-treated cells. These studies support the hypothesis that PMA stimulation of neutrophils results in assembly of oxidase components from the cytosol and the specific granules in the plasma membrane with subsequent expression of NADPH oxidase activity.
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PMID:Assembly and activation of the NADPH:O2 oxidoreductase in human neutrophils after stimulation with phorbol myristate acetate. 215 19

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

Cytoplasmic pH (pHi) has been shown to be an important determinant of the activity of the NADPH oxidase in phagocytic cells. We hypothesized that a difference in pHi and/or its regulation existed between activated and resident macrophages (RES MOs) which might explain the increased NADPH oxidase activity observed in the former. The pHi of RES and lipopolysaccharide (LPS)-elicited MOs was examined using the fluorescent dye BCECF. Resting pHi did not differ between resident (RES) and elicited (ELI) MOs (7.16 +/- 0.05 and 7.20 +/- 0.05, respectively). pHi recovery after intracellular acid loading was partially dependent on the presence of Na+ in the extracellular medium, and was partially inhibited by the Na+/H+ antiport inhibitor, amiloride. At comparable pHi, the rate of acid extrusion during recovery was not different in RES and ELI MOs (1.48 +/- 0.12 and 1.53 +/- 0.06 mM/min, respectively). In both RES and ELI MOs, approx. 40% of total pHi recovery was insensitive to amiloride and independent of extracellular Na+. In both RES and ELI MOs, stimulation with TPA resulted in a biphasic pHi response: an initial acidification followed by a sustained alkalinization to a new steady-state pHi. This alkalinization was Na(+)-dependent and amiloride-sensitive, consistent with a TPA-induced increase in Na+/H+ antiport activity. The new steady-state pHi attained after TPA stimulation was equivalent in RES and ELI MOs (7.28 +/- 0.04 and 7.31 +/- 0.06, respectively), indicating comparable stimulated Na+/H+ antiport activity. However, the initial acidification induced by TPA was greater in ELI than in RES MOs (0.18 +/- 0.02 vs. 0.06 +/- 0.02 pH unit, respectively, P less than 0.05). The specific NADPH oxidase inhibitor diphenylene iodonium (DPI) completely inhibited the respiratory burst but reduced the magnitude of this pHi reduction by only about 50%. This suggested that the TPA-induced pHi reduction was due in part to acid produced via the respiratory burst, and in part to other acid-generating pathways stimulated by TPA.
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PMID:Regulation of cytoplasmic pH in resident and activated peritoneal macrophages. 215 21

The kinetics of sodium dodecyl sulfate-induced activation of respiratory burst oxidase (NADPH oxidase) in a fully soluble cell-free system from resting (control) or phorbol myristate acetate (PMA)-stimulated human neutrophils were investigated. In a cell-free system containing solubilized membranes and cytosol fractions (cytosol) derived from control neutrophils (control cell-free system), the values of Km and Vmax for NADPH of the NADPH oxidase from control neutrophils continuously increased with increasing concentrations of cytosol, but with increasing concentrations of solubilized membranes from the control neutrophils, Km values continuously decreased, suggesting cytosolic activation factor-dependent continuous changes in the affinity of NADPH oxidase to NADPH. In a cell-free system containing solubilized membranes and cytosol prepared from PMA-stimulated neutrophils, NADPH oxidase was not activated after the addition of NADPH. However, cytosol from control neutrophils activated the NADPH oxidase of PMA-stimulated neutrophils in a cell-free system. Cytosol from PMA-stimulated neutrophils did not activate the control neutrophil oxidase, although it contained no inhibitors of NADPH oxidase activation. The results suggest that, in PMA-stimulated neutrophils, cytosolic activation factors may be consumed or exhausted with an increasing period of time after the stimulation of neutrophils, and that the affinity of PMA-stimulated neutrophil NADPH oxidase to NADPH may almost be the same as that of control neutrophil oxidase. It was concluded that the affinity of NADPH oxidase to NADPH was closely associated with interaction between solubilized membranes and cytosolic activation factors, as indicated by the concentration ratio.
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PMID:Human neutrophil cytosolic activation factor of the NADPH oxidase. Characterization of activation kinetics. 215 61

The effects of hydrocortisone on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole-cell and full soluble (cell-free) systems were investigated. In the whole-cell system, hydrocortisone inhibited the generation of superoxide by neutrophils exposed to phorbol myristate acetate, suggesting that steroids inhibit the bactericidal capacity of the body in an acute inflammatory phase. Hydrocortisone, which was added to the cuvette after the addition of NADPH and before the addition of sodium dodecyl sulfate, in a cell-free system, was found to inhibit the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate. The concentration of hydrocortisone required for 50% inhibition of oxidase was 40 microM. Its inhibition was dose- and time-dependent in the cell-free system. However, hydrocortisone did not alter the Km of the oxidase for NADPH. These results suggest that steroids inhibit the reconstitution of NADPH oxidase by sodium dodecyl sulfate in the cell-free system, and that they do not alter the affinity to NADPH of the oxidase.
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PMID:Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems. 215 98

The superoxide (O2-)-generating NADPH oxidase of resting macrophages can be activated in a soluble cell-free system by certain anionic amphiphiles, such as sodium dodecyl sulfate (SDS). We demonstrate that cell-free activation is specifically enhanced by nonhydrolyzable guanosine 5'-triphosphate (GTP) analogues. Guanosine 5'-diphosphate (GDP) and guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) prevent cell-free oxidase activation and reverse the activated state when added to preactivated oxidase preparations. Nonhydrolyzable GTP analogues have a protective effect on the SDS-stimulated NADPH oxidase, as shown by the maintenance of more than 90% and close to 60% of enzyme activity 6 and 24 hr, respectively, after the addition of SDS to the cell-free preparation. A novel procedure is described for separating the activated NADPH oxidase from SDS and added nucleotides by gel filtration of the SDS-stimulated solubilized membrane-cytosol mixtures through a Sephadex G-25 column. By utilizing this method, it was found that the presence of micromolar concentrations of nonhydrolyzable GTP analogues during activation by SDS results in a marked increase in the recovery of SDS-independent, NADPH-dependent O2(-)-producing activity in the excluded volume of the column. It is suggested that GTP stabilizes the SDS-induced complex between membrane and cytosolic components of the oxidase. Cell-free activation of NADPH oxidase by SDS was found to be cholera and pertussis toxin insensitive. These results serve as evidence of the participation of a G protein in the activation of the O2(-)-generating NADPH oxidase of macrophages by SDS.
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PMID:Activation of the superoxide-generating NADPH oxidase of macrophages by sodium dodecyl sulfate in a soluble cell-free system: evidence for involvement of a G protein. 216 54

The effects of non-steroidal anti-inflammatory drugs (NSAIDs) on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole cell and fully soluble (cell-free) systems were investigated. Three NSAIDs, indomethacin, salicylic acid and acetylsalicylic acid (aspirin), were found to inhibit the superoxide generation by human neutrophils exposed to phorbol myristate acetate in a whole cell system and the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate in a cell-free system. Concentrations of these NSAIDs required for 50% inhibition of the oxidase (IC50) were: indomethacin (180 microM in both systems), salicylic acid (1.30 mM in the cell-free system, and more than 3.0 mM in the whole cell system) and acetylsalicylic acid (1.35 mM in the cell-free system, and more than 3.0 mM in the whole cell system). In addition, in the cell-free system, these NSAIDs did not change the Km values for NADPH of the oxidase. These results suggest that these NSAIDs, especially indomethacin, concentration-dependently inhibit the reconstitution of the solubilized membrane-bound enzyme by sodium dodecyl sulfate in the cell-free system.
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PMID:Effects of non-steroidal anti-inflammatory drugs on human neutrophil NADPH oxidase in both whole cell and cell-free systems. 216 17


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