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 enzyme 4-hydroxyphenylacetate, NAD(P)H:oxygen oxidoreductase (1-hydroxylating) (EC 1.14.13 ...; 4-hydroxyphenylacetate 1-monooxygenase; referred to here as 4-HPA 1-hydroxylase) was induced in Pseudomonas acidovorans when 4-hydroxyphenylacetate (4-PHA) was utilized as carbon source for growth; homogentisate and maleylacetoacetate were intermediates in the degradation of 4-HPA. A preparation of the hydroxylase that was free from homogentisate dioxygenase and could be stored at 4 C in the presence of dithioerythritol with little loss of activity was obtained by ultracentrifuging cell extracts; but when purified 18-fold by affinity chromatography the enzyme became unstable. Flavin adenine dinucleotide and Mg2+ ions were required for full activity. 4-HPA 1-hydrocylase was inhibited by KCl, which was uncompetitive with 4-HPA. Values of Ki determined for inhibitors competitive with 4-HPA were 17 muM dl-4-hydroxymandelic acid, 43 muM 3,4-dihydroxyphenylacetic acid, 87 muM 4-hydroxy-3-methylphenylacetic acid, and 440 muM 4-hydroxyphenylpropionic acid. Apparent Km values for substrates of 4-HPA 1-hydroxylase were 31 muM 4-HPA, 67 muM oxygen, 95 muM reduced nicotinamide adenine dinucleotide (NADH); AND 250 muM reduced nicotinamide adenine dinucleotide phosphate (NADPH). The same maximum velocity was given by NADH and NADPH. A chemical synthesis is described for 2-deutero-4-hydroxyphenylacetic acid. This compound was enzymatically hydroxylated with retention of half the deuterium in the homogentisic acid formed. Activity as substrate or inhibitor of 4-HPA 1-hydroxylase was shown only by those analogues of 4-HPA that possessed a hydroxyl group substituent at C-4 of the benze nucleus. A mechanism is suggested that accounts for this structural requirement and also for the observation that when 4-hydroxyphenoxyacetic acid was attacked by the enzyme, hydroquinone was formed by release of the side chain, probably as glycolic acid. Only one enantiometer of racemic 4-hydroxyhydratropic acid was attacked by 4-HPA 1-hydroxylase; the product, alpha-methylhomogentisic acid (2-(2,5-dihydroxyphenyl)-propionic acid), exhibited optical activity. This observation suggests that, during its shift from C-1 to C-2 of the nucleus, the side chain of the substrate remains bound to a site on the enzyme while a conformational change of the protein permits the necessary movement of the benzene ring.
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PMID:Metabolic function and properties of 4-hydroxyphenylacetic acid 1-hydroxylase from Pseudomonas acidovorans. 23 37

The activity of the hepatic microsomal ethanol-oxidizing system (MEOS) was compared with the content of three forms of cytochrome P-450. Measurements were also made of the activity of microsomal reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, the enzyme which generates H2O2 in microsomes and which has been considered by some to be the rate-limiting step of MEOS activity. Ethanol feeding to rats for 4 to 5 weeks significantly enhanced the activities of MEOS and NADPH oxidase by 102 and 62%, respectively. Concomitantly, form I of cytochrome P-450 was increased by 88% (P less than .001). Acute administration of a large dose of ethanol to animals pretreated chronically with ethanol enhanced MEOS activity by 21% (P less than .05), whereas NADPH oxidase activity remained unchanged. In addition, an acute dose of ethanol enhanced form I of cytochrome P-450 by 20% (P less than .05); thus its increase was comparable to that of MEOS activity. Pretreatment of rats with phenobarbital increased the specific activity of microsomal NADPH oxidase by 40% (P less than .05) but not that of MEOS. By contrast, CCl4 administration to rats diminished MEOS activity by 33% (P less than .01), whereas NADPH oxidase activity remained unchanged. The CCl4 treatment was found to decrease significantly all three forms of cytochrome P-450: form I by 45%, form II by 56% and form III by 24%. These results suggest that in the presence of NADPH microsomes oxidize ethanol to acetaldehyde by a process which involves, at least in part, the form I of cytochrome P-450 and in which H2O2 generation by NADPH oxidase is not the rate-limiting step.
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PMID:Hepatic microsomal ethanol-oxidizing system (MEOS): dissociation from reduced nicotinamide adenine dinucleotide phosphate oxidase and possible role of form I of cytochrome P-450. 115 72

Peripheral blood leukocytes from patients given corticosteroid or radiation therapy, as well as patients with bacterial or viral infections, were studied with regard to the selected enzyme activities of the hexose monophosphate shunt (HMS). Glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD) and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase were assayed spectrophotometrically on mixed leukocyte suspensions in isotonic glycerol. Enzyme activities of G-6PD and NADPH oxidase in patients receiving corticosteroid or radiation therapy were significantly lower than the enzyme activity of 6-PGD. In patients with bacterial infections, activities of the three enzymes increased but in patients with viral infections, only the activities of NADPH oxidase and G-6PD were slightly decreased. Nitroblue tetrazolium (NBT) dyereducing activities of neutrophils from patients receiving corticosteroid or radiation therapy were attenuated which coincides with the reduced activities of HMS enzymes. From these results, it is likely that the reduced activities of intraleukocytic HMS enzymes of patients receiving corticosteroid or radiation therapy are correlated with intracellular bactericidal activities which might result from the attenuated level of hydrogen peroxide production.
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PMID:The metabolic and phagocytic activities of leukocytes from patients receiving corticosteroid and radiation therapy, and patients with bacterial infections. 117 10

The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47phox, p67phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b558. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b558. Alignment of the amino acid sequence of the beta subunit of cytochrome b558 (gp91phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b558 is the only obligate electron transporting component of the NADPH oxidase.
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PMID:Cytochrome b558: the flavin-binding component of the phagocyte NADPH oxidase. 131 79

Low-temperature electron paramagnetic resonance (EPR) spectrometry on granulocytes prepared from pig blood was carried out with concentrated cellular and subcellular fractions to characterize EPR signals of cytochrome b-558 (cyt b-558). A thick cell suspension (approximately 2 x 10(9) cells/ml), containing mostly neutrophils, showed typical high-spin EPR signals due to myeloperoxidase (MPO) and a low spin signal at a g value of around 3.2. A similar thick granulocyte suspension containing eosinophils showed not only these signals but also low spin heme signals at g values of 2.86, 2.13, and 1.66, which have been reported to be of cyt b-558 (Ueno et al. 1991, FEBS Lett. 281, 130-132). MPO and eosinophil peroxidase (EPO) were released from the membrane fractions with 50 mM phosphate buffer (pH 7.0) containing 1 M NaCl, and then were highly concentrated, in which no cyt b-558 was detected by absorption spectra. The signal at a g value of 2.86 was found only in the EPO fraction, suggesting that this signal is derived from a low-spin form of an EPO-complex, but neither from MPO nor cyt b-558. The O2(-)-forming NADPH oxidase associated in the membranes was solubilized with heptyl-thio-glucoside at 0 degree C and concentrated up to 45 microM cyt b-558 with no modification of the heme moiety confirmed by its O2(-)-generating activity and lack of carbon monoxide-binding capacity. Cyt b-558 showed an anisotropic signal at a g value of 3.2 +/- 0.05, which was cyanide-insensitive and reducible with reductants. The signal intensity was concentration dependent, suggesting that the g = 3.2 signal is characteristic of the low-spin heme iron in cyt b-558.
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PMID:Electron paramagnetic resonance studies on cytochrome b-558 and peroxidases of pig blood granulocytes. 132 37

The mechanism of cAMP regulation of the respiratory burst was studied with HL-60 cells that had been DMSO-differentiated to a neutrophil-like cell. To evaluate the effects of known cAMP concentrations, cells were permeabilized with streptolysin-O. Chemotactic peptide (FMLP)-stimulated NADPH oxidase activity was inhibited by cAMP at concentrations higher than 3 microM. Because intracellular calcium was buffered, inhibitory actions of cAMP were not mediated by modulation of calcium concentration. Effects of cAMP on chemotactic peptide signal transduction mediated by phospholipase C, phospholipase D, and phospholipase A2 were then determined. Neither inositol phosphate generation (phospholipase C) nor phosphatidylethanol generation (phospholipase D activity in presence of 1.6% ethanol) induced by FMLP were significantly affected by cAMP. In contrast, cAMP potently inhibited FMLP-induced arachidonic acid mobilization (phospholipase A2). NADPH oxidase activity induced by exogenous arachidonic acid was not inhibited by cAMP. These results indicate that cAMP-mediated inhibition of arachidonic acid mobilization may be important in regulation of the respiratory burst.
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PMID:Regulation of the respiratory burst by cyclic 3',5'-AMP, an association with inhibition of arachidonic acid release. 133 10

Cytosolic low molecular components in guinea pig neutrophils were examined for the activity to enhance superoxide anion (O2-)-generating NADPH oxidase activity. A component was separated by Sephadex G-25 gel filtration from high molecular weight components, the latter revealed NADPH oxidase activity in a cell-free system in combination with the membrane fraction and arachidonic acid. Addition of this cytosolic low molecular weight component to the cell-free system significantly enhanced NADPH oxidase activity, though this component did not substitute the high molecular weight components in constituting the system. The low molecular weight NADPH oxidase activation factor (LMWAF) found here was not of protein nature, since protease treatment failed to reduce its activity. This factor did not contain phosphate, and was neither flavin nor guanine nucleotide. Though LMWAF was extractable with chloroform-methanol, it was not identical with diacylglycerol.
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PMID:A novel low molecular weight factor detected in the cytosol of guinea pig neutrophils to enhance superoxide anion production. 133 84

Effects of non-steroidal anti-inflammatory drugs (NSAID: amfenac sodium, diclofenac sodium, indomethacin and ketoprofen) on the generation of superoxide anion (O2-) by isolated rat polymorphonuclear leukocytes (PMN) were studied spectrophotometrically using cytochrome c. The effects of these drugs were also studied on O2- production by the xanthine-xanthine oxidase and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-NADPH oxidase systems. Amfenac sodium, at 0.1 mM, inhibited significantly O2- generation in rat PMN induced by opsonized zymosan. At 0.5 mM, diclofenac sodium and indomethacin inhibited the O2- generation in rat PMN. All of the above drugs slightly inhibited O2- production by the xanthine-xanthine oxidase system. On the other hand, O2- production by the NADPH-NADPH oxidase system was significantly inhibited by the addition of amfenac sodium, ketoprofen or indomethacin. These results suggest that non-steroidal anti-inflammatory drugs do not work as an O2- scavenger and block O2- production by the NADPH-NADPH oxidase system of rat PMN. It is concluded that amfenac sodium and the other drugs are able to inhibit granulocyte O2- production by blocking the activation of NADPH-oxidase.
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PMID:Inhibitory effects of non-steroidal anti-inflammatory drugs on superoxide generation. 165 19

A major action of the microbicidal system of human neutrophils is the formation of superoxide anion (O2-) by a multicomponent oxidase that transfers electrons from the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) to molecular oxygen. The mechanism of assembly and activation of the oxidase from its cytosolic and membrane-bound components is unknown, but may require the activity of a guanosine 5'-triphosphate (GTP)-binding component. A cytosolic GTP-binding protein (Gox) that regulates the NADPH oxidase of neutrophils was identified. Gox was purified and shown to augment the rate of O2- production in a cell-free oxidase activation system. Sequence analysis of peptide fragments from Gox identified it as Rac 2, a member of the Ras superfamily of GTP-binding proteins. Antibody to a peptide derived from the COOH-terminus of Rac 2 inhibited O2- generation in a concentration-dependent manner. These results suggest that Rac 2 is a regulatory component of the human neutrophil NADPH oxidase, and provide new insights into the mechanism by which this oxygen radical-generating system is regulated.
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PMID:Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. 166 Jan 88

Chronic granulomatous diseases (CGDs) are characterized by recurrent infections resulting from impaired superoxide production by a phagocytic cell, nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) oxidase. Complementary DNAs were cloned that encode the 67-kilodalton (kD) cytosolic oxidase factor (p67), which is deficient in 5% of CGD patients. Recombinant p67 (r-p67) partially restored NADPH oxidase activity to p67-deficient neutrophil cytosol from these patients. The p67 cDNA encodes a 526-amino acid protein with acidic middle and carboxyl-terminal domains that are similar to a sequence motif found in the noncatalytic domain of src-related tyrosine kinases. This motif was recently noted in phospholipase C-gamma, nonerythroid alpha-spectrin (fodrin), p21ras-guanosine triphophatase-activating protein (GAP), myosin-1 isoforms, yeast proteins cdc-25 and fus-1, and the 47-kD phagocyte oxidase factor (p47), which suggests the possibility of common regulatory features.
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PMID:Cloning of a 67-kD neutrophil oxidase factor with similarity to a noncatalytic region of p60c-src. 169 59


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