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:1.17.3.2 (xanthine oxidase)
8,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The murine malaria parasite Plasmodium yoelii was killed in vitro when incubated with glucose and glucose oxidase, a system generating hydrogen peroxide, or with xanthine and xanthine oxidase, a system which produces the superoxide anion and subsequently other products of the oxidative burst. Catalase blocked the killing in both cases; superoxide dismutase and scavengers of hydroxyl radicals or singlet oxygen were ineffective in the xanthine oxidase system. Thus, hydrogen peroxide appears to be the main reactive oxygen species killing P. yoelii.
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PMID:Killing of Plasmodium yoelii by enzyme-induced products of the oxidative burst. 654 75

In addition to the phosphate residues contained in the acid-dissociable FAD and the molybdenum cofactor moieties, milk xanthine oxidase contains one mole of covalently bound phosphorus per active-center molybdenum. Acid hydrolysis of the apoprotein moiety and subsequent analysis by high-voltage thin-layer electrophoresis has identified the phosphorylated amino acid residue to be phosphoserine. 31P NMR data show the phosphopeptide to be monosubstituted, in agreement with the chemical analysis. A pH-dependent chemical shift of the phosphorus residue in the molybdenum cofactor moiety is also observed which provides unequivocal support for suggestions in the literature that this cofactor contains a monosubstituted phosphate. 31P NMR studies on the intact enzyme show phosphorus resonances at about -3 ppm, +1 ppm, +8.8 ppm and at +13.5 ppm. The resonances at +8.8 ppm and at +13.5 ppm are assigned to those of the pyrophosphate linkage of the FAD moiety by analogy with chemical shift data of the FAD on glucose oxidase [James, T.L., Edmondson, D.E., and Husain, M. (1981) Biochemistry 20, 617] and from the absence of any resonances in this region upon examination of preparations of deflavo xanthine oxidase. The intensity and resolution of the resonance at about -3 ppm is dependent on the degree of functionality of the enzyme. This resonance has a small amplitude relative to the FAD resonances in 50-60% functional enzyme, but increases dramatically in intensity in the desulpho enzyme. This resonance is the only one exposed to solvent as it is the only one susceptible to paramagnetic line-broadening on the addition of Mn(II) to the enzyme solution. Treatment of the enzyme with allopurinol leads to alteration of the approximately equal to -3-ppm resonance, but does not significantly affect the other resonances. Formation of the stable Mo(V) 'inhibited' form of the enzyme with ethylene glycol results in extensive line-broadening of the resonances at -3 ppm and +1 ppm, but has no observable affect on the FAD resonances. These data suggest that in addition to the phosphate on the molybdenum cofactor, the phosphoserine residue in xanthine oxidase is also in close proximity to the activesite molybdenum center of this enzyme. These results are discussed with respect to possible implications on the catalytic mechanism of the enzyme.
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PMID:31P nuclear magnetic resonance and chemical studies of the phosphorus residues in bovine milk xanthine oxidase. 654 6

In order to understand why different stages of Trichinella spiralis vary in their susceptibility to killing by leukocytes, the effects of artificially generated oxidants on different stages of this parasite were compared. More than 90% newborn larvae were killed after incubation in acetaldehyde-xanthine oxidase or glucose-glucose oxidase. On the other hand, fewer than 10% of adult worms or muscle larvae were killed when incubated under identical conditions. Thus, only the stages which are resistant to killing by leukocytes are resistant to killing by oxidants. The larvicidal effect of acetaldehyde-xanthine oxidase was blocked by the addition of either superoxide dismutase or catalase and was partially inhibited by radical scavengers and singlet oxygen quenchers. The oxidant resistant adults and muscle larvae contained 3-5 times more superoxide dismutase and at least five times more glutathione peroxidase than the oxidant sensitive newborn larvae. In contrast, all 3 stages lacked detectable amounts of catalase and contained roughly equivalent amounts of reduced glutathione. Accordingly, adults and muscle larvae may be more resistant to killing by leukocytes than newborn larvae because they contain better oxidant defenses.
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PMID:Scavenger enzymes and resistance to oxygen mediated damage in Trichinella spiralis. 669 69

Mechanisms of H2O2-induced cell injury were explored in primary cultures of rat hepatocytes. Cells prepared from male rats and cultured for 1 day prior to treatment were killed by H2O2 either added directly to the medium at 0.25-2 mM or generated in situ by glucose oxidase (0.25-2 U/ml) or xanthine oxidase (20-120 mM/ml) and 2 mM xanthine. Catalase protected the cells in each case. Lipid peroxidation as measured by the accumulation of malondialdehyde (MDA) preceded the cell death due to H2O2 added directly to the cultures or generated in the medium. The antioxidants N,N'-diphenyl-p-phenylenediamine (DPPD) and promethazine prevented the accumulation of MDA in both cases and protected the cells treated with H2O2 directly. DPPD and promethazine did not react directly with H2O2. Other antioxidants including butylated hydroxytoluene, vitamin E, and N-propylgallate had varied protective activity against the addition of H2O2 in proportion to their ability to reduce MDA accumulation. In glucose oxidase-treated cultures, DPPD and promethazine prevented the cell killing during the first hour but failed to protect between 1 and 3 h despite prevention of lipid peroxidation. The cell killing between 1 and 3 h in the presence of DPPD was prevented by catalase indicating its dependence upon continued generation of H2O2. Further addition of H2O2 in the presence of DPPD also increased the number of dead cells without lipid peroxidation. The data are consistent with at least two mechanisms of hepatocyte killing by H2O2. The first pathway is prevented by the antioxidants DPPD and promethazine and is very likely related to the peroxidation of membrane phospholipids. The second is independent of lipid peroxidation yet dependent upon the continued presence of H2O2.
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PMID:Mechanisms of the killing of cultured hepatocytes by hydrogen peroxide. 669 41

We have developed a quantitative assay to monitor the oxidative burst (H2O2 production) of polymorphonuclear leukocytes (PMNL) using single cell analysis by flow cytometry, and have examined whether PMNL respond to membrane stimulation with an all-or-none oxidative burst. During incubation with normal neutrophils, dichlorofluorescin diacetate diffused into the cells, was hydrolyzed to 2',7'-dichlorofluorescin (DCFH) and was thereby trapped within the cells. The intracellular DCFH, a nonfluorescent fluorescein analogue, was oxidized to highly fluorescent 2',7'-dichlorofluorescein (DCF) by PMNL stimulated by phorbol myristate acetate (PMA). That the oxidative product was DCF was shown by excitation/emission spectra and by mass spectrometry of the product from PMA-stimulated PMNL. Normal resting and PMA-stimulated PMNL oxidized 6.9 +/- 0.7 and 160 +/- 13 attomoles DCF per cell, respectively, in 15 min. Absence of calcium and magnesium ions and/or addition of 2 mM EDTA did not inhibit DCF formation by PMNL stimulated by 100 ng/ml PMA. Since EDTA prevented aggregation of PMNL (even when stimulated by 100 ng/ml PMA), which would prevent accurate flow cytometric analysis, further experiments were performed with EDTA in the medium. A close correlation between average DCFH oxidation and hexose monophosphate shunt stimulation was demonstrated using cells from patients whose PMNL had oxidative metabolic defects of varying severity. Intracellular DCFH was also oxidized by reagent H2O2 or oxygen derivatives generated by glucose oxidase + glucose or by xanthine oxidase + acetaldehyde; DCFH oxidation by these systems was inhibited by catalase but unchanged by superoxide dismutase. The data indicate that the DCFH oxidation assay is quantitatively related to the oxidative metabolic burst of PMNL, and they strongly suggest that the reaction is mediated by H2O2 generated by the PMNL. Incubation of PMNL with varying concentrations of PMA caused graded responses by all PMNL present; i.e., 1 ng/ml PMA caused a mean response of 34% maximal with a single population of responding PMNL (rather than 66% resting and 34% fully stimulated as predicted by the all-or-none hypothesis). Thus, with these assay conditions, oxidative product formation by PMNL occurs as a graded response to membrane stimulation by PMA.
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PMID:Flow cytometric studies of oxidative product formation by neutrophils: a graded response to membrane stimulation. 683 55

Respiratory activity of isolated rat brain mitochondria was measured following in vitro exposure to oxygen radicals. The radicals were generated by hypoxanthine and xanthine oxidase in the presence of a suitable iron chelate and caused a severe inhibition of respiration stimulated by phosphate plus ADP (with malate + glutamate as substrate). The damage could be prevented by catalase or high concentrations of mannitol, but not by superoxide dismutase. A similar effect was observed when hypoxanthine and xanthine oxidase were replaced by glucose and glucose oxidase or by hydrogen peroxide. Most of the findings indicate that the hydroxyl radical is the damaging agent. It is concluded that brain mitochondria exposed to oxygen radicals in vitro show an inhibition of respiratory activity similar to that reported by other investigators as occurring in mitochondria in vivo following transient cerebral ischemia. Therefore, oxygen radicals may contribute to this type of cell damage.
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PMID:Respiratory activity of isolated rat brain mitochondria following in vitro exposure to oxygen radicals. 684 68

The catalytic oxidation of [14C]-formate to 14CO2 was adapted to measure H2O2 formation in cellfree system. Standard curves employing glucose-glucose oxidase and xanthine-xanthine oxidase demonstrated linearity between 14CO2 evolution and enzyme concentration. A particulate fraction from human neutrophils was capable of oxidizing [14C]-formate; this reaction was dependent upon the presence of catalase, reduced pyridine nucleotide, and cellular material. Reaction increased with time of incubation and protein concentration, although not in a strictly linear fashion. The pH optimum was approximately 5.5 NADPH was a significantly better substrate than NADH, although both were capable of generating H2O2. The particulate fraction derived from phagocytizing cells was more active than a corresponding fraction from resting cells with either substrate. H2O2 production was abnormal in particulate fractions derived from 2 patients with chronic granulomatous disease. H2O2 production was markedly inhibited by superoxide dismutase or cytochrome c (scavengers of superoxide anion) but not by scavengers of singlet oxygen or hydroxyl radical. Reaction was greatly stimulated by the addition of manganous ion. These results are consistent with the hypothesis that the respiratory burst in human neutrophils is initiated by an oxidase that can utilize either NADPH or NADH but exhibits a marked preference for the former. Further, the inhibitor studies strongly support a mechanism involving an initial enzymatic reaction followed by a self-sustaining free radical reaction involving superoxide anion.
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PMID:Pyridine nucleotide-dependent generation of hydrogen peroxide by a particulate fraction from human neutrophils. 689 95

The intrapulmonary instillation into rat lung of enzymes that generate oxygen metabolites results in acute lung injury. The injection of xanthine oxidase and xanthine produces acute lung injury that, in the presence of superoxide dismutase, but not in the presence of catalase, can be significantly diminished, suggesting that O2- has the capacity to injure the lung. Instillation of a generator of H2O2, namely glucose oxidase, will, in sufficient quantities, produce acute injury that is not neutrophil-dependent. When either a low dose of glucose oxidase alone or lactoperoxidase alone is employed, little lung injury occurs. However, instilling the combination of the two enzymes produces severe, acute injury that can be blocked in a dose-dependent manner by catalase, but not by superoxide dismutase. Purified human leukocytic myeloperoxidase, but not horseradish peroxidase, will substitute for lactoperoxidase in the model of lung injury. The lung damaging effects of these enzymes cannot be attributed to the presence of contaminating proteases. Acute lung injury produced by the instillation of glucose oxidase and lactoperioxidase progresses to interstitial fibrosis. These studies represent a direct application of generators of oxygen metabolites to the in vivo induction of lung injury. The data suggest that rat lung is susceptible to injury by a variety of oxygen metabolites, including O2-, H2O2 and its lactoperoxidase or myeloperoxidase-produced derivatives. The studies also indicate that lung injury produced by oxygen metabolites can result in interstitial pulmonary fibrosis.
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PMID:In vivo damage of rat lungs by oxygen metabolites. 689 54

The chemical reactivity of 8-chloroflavins and 8-mercaptoflavins has been exploited in order to examine the orientation of protein-bound flavins relative to solvent. The apoprotein form of a series of flavoproteins was prepared and the native flavin was replaced by either 8-Cl-flavin or 8-mercaptoflavin (FAD, FMN, or riboflavin form as was appropriate). The reconstituted proteins were exposed to reagents capable of reacting with the group at position 8. The 8-Cl-proteins were challenged with sodium sulfide and thiophenol, while the 8-mercaptoproteins were faced with iodoacetamide and iodoacetic acid. The kinetics of the ensuing reactions served as a measure of the solvent availability of position 8 for the protein-bound flavin. These studies indicated that position 8 of flavin bound to melilotate hydroxylase, D-amino acid oxidase, old yellow enzyme, p-OH-benzoate hydroxylase, and flavodoxin is accessible to solvent, while position 8 on L-lactate oxidase, glucose oxidase, putrescine oxidase, and riboflavin-binding protein appears to be inaccessible. For luciferase, D-lactate dehydrogenase, and xanthine oxidase, the data suggest that position 8 is exposed but the results are inconclusive. The effect of ligand binding on the accessibility of position 8 was also studied. NADPH binding to 8-mercapto old yellow enzyme and benzoate binding to 8-Cl-D-amino acid oxidase results in complete blockage of previously available position 8. On the other hand, p-OH-benzoate hydroxylase and melilotate hydroxylase bind their respective substrates (p-OH-benzoate and melilotate) without significantly altering the reactivity of position 8.
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PMID:Active site probes of flavoproteins. Determination of the solvent accessibility of the flavin position 8 for a series of flavoproteins. 689 55

Natural killer cells spontaneously lyse certain tumor cells and may defend against malignancy. We have previously shown that natural killing (NK) by human peripheral blood mononuclear cells (PBMC) is suppressed in vitro by phorbol diester tumor promoters, including 12-O-tetradecanoylphorbol-13-acetate (TPA). We here demonstrate that suppression of NK is mediated by monocytes or polymorphonuclear leukocytes (PMN) and that suppression is dependent on the generation of reactive forms of molecular oxygen (RO), particularly hydrogen peroxide (H2O2). NK was suppressed not only by TPA but also by opsonized zymosan (yeast cell walls), which, like TPA, was not toxic to PBMC. Both TPA and zymosan stimulated the production of superoxide anion (O2-) and H2O2 by PBMC. Production of RO correlated with suppression of NK. When PBMC were depleted of monocytes, the production of RO and the suppression of NK were both markedly reduced. Suppression could be restored by monocytes or PMN, both of which produced RO in response to TPA or zymosan. Suppression of NK was dependent on RO. Monocytes or PMN from a patient with chronic granulomatous disease, whose cells cannot generate RO, did not mediate suppression of NK. Suppression was also reduced in glucose-free medium, which did not support the generation of RO. Suppression of NK by TPA was inhibited by catalase. Bovine superoxide dismutase had a limited effect on suppression, even in high concentration, and tyrosine-copper (II) complex, which also enhances dismutation of O2- to H2O2, had almost no effect on suppression. When H2O2 was directly generated enzymatically from glucose oxidase and glucose, NK was suppressed and suppression was reversed by catalase. NK was also suppressed by the enzymatic generation of O2- from xanthine oxidase and xanthine, but suppression under these conditions was again inhibited by catalase and not by superoxide dismutase, indicating that suppression was due to the secondary formation of H2O2 from O2-. These results indicate that H2O2 is important in suppression of NK. Myeloperoxidase did not appear to play a role in suppression because inhibition of this enzyme by sodium azide, cyanide, or aminotriazole did not prevent suppression of NK. Suppression of NK was reversible; after exposure to zymosan, NK could be partially restored by the addition of catalase and superoxide dismutase or by the removal of zymosan. These studies demonstrate cellular regulation of NK by monocytes or polymorphonuclear leukocytes and indicate a role for RO in immunoregulation.
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PMID:Suppression of natural killing in vitro by monocytes and polymorphonuclear leukocytes: requirement for reactive metabolites of oxygen. 707 51


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