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)

Egg yolk phosphatidylcholine monolamellar liposomes (1000 A in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O2-., were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O2-. (P'O-2 = (7.6 +/- 0.3) X 10(-8) cm/s), or HO.2 (P'HO.2 = 4.9 X 10(-4) cm/s) were determined. The effect of the transmembrane electric potential (K+ concentration gradient, valinomycin) on the permeability of liposomal membranes for O2-. were investigated. It was found that O2-. can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.
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PMID:Permeability of bilayer lipid membranes for superoxide (O2-.) radicals. 609 12

Superoxide dismutase (SOD) activity was measured by seven assay methods. The nitrite method was found to be the best for our SOD assay kit. This method was then modified to give better sensitivity and minimize interference by coexisting protein, a factor which has been previously ignored. Hydroxylamine or its O-sulfonic acid, xanthine oxidase, hypoxanthine, EDTA, and the sample were incubated with or without KCN at pH 8.2, 37 degrees C, for 30 min. Diazo dye-forming reagent was added and the absorption was measured at 550 nm. Human plasma and erythrocyte lysate from healthy adults and Down's syndrome patients were assayed by this SOD kit and by the cytochrome c method. Our kit gave 8.5 times higher sensitivity than the cytochrome c method. This high sensitivity allowed the use of a simple spectrophotometer and, moreover, only one dilution was needed to determine the SOD unit with the help of our formulas. Good recovery, reproducibility, and stability of reagents were demonstrated.
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PMID:Reevaluation of assay methods and establishment of kit for superoxide dismutase activity. 609 57

Changes in superoxide dismutase (SOD) activity were studied in vitro at increasing NaCl or KCl concentrations. SOD activity was measured using two different systems of superoxide radical generation: pyrogallol autoxidation, and xanthine-xanthine oxidase reaction. Pyrogallol autoxidation was directly measured by spectrophotometry, whereas in the second case cytochrome c reduction was followed at 550 nm. The inhibition of SOD on those parameters was taken as measure of SOD activity. Increasing concentrations of NaCl and KCI significantly increased the rate of pyrogallol autoxidation. The inhibitory effect of SOD significantly decreased under the influence of these salts and followed an exponential curve. The two salts studied resulted in essentially identical changes in SOD activity. Increasing concentrations of NaCl and KCl decreased the rate of cytochrome c reduction in the xanthine-xanthine oxidase system. When correcting the results for these primary effects, SOD activity also displayed in this system an exponential decay with increasing salt concentrations. The results are interpreted in terms of the known charge distribution pattern on the surface of the SOD molecule, and of the age-dependent increase of the intracellular potassium and sodium concentrations in the postmitotic cells.
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PMID:Effects of ionic strength on the activity of superoxide dismutase in vitro. 609 11

The relationship between the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin was investigated. The rates of reduction of phosvitin-bound ferricytochrome c by cytochrome b2, ascorbate and the superoxide radical generated by xanthine oxidase wer repressed where the binding ratio was less than half the maximum, but at higher ratios they were restored gradually with increase in the ratio. The affinity of cytochrome b2 for cytochrome c was not affected by binding of cytochrome c to phosvitin. The redox potential of the bond form was lower than that of the free form and only decreased with decrease in the ratio. The conformatin around the heme moiety and the electronic structure of the heme group of bound ferricytochrome c were similar to those of free ferricytochrome c, but the conformational stability in the vicinity of the prosthetic group was related to the binding ratio as ratios above half the maximum and was well correlated with the reduction rate. Since the binding of cytochrome c to phosvitin is much stronger at binding ratios below half the maximum, these results suggest that this binding strength exclusively affects the conformational flexibility of the heme crevice in the cytochrome molecule, thus altering the reduction rate.
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PMID:Relation of the structure and function of ferricytochrome c bound to the phosphoprotein phosvitin. 625 2

1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH-cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH-cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH-cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH-cytochrome P-450 reductase and cytosolically orientated membrane factor(s).
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PMID:Asymmetric distribution of cytochrome P-450 and NADPH--cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver. 625 76

Product formation during the oxidation of xanthine oxidase has been examined directly by using cytochrome c peroxidase as a trapping agent for hydrogen peroxide and the reduction of cytochrome c as a measure of superoxide formation. When fully reduced enzyme is mixed with high concentrations of oxygen, 2 molecules of H2O2/flavin are produced rapidly, while 1 molecule of O2-/flavin is produced rapidly and another produced much more slowly. Time courses for superoxide formation and those for the absorbance changes due to enzyme oxidation were fitted successfully to the mechanism proposed earlier (Olson, J. S., Ballou, D. P., Palmer, G., and Massey, V. (1974) J. Biol. Chem. 249, 4363-4382). In this scheme, each oxidative step is initiated by the very rapid and reversible formation of an oxygen.FADH2 complex (the apparent KD = 2.2 X 10(-4) M at 20 degrees C, pH 8.3). In the cases of 6- and 4-electron-reduced enzyme, 2 electrons are transferred rapidly (ke = 60 s-1) to generate hydrogen peroxide and partially oxidized xanthine oxidase. In the case of the 2-electron-reduced enzyme, only 1 electron is transferred rapidly and superoxide is produced. The remaining electron remains in the iron-sulfur centers and is removed slowly by a second order process (ks = 1 X 10(4) M-1 s-1). When the pH is decreased from 9.9 to 6.2, both the apparent KD for oxygen binding and the rapid rate of electron transfer are decreased about 20-fold. This result is suggestive of uncompetitive inhibition and implies that proton binding to the enzyme-flavin active site affects primarily the rate of electron transfer, not the formation of the initial oxygen complex.
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PMID:The reaction of reduced xanthine oxidase with oxygen. Kinetics of peroxide and superoxide formation. 626 59

In the course of studying the effects on platelets of the oxidant species superoxide (O2.-), O2.- was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O2.-), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase. In addition to generating O2.-, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggregation is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O2.0, or that O2.- itself exerts no important direct effect on platelet function under these experimental conditions. The ability of C2.- to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O2.- in platelet studies because of its own effects on platelets.
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PMID:Superoxide, xanthine oxidase and platelet reactions: further studies on mechanisms by which oxidants influence platelets. 626 49

Carbon monoxide:methylene blue oxidoreductase, the key enzyme of CO-oxidation in energy metabolism of the carboxydobacterium Pseudomonas carboxydovorans, has been isolated in good yield and purity and found to contain FAD, molybdenum, iron, and labile sulfide in the ratio of 1:1:4:4. The enzyme is, therefore, a new molybdenum-containing iron-sulfur flavoprotein, exhibiting chemical and spectral properties quite similar to those of xanthine oxidase. Analytical data on the spectral characteristics of the enzyme in the oxidized and various reduced states are presented. Carbon monoxide:methylene blue oxidoreductase turned out to be photoreducible in the presence of EDTA and urea and was subject to reoxidation by air oxygen; no flavoprotein semiquinone was formed. Unphysiological electron acceptors, e.g. methylene blue, were used as oxidizing substrates whereas NAD or NADP turned out to be ineffective. Methylene blue reduction with CO was not affected by the presence of allopurinol, and carbon monoxide:methylene blue oxidoreductase was not able to catalyze the reduction of methylene blue with xanthine, adenine, or aldehydes. CO was the only reducing substrate used by the enzyme. Carbon monoxide:methylene blue oxidoreductase formed no sulfite adduct, and the reactivity with ferricyanide or cytochrome c was significant but slow. As known for other molybdenum hydroxylases, carbon monoxide:methylene blue oxidoreductase was rapidly inactivated by methanol, but the enzyme exhibited no ability to catalyze the oxidation of NADH with methylene blue, and NAD was not able to overcome methanol inhibition.
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PMID:Chemical and spectral properties of carbon monoxide: methylene blue oxidoreductase. The molybdenum-containing iron-sulfur flavoprotein from Pseudomonas carboxydovorans. 627 81

Superoxide anion radicals have been implicated recently as mediators of inflammation and tissue injury. Protection from superoxide anion radicals is provided primarily by a copper-containing, intracellular enzyme (superoxide dismutase) (SOD) that catalyzes the dismutation of superoxide to hydrogen peroxide and oxygen. We have found that the action of cytoplasmic SOD to scavenge superoxide and thereby to inhibit superoxide-mediated reactions can be mimicked by the copper-containing plasma protein and acute-phase reactant, ceruloplasmin. Ceruloplasmin, at concentrations present in normal plasma, inhibited reduction of both cytochrome c and nitroblue tetrazolium (NBT) mediated by the aerobic action of xanthine oxidase on hypoxanthine (a superoxide-generating system). Ceruloplasmin neither inhibited formation of uric acid by xanthine oxidase nor accelerated autooxidation of cytochrome c. Furthermore, in an experimental system in which contact between ceruloplasmin and indicator was prevented by a relatively impermeable lipid membrane barrier, ceruloplasmin inhibited reduction of NBT trapped within liposomes exposed to xanthine oxidase and hypoxanthine. Ceruloplasmin also inhibited reduction of cytochrome c and NBT mediated by the aerobic action of xanthine oxidase on acetaldehyde (another superoxide-generating system) and mimicked the activity of purified human erythrocyte SOD by inhibiting photoreduction of NBT and by accelerating aerobic photooxidation of dianisidine. Ceruloplasmin could be separated from purified human erythrocyte SOD by electrophoresis on alkaline 12% polyacrylamide gels and identified by its superoxide-scavenging activity. These results suggest that ceruloplasmin may function as a circulating scavenger of oxygen-derived free radicals.
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PMID:Ceruloplasmin: an acute phase reactant that scavenges oxygen-derived free radicals. 628 6

The stoichiometry of reducing equivalents per protomer for the complex molybdoflavoprotein xanthine oxidase has been re-examined by reductive titrations with sodium dithionite and anaerobic reoxidation with cytochrome c and phenazine methosulfate of dithionite- or photo-reduced enzyme. It is found that 8.0 +/- 0.1 reducing equivalents are taken up (or given up) by the enzyme, a value of 2 eq greater than expected on the basis of the known oxidation-reduction centers in the enzyme. The reaction of reduced xanthine oxidase with [14C]iodoacetate indicates that, in the reduced form of the enzyme, additional cysteine residues are available for reaction. These results, in conjunction with the observation that reaction of oxidized enzyme with sulfite results in the appearance of an additional equivalent of thiol capable of reacting with 5,5'-dithiobis-(2-nitrobenzoic acid) or iodoacetate, indicate the presence of a disulfide linkage in the enzyme that can be reduced by dithionite or photochemically employing EDTA and 5-deazaflavin. Neither xanthine nor lumazine, however, is capable of reducing this oxidation-reduction center, suggesting that the disulfide does not play a role in the catalytic reactions of the enzyme. These results resolve discrepancies in the literature which indicated that greater than 6 reducing equivalents were consistently needed to bring about the complete reduction of xanthine oxidase.
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PMID:The presence of a reducible disulfide bond in milk xanthine oxidase. 628 47


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