Myeloperoxidase-derived hypochlorous acid antagonizes the oxidative stress-mediated activation of iron regulatory protein 1.
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Hypochlorous acid (HOCl) is a highly reactive product generated by the myeloperoxidase reaction during the oxidative burst of activated neutrophils, which is implicated in many bactericidal and cytotoxic responses. Recent evidence suggests that HOCl may also play a role in the modulation of redox sensitive signaling pathways. The short half-life of HOCl and the requirement for a continuous presence of H2O2 as a substrate for its myeloperoxidase-catalyzed generation make the study of HOCl-mediated responses very difficult. We describe here an enzymatic model consisting of glucose/glucose oxidase, catalase, and myeloperoxidase (GOX/CAT/MPO) that allows the controlled generation of both HOCl and H2O2 and thus, mimics the oxidative burst of activated neutrophils. By employing this model we show that HOCl prevents the H2O2-mediated activation of iron regulatory protein 1 (IRP1), a central post-transcriptional regulator of mammalian iron metabolism. Activated IRP1 binds to (R)iron-responsive elements" (IREs) within the mRNAs encoding proteins of iron metabolism and thereby controls their translation or stability. The inhibitory effect of HOCl is not a result of a direct modification of IRP1 by this oxidant. Kinetics experiments provide evidence that HOCl intervenes with the signaling cascade, which results in the activation of IRP1. We further demonstrate that HOCl antagonizes the H2O2-mediated increase in the levels of transferrin receptor, which is a downstream target of IRP1. Our findings suggest that HOCl can modulate signaling pathways in a concerted action with H2O2. The GOX/CAT/MPO system provides a valuable tool for studying the regulatory function of HOCl. (+info)
Improving accuracy of glucose oxidase procedure for glucose determinations on discrete analyzers.
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We describe a rapid kinetic glucose oxidase (EC 1.1.3.4) procedure for quantifying glucose. Glucose oxidase concentration was reduced from the more usual 20 kU/L to 4 kU/L, and pH was reduced from 7.0 to 6.6. Potassium ferrocyanide (20 mumol/L) and ascorbate oxidase (1 kU/L) were incorporated in the procedure. The assay results vary linearly with glucose concentration from 0 to 50 mmol/L and are unaffected by bilirubin concentrations less than or equal to 600 mumol/L, hemoglobin less than or equal to 12 g/L, Intralipid less than or equal to 4 g/L, urate less than or equal to 1 mmol/L, and ascorbate less than or equal to 2.0 mmol/L. The assay is readily adaptable to most open-system analyzers. (+info)
New chromogen for assay of glucose in serum.
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We describe a colorimetric assay for glucose determination in human serum, with use of the chromogen 2-amino-4-hydroxybenzenesulfonic acid (AHBS), glucose oxidase, and peroxidase. With this assay, glucose concentrations less than or equal to 27.8 mmol/L can be measured in serum, with a sample/reagent volume ratio as low as 0.0025. The chromogen itself is easily soluble in water and does not require other components for the color change, making the reagent composition less complex. A single working reagent is used, and the reaction is completed within 10 min at 37 degrees C. The absorbance of the yellow reaction product is measured at 415 nm, and a blank sample measurement is not needed. The average analytical recovery of glucose in different human sera was 97.6%, with no significant interference of reducing compounds in serum. The results of the recommended procedure correlated well with those of the phenol/4-aminoantipyrine method of Trinder. (+info)
Lethal oxidative damage to human immunodeficiency virus by human recombinant myeloperoxidase.
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Human recombinant myeloperoxidase was evaluated in a cell-free system for its inactivation properties on the replication of human immunodeficiency virus, HTLV-IIIB. In the presence of a hydrogen peroxide generating system (glucose and glucose oxidase) and sodium thiocyanate, the recombinant enzyme inhibited virus-induced syncytium formation and viral replication without causing any cytopathic effects on SupT1 reporter cells. In addition, U937 monocytoid cells, chronically infected with HIV1, were exposed to recombinant myeloperoxidase (10 U/ml) and monitored during 48 h for the accumulation of intracellular p24 viral antigen. Under these conditions, the recombinant enzyme significantly reduced intracellular viral replication without affecting cell viability. (+info)
Role of cellular superoxide dismutase against reactive oxygen metabolite injury in cultured bovine aortic endothelial cells.
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We examined the protective effect of cellular superoxide dismutase against extracellular hydrogen peroxide in cultured bovine aortic endothelial cells. 51Cr-labeled cells were exposed to hydrogen peroxide generated by glucose oxidase/glucose. Glucose oxidase caused a dose-dependent increase of 51Cr release. Pretreatment with diethyldithiocarbamate enhanced injury induced by glucose oxidase, corresponding with the degree of inhibition of endogenous superoxide dismutase activity. Inhibition of cellular superoxide dismutase by diethyldithiocarbamate was not associated either with alteration of other antioxidant defenses or with potentiation of nonoxidant injury. Enhanced glucose oxidase damage by diethyldithiocarbamate was prevented by chelating cellular iron. Inhibition of cellular xanthine oxidase neither prevented lysis by hydrogen peroxide nor diminished enhanced susceptibility by diethyldithiocarbamate. These results suggest that, in cultured endothelial cells: 1) cellular superoxide is involved in mediating hydrogen peroxide-induced damage; 2) superoxide, which would be generated upon exposure to excess hydrogen peroxide independently of cellular xanthine oxidase, promotes the Haber-Weiss reaction by initiating reduction of stored iron (Fe3+) to Fe2+; 3) cellular iron catalyzes the production of a more toxic species from these two oxygen metabolites; 4) cellular superoxide dismutase plays a critical role in preventing hydrogen peroxide damage by scavenging superoxide and consequently by inhibiting the generation of the toxic species. (+info)
The role of cationized catalase and cationized glucose oxidase in mucosal oxidative damage induced in the rat jejunum.
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The successful prevention of hydrogen peroxide-induced damage to the rat jejunal mucosa by cationized catalase is described in this study. Biological damage was induced in a closed circulating intestinal loop of the rat by hydrogen peroxide and by hydroxyl radicals induced in situ via the metal-mediated Haber-Wiess reaction. The mucosal activity of lactate dehydrogenase and the amount of potassium ions were used to quantitatively characterize the tissue damage. Catalase was cationized by reacting it with N,N'-dimethyl-1,3-propanediamine to give a soluble product or with polyhistidine to give an insoluble product. The activity of the modified enzymes was assessed, and their ability to protect the rat jejunal mucosa against oxidative stress was studied. It was found that in all cases the cationized enzymes were superior to the native catalase in their shield capability. A significant protection against Fe(II)/H2O2 and ascorbic acid/copper ion-mediated damage was obtained when the cationized enzymes were used. In the presence of glucose, native glucose oxidase failed to cause damage in the rat jejunal mucosa; however, the cationized enzyme caused profound tissue injury. These findings indicate the potential therapeutic merit of cationized enzymes for the treatment of pathological processes in the intestine, whenever oxidative stress is involved. (+info)
ENZYMES RELEASED FROM ESCHERICHIA COLI WITH THE AID OF A SERVALL CELL FRACTIONATOR.
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The release and stability of the enzymes S-adenosylhomocysteine nucleosidase, lysine decarboxylase, arginine decarboxylase, glutamic decarboxylase, formic hydrogenlyase, formic oxidase, and glucose oxidase from Escherichia coli during disruption of the organisms in a Servall-Ribi refrigerated cell fractionator were examined. With the possible exception of arginine decarboxylase, maximal activity was retained by all the enzymes reported here when the cell suspensions were processed at pressures necessary for rupture of all the organisms (15,000 to 25,000 psi). Considerable variation in the stability of different enzymes liberated by disruption at higher pressures (45,000 to 55,000 psi) was observed. It is reasonable to assume that mechanical forces rather than effects of temperature are responsible for inactivation of these enzymes. (+info)
AN INDUCIBLE SYSTEM FOR THE HYDROLYSIS AND TRANSPORT OF BETA-GLUCOSIDES IN YEAST. I. CHARACTERISTICS OF THE BETA-GLUCOSIDASE ACTIVITY OF INTACT AND OF LYSED CELLS.
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A strain of bakers' yeast was isolated which could utilize cellobiose and other beta-D-glucosides quantitatively as carbon and energy sources for growth. Cellobiose-grown cells contained a largely cryptic enzyme active against the chromogenic substrate p-nitrophenyl-beta-D-glucoside. The patent (intact cell) activity of such cells was inhibited by azide and, competitively, by cellobiose; neither agent inhibited the beta-glucosidase activity of lysed cells or of extracts. The enzyme induced by growth in cellobiose medium had no affinity for cellobiose as either substrate or inhibitor; its substrate specificity classifies it as an aryl-beta-glucosidase. It was concluded that growth in cellobiose also induced the formation of a stereospecific and energy-dependent system whose function determined the rate at which intact cells could hydrolyze substrates of the intracellular beta-glucosidase. (+info)