Biomedical subjects

R Radi

Publications and source records attributed to R Radi.

At least 73 records · Page 4Linked to original sources

PubMed1994

Aconitase is readily inactivated by peroxynitrite, but not by its precursor, nitric oxide.

Mitochondrial and cytosolic aconitases have been indicated as major targets of .NO- and O2-.-mediated toxicity in cells due to the oxidant-mediated disruption of the [4Fe-4S] prosthetic group. However, under circumstances in which both .NO and O2-. are generated, their almost diffusion-controlled combination reaction (k = 6.7 x 10(9) M-1 s-1), leading to the formation of peroxynitrite anion (ONOO-), can out-compete the direct reactions of .NO and O2-. with aconitase and even the enzymatic dismutation of O2-. by superoxide dismutase. In this work, we report that ONOO- reacts with isolated pig heart mitochondrial aconitase at 1.4 x 10(5) M-1 s-1, resulting in a significant loss of enzymatic activity. Aconitase activity was totally recovered after postincubation with thiols and ferrous iron, indicating that ONOO- reactions with the enzyme involve the perturbation of the labile Fe alpha to yield the inactive [3Fe-4S] cluster, which is also evident by spectral changes. On the other hand, anaerobic exposure of isolated aconitase to high concentrations of .NO (> 100 microM) led to a moderate inhibition of the enzyme, which could be fully overcome by .NO displacement under an argon-saturated atmosphere, in agreement with the formation of a reversible inhibitory complex between .NO and the active site of aconitase. Superoxide inactivated mitochondrial aconitase at (3.5 +/- 2) x 10(6) M-1 s-1, a reaction rate 3 orders of magnitude slower than its reaction rate with .NO. O2-. could represent the main mechanism of inactivation of the enzyme in systems in which it is formed without significant concomitant production of .NO. Our results imply that the mechanisms by which .NO and O2-. inactivate aconitase in cell systems may not be simple due to their direct reactions with the iron-sulfur cluster, but may rely on the formation of ONOO-.

Aconitate Hydratase↗

PubMed1994

Nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. Formation of novel nitrogen-containing oxidized lipid derivatives.

Superoxide (O2-.), nitric oxide (.NO), and their reaction product peroxynitrite (ONOO-) have all been shown to independently exert toxic target molecule reactions. Because these reactive species are often generated in excess during diverse inflammatory and other pathologic circumstances, we assessed the influence of .NO on membrane lipid peroxidation induced by O2-., H2O2, and .OH derived from xanthine oxidase (XO) and by ONOO-. Experimental conditions in lipid oxidation systems were adjusted to yield different rates of delivery of .NO, relative to rates of O2-. and H2O2 generation, by infusion of either .NO or via .NO released from S-nitroso-N-acetylpenicillamine or S-nitrosoglutathione. Peroxidation of phosphatidylcholine liposomes was assessed by formation of thiobarbituric acid-reactive products and by liquid chromatography-mass spectrometry. Liposomes exposed to XO-derived reactive species in the presence of .NO exhibited both stimulation and inhibition of lipid peroxidation, depending on the ratio of the rates of reactive oxygen species production and .NO introduction into reaction systems. Nitric oxide alone did not induce lipid peroxidation. Linolenic acid emulsions peroxidized by XO-derived reactive species showed similar dose-dependent regulation of lipid peroxidation by .NO. Mass spectral analysis of oxidation products showed formation of nitrito-, nitro-, nitrosoperoxo-, and/or nitrated lipid oxidation adducts, demonstrating that .NO serves as a potent terminator of radical chain propagation reactions. Electron spin resonance (ESR) analysis of incubation mixtures provided no evidence for formation of paramagnetic iron-lipid-nitric oxide complexes in reaction systems. Peroxynitrite-dependent lipid peroxidation, which predominantly occurs by metal-independent mechanisms, was also inhibited by .NO. Peroxynitrite-mediated benzoate hydroxylation was partially inhibited by .NO, inferring reaction between .NO and ONOOH. It is concluded that .NO can both stimulate O2-./H2O2/.OH-induced lipid oxidation and mediate oxidant-protective reactions in membranes at higher rates of .NO production, with the prooxidant versus antioxidant outcome critically dependent on relative concentrations of individual reactive species. Prooxidant reactions of .NO will occur after O2-. reaction with .NO to yield potent secondary oxidants such as ONOO- and the antioxidant effects of .NO a consequence of direct reaction with alkoxyl and peroxyl radical intermediates during lipid peroxidation, thus terminating lipid radical chain propagation reactions.

Hydroxyl Radical↗

PubMed1994

Peroxynitrite-mediated oxidation of albumin to the protein-thiyl free radical.

Nitric oxide reacts with superoxide to produce peroxynitrite, which may be an important mediator of oxidant-induced cellular injury. Here we report that peroxynitrite is able to oxidize a protein, bovine serum albumin (BSA), to the corresponding protein-thiyl free radical as demonstrated by electron paramagnetic resonance (EPR)-spin-trapping experiments with both alpha-phenyl-N-tert-butyl nitrone (PBN) and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). BSA radical adduct yields increased with pH indicating peroxynitrite anion as its main forming agent. Reaction with peroxynitrite may be another aspect of the antioxidant action of albumin in extracellular fluids.

Cyclic N-Oxides↗

PubMed1994

Inhibition of mitochondrial electron transport by peroxynitrite.

Mammalian mitochondria are sensitive targets of the cytotoxic effects of superoxide (O.2-) and nitric oxide (.NO). In turn, when superoxide and nitric oxide are simultaneously produced, they rapidly react with each other yielding the highly oxidizing peroxynitrite anion (ONOO-) which may be also toxic to mammalian mitochondria. In this study we report that peroxynitrite exposure to rat heart mitochondria resulted in significant inactivation of electron carriers such as succinate dehydrogenase and NADH dehydrogenase as well as the mitochondrial ATPase. As a result of enzyme inactivation, peroxynitrite lead to a profound inhibition of glutamate/malate- and succinate-supported oxygen consumption but did not cause mitochondrial uncoupling. Secondary to inhibiting mitochondrial electron transport, peroxynitrite induced an enhanced succinate-stimulated hydrogen peroxide formation by heart mitochondria. Most of the damaging effects against mitochondria can be ascribed to peroxynitrite anion itself and not to hydroxyl radical-like oxidant yielded during the proton-catalyzed decomposition of peroxynitrite, as hydroxyl radical scavengers provided a rather modest protection. Our observations indicate that mitochondria may constitute a key intracellular loci for the toxic effects of peroxynitrite under the various pathological conditions in which peroxynitrite appears to play a contributory role.

Animals↗

PubMed1994

Peroxynitrite inactivates thiol-containing enzymes of Trypanosoma cruzi energetic metabolism and inhibits cell respiration.

Activated macrophages release peroxynitrite anion (ONOO-), which has been recently shown to be highly cytotoxic against Trypanosoma cruzi epimastigotes. In this work, we report that two critical enzymes for the energetic metabolism of the parasite, succinate dehydrogenase and fumarate reductase, are inactivated by biologically relevant concentrations of peroxynitrite. Enzyme inactivation was accompanied by a significant inhibition of succinate-dependent respiration in intact cells as well as in the membrane-rich fraction. Peroxynitrite also inhibited NADH-dependent oxygen consumption which depends almost exclusively on fumarate reductase activity in T. cruzi epimastigotes. Direct reactions of peroxynitrite anion with critical sulfhydryl residues of the two enzymes were responsible for most of the observed inactivation as indicated by the protection afforded by peroxynitrite scavengers and the reactivation of the enzymes by dithiothreitol. We propose that peroxynitrite-mediated inactivation of succinate dehydrogenase and fumarate reductase may be a key mechanism of macrophage-mediated cytotoxicity to T. cruzi, through inhibition of the energetic metabolism of the parasite.

Adenosine Triphosphatases↗

PubMed1994

Spin-trapping studies of peroxynitrite decomposition and of 3-morpholinosydnonimine N-ethylcarbamide autooxidation: direct evidence for metal-independent formation of free radical intermediates.

Decomposition of peroxynitrite, the reaction product of superoxide and nitric oxide, was studied by electron paramagnetic resonance (EPR)-spin-trapping experiments with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Proton-catalyzed decomposition of peroxynitrite at pH 7.5 resulted in the formation of the DMPO-hydroxyl radical adduct (DMPO-OH). Yields were low as DMPO-OH decomposes by direct reactions with peroxynitrite anion and nitrogen dioxide. The yield of DMPO-OH greatly increased in the presence of glutathione or cysteine. Both thiols inhibited the DMPO-OH signal decay by scavenging excess peroxynitrite anion and presumably nitrogen dioxide yielded during peroxynitrite decomposition. In turn, the reactions of peroxynitrite with either glutathione or cysteine resulted in the formation of thiyl radicals, detectable as the corresponding DMPO adduct. Systematic spin-trapping studies of peroxynitrite decomposition in the presence of glutathione established that DMPO-hydroxyl radical adduct formation was metal independent, occurring in a metal-free buffer and being unaffected by diethylene-triaminepentaacetic acid. Also, quantitative competition experiments with ethanol and formate demonstrated that the oxidant generated during peroxynitrite decomposition reacts with rate constants similar to those expected for free hydroxyl radical and forming the same free radical intermediates, alpha-ethyl-hydroxy and carbon dioxide radicals, respectively. Similar spin-trapping results were obtained in studies of the autooxidation of 3-morpholinosydnonimine, a sydnonimine which generates a flux of both superoxide and nitric oxide. The obtained results contribute for the understanding of the reactivity of peroxynitrite, a transient intermediate of emerging biological significance.

Benzoates↗

PubMed1994

On the pH-dependent yield of hydroxyl radical products from peroxynitrite.

Nitric oxide reacts rapidly with superoxide to give the strongly oxidizing peroxynitrite anion (ONOO-), which undergoes spontaneous first-order decomposition when protonated. The oxidative chemistry of peroxynitrite (ONOO-) is highly pH-dependent. At acidic pH, peroxynitrous acid (ONOOH) oxidizes dimethylsulfoxide to formaldehyde and 2,2'-azino-bis-(3-ethyl-1,2- dihydrobenzothiazoline 6-sulfonate) (ABTS) to the greenish-colored ABTS+ radical cation. The product yield from dimethylsulfoxide and ABTS decreased at more alkaline pH with apparent pK(a)s of 7.9 and 8.2, respectively. Decreasing yield with increasing pH could not be explained by the oxidation of either formaldehyde or ABTS+ by peroxynitrite. In the presence of 50 mM dimethylsulfoxide, nitrogen dioxide was formed in approximately equimolar amounts to the other reaction product, formaldehyde. The yield of nitrogen dioxide also decreased with an apparent pK(a) of 8.0. We propose that the complex oxidative chemistry of peroxynitrite is controlled by the pH-dependent isomerization of the relatively stable cis-configuration (predominant at high pH) to the trans-configuration. Trans-peroxynitrous acid can form a vibrationally excited intermediate capable of reacting like hydroxyl radical. The vibrationally excited intermediate can also directly rearrange to nitric acid, reducing the apparent hydroxyl radical yield to less than 30%. The loss of hydroxyl radical-like reactivity can be explained on the basis of ionization of trans-peroxynitrous acid to the trans-anion, which in turn undergoes internal rearrangement to nitrate without forming a strong oxidant.(ABSTRACT TRUNCATED AT 250 WORDS)

Benzothiazoles↗

PubMed1993

Peroxynitrite-induced luminol chemiluminescence.

Vascular endothelial cells, smooth muscle cells, macrophages, neutrophils, Kupffer cells and other diverse cell types generate superoxide (O2.-) and nitric oxide (.NO), which can react to form the potent oxidant peroxynitrite anion (ONOO-). Peroxynitrite reacted with luminol to yield chemiluminescence which was greatly enhanced by bicarbonate. The quantum chemiluminescence yield of the ONOO- reaction with luminol in bicarbonate was approx. 10(-3). Chemiluminescence was superoxide dismutase-inhibitable, indicating that O2.- was a key intermediate for chemiexcitation. O2.- appears to be formed secondarily to the reaction of a bicarbonate-peroxynitrite complex with luminol, yielding luminol radical and O2.-. Luminol radical reacts with O2.- to form the unstable luminol endoperoxide, which follows the light-emitting pathway. Neither .NO nor O2.- alone were capable of directly inducing significant luminol chemiluminescence in our assay systems. These results suggest that ONOO- can be a critical unrecognized mediator of cell-derived luminol chemiluminescence reported in previous studies. In addition, it is shown that bicarbonate can participate in secondary oxidation reactions after reacting with ONOO-.

Antioxidants↗

PubMed1993

The role of cytochrome c and mitochondrial catalase in hydroperoxide-induced heart mitochondrial lipid peroxidation.

The role of cytochrome c and catalase in hydroperoxide-induced lipid peroxidation of rat heart mitochondria was investigated. Mitoplasts were prepared from hearts of aminotriazole-treated rats which displayed both an 80-90% reduction in matrix catalase activity and rate of H2O2 consumption. Catalase-depleted mitochondria were more susceptible to H2O2-dependent lipid peroxidation and had similar extents of tert-butyl hydroperoxide (t-BuOOH)-induced lipid peroxidation compared with control mitochondria. The magnitude of lipid peroxidation induced by H2O2 was greater than that for t-BuOOH in catalase-depleted mitochondria, while t-BuOOH induced soybean phosphatidylcholine (PC) liposome lipid peroxidation to a greater extent than H2O2. The t-BuOOH- and H2O2-dependent mitochondrial lipid peroxidation was inhibited 50 and 7%, respectively, by cytochrome c3+ depletion of mitochondria. Similar relative sensitivities to t-BuOOH- and H2O2-dependent peroxidation occurred for cytochrome c(3+)-supplemented soybean PC liposomes. These data show a critical role for cytochrome c3+ in hydroperoxide-induced mitochondrial lipid peroxidation and demonstrate the importance of matrix catalase in protecting heart mitochondria from the toxicity of H2O2.

Animals↗

PubMed1993

Peroxynitrite-mediated cytotoxicity to Trypanosoma cruzi.

Macrophages produce and release superoxide anion (O2.-) and nitric oxide (.NO) as part of their microbicidal effector molecules. The simultaneous production of O2.- and .NO results in the rapid formation of peroxynitrite anion (ONOO-) by macrophages. Peroxynitrite is a strong oxidant with a half-life of less than 1 s in biological systems. There is solid experimental evidence implicating .NO and O2.- in macrophage-induced cytotoxicity against bacteria, parasites, and tumor cells. However, the cytotoxic role of peroxynitrite in these processes remains to be studied. In this work we demonstrate the parasiticidal activity of ONOO- against Trypanosoma cruzi. Peroxynitrite was highly trypanocidal, killing T. cruzi in a dose-dependent manner. Addition of 500 microM ONOO- as a single bolus resulted in 50% inhibition of cell proliferation as followed by growth curves. Fifty percent inhibition of [3H]thymidine incorporation measured at 6 h postaddition of ONOO- was obtained at 150 microM. Addition of ONOO- as a continuous infusion rather than a single bolus resulted in an even stronger inhibition in cell growth. Other cytotoxic effects of ONOO- included cellular swelling and inhibition of cell motility. Classical hydroxyl radical scavengers and metal chelators afforded minimal protection against ONOO(-)-mediated cytotoxicity, indicating that peroxynitrite anion itself, rather than the .OH-like oxidant derived from its proton-catalyzed decomposition, was the main damaging species. From literature data we estimated the production of ONOO- by activated macrophages inside phagolysosomes to be around 500 microM/min. Therefore, our results demonstrate that ONOO- may operate in vivo as a critical macrophage-derived reactive intermediate against T. cruzi.

Animals↗

PubMed1993

Detection of H2O2 release from vascular endothelial cells.

Endothelial cells are both significant sources and targets of reactive oxygen species, including O2.-, H2O2, .OH, .NO, and ONOO-, which play important roles in vascular homeostatic mechanisms and pathogenic processes. To better quantify cell oxidant metabolism processes, a fluorescence technique has been developed to measure H2O2 release from bovine aortic endothelial cells. Incubation of H2O2 with horseradish peroxidase (HRP) results in HRP-mediated oxidation of p-hydroxy-phenylacetic acid (PHPA) to the fluorescent PHPA dimer, 2,2'-dihydroxy-biphenyl-5,5' diacetate [(PHPA)2]. The HRP-mediated dimerization of 5 mM PHPA with concentrations of H2O2 up to 2.5 mM resulted in a linear increase in fluorescence (R = .995, n = 8). Maximal fluorescence occurred at 2.9 mM H2O2, with greater H2O2 concentrations yielding products with altered spectrophotometric characteristics and decreased fluorescent yield. The fluorescence of (PHPA)2 was pH sensitive and increased 500-fold from pH to 9. Fluorescence versus pH profiles were adjusted to a Henderson-Hasselbalch fitting, with a 50% maximal emission at pH = 8.1 +/- 0.2. The apparent pKa of fluorescence emission correlated well with a weak range of buffering capacity of (PHPA)2, which had a pKa = 8.0 +/- 0.1. With cells maintained in Hank's balanced salt solution (HBSS), the pH can increase to 7.90 during 3 h, with the increased pH due to the loss of HCO3- from HBSS. After adjustment for pH changes, a linear cellular H2O2 release of 217 pmol H2O2.min-1.mg protein-1 was observed. When bovine aortic endothelial cells (BAEC) were incubated with HBSS and PHPA alone, 50% less fluorescence was observed than when HRP was added.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Roles of catalase and cytochrome c in hydroperoxide-dependent lipid peroxidation and chemiluminescence in rat heart and kidney mitochondria.

A recent report (Radi et al., J. Biol. Chem. 266:22028-22034, 1991) showed that rat heart mitochondria contain catalase. The protective role of mitochondrial catalase was tested by exposing heart or kidney mitochondria and mitoplasts to two oxidants (H2O2) or tert-butyl hydroperoxide, t-BOOH), estimating lipid peroxidation (as thiobarbituric acid-reactive substances, TBARS) and overall oxidative stress (as chemiluminescence). Additional controls included heart and kidney preparations from aminotriazole-treated (catalase-depleted) rats. Both oxidants increased TBARS in catalase-free preparations to similar extents over their respective controls (between 200 to 350%). In catalase-containing preparations, H2O2 lipid peroxidation increased by only 40 to 96% over controls. Similar qualitative results were obtained when measuring chemiluminescence. The catalytic role of cytochrome c in mitochondrial lipid peroxidation was investigated by exposing either control or cytochrome-c-depleted kidney mitoplasts (catalase free) to either H2O2 or t-BOOH. Hydrogen-peroxide-dependent mitochondrial lipid peroxidation varied with cytochrome c concentration, remaining close to controls when cytochrome c concentration decreased by 66%, even though there was no catalase present. Tert-butyl hydroperoxide-dependent lipid peroxidation was less affected by cytochrome c remaining 2.3-fold above controls under the same conditions, suggesting that organic peroxides are more likely to remain in the less polar membrane environment being decomposed by heme or nonheme iron imbedded in the inner mitochondrial membrane. Chemiluminescence was less affected by cytochrome c depletion. Comparing control and cytochrome-c-deficient mitochondria, chemiluminescence was 1.7-fold and 2.8-fold higher when control preparations were challenged with t-BOOH or H2O2, respectively.

Animals↗

PubMed1993

Physiologic levels of uric acid inhibit xanthine oxidase in human plasma.

Xanthine oxidase, a key source of reactive oxygen species, and purine substrates are detected in the circulation after ischemia-reperfusion. High levels of uric acid, produced by a xanthine oxidase-catalyzed reaction, are found in human plasma. We studied whether uric acid could alter xanthine oxidase activity in plasma obtained from eight adults and eight neonates. Known amounts of uric acid were added to xanthine and xanthine oxidase-supplemented buffer and plasma, and the production of uric acid and superoxide was determined. Uric acid, 150 and 300 microM, decreased the oxidation of xanthine to uric acid in adult plasma by 37.5 +/- 5.6 and 48.9 +/- 6.1% and formation of superoxide by 23.2 +/- 1.9 and 32.0 +/- 2.3%, respectively, compared with plasma without uric acid. In newborn plasma, a similar pattern and extent of inhibition was observed. Superoxide formation, however, was inhibited to a greater extent than in adult plasma. Endogenous xanthine oxidase was detected in newborn plasma in nine additional neonates using HPLC. These results indicate that uric acid is an effective inhibitor of the formation of superoxide and hydrogen peroxide by xanthine oxidase at the levels found in human plasma. Plasma uric acid may play an important role in attenuating the oxidant-mediated tissue damage caused by xanthine oxidase released into the circulation during ischemia-reperfusion.

Adult↗

PubMed1993

Intracellular catalase inhibition does not predispose rat heart to ischemia-reperfusion and hydrogen peroxide-induced injuries.

The objective of this study was to determine whether inhibition of intracellular catalase would decrease the tolerance of the heart to ischemia-reperfusion and hydrogen peroxide-induced injuries. Isolated bicarbonate buffer-perfused rat hearts were used in the study. Intracellular catalase was inhibited with 3-amino-1,2,4-triazole (ATZ, 1.5 g/kg body weight, two hours prior to heart perfusion). In the ischemia-reperfusion protocol, hearts were arrested with St. Thomas'II cardioplegic solution, made ischemic for 35 min at 37 degrees C, and reperfused with Krebs-Henseleit buffer for 30 min. The extent of ischemic injury was assessed using postischemic contractile recovery and lactate dehydrogenase (LDH) leakage into reperfusate. In the hydrogen peroxide infusion protocol, hearts were perfused with increasing concentrations of hydrogen peroxide (inflow rates 0.05-1.25 mumol/min). Inhibition of catalase activity (30.4 +/- 1.8 mU/mg protein in control vs 2.4 +/- 0.3 mU/mg in ATZ-treated hearts) affected neither pre-ischemic aerobic cardiac function nor post-ischemic functional recovery and LDH release in hearts subjected to 35 min cardioplegic ischemic arrest. Myocardial contents of lipid hydroperoxides were similar in control and ATZ-treated animals after 20 min aerobic perfusion, ischemia, and ischemia-reperfusion. During hydrogen peroxide perfusion, there was an increase in coronary flow rate followed by an elevation in diastolic pressure and inhibition of contractile function in comparison with control hearts. The functional parameters between control and ATZ-treated groups remained unchanged. The concentrations of myocardial lipid hydroperoxides were the same in both groups. We conclude that inhibition of myocardial catalase activity with ATZ does not predispose the rat heart to ischemia-reperfusion and hydrogen peroxide-induced injury.

Amitrole↗

PubMed1992

Inhibition of xanthine oxidase by uric acid and its influence on superoxide radical production.

The inhibition of xanthine oxidase by its reaction product, uric acid, was studied by steady state kinetic analysis. Uric acid behaved as an uncompetitive inhibitor of xanthine oxidase with respect to the reducing substrate, xanthine. Under 50 microM xanthine and 210 microM oxygen, the apparent K(i) for uric acid was 70 microM. Uric acid-mediated xanthine oxidase inhibition also caused an increase in the percentage of univalent reoxidation of the enzyme (superoxide radical production). Steady-state rate equations derived by the King-Altman method support the formation of an abortive-inhibitory enzyme-uric acid complex (dead-end product inhibition). Alternatively, inhibition could also depend on the reversibility of the classical ping-pong mechanism present in xanthine oxidase-catalyzed reactions.

Free Radicals↗

PubMed1992

The contribution of vascular endothelial xanthine dehydrogenase/oxidase to oxygen-mediated cell injury.

The conversion of xanthine dehydrogenase (XDH) to xanthine oxidase (XO) and the reaction of XO-derived partially reduced oxygen species (PROS) have been suggested to be important in diverse mechanisms of tissue pathophysiology, including oxygen toxicity. Bovine aortic endothelial cells expressed variable amounts of XDH and XO activity in culture. Xanthine dehydrogenase plus xanthine oxidase specific activity increased in dividing cells, peaked after achieving confluency, and decreased in postconfluent cells. Exposure of BAEC to hyperoxia (95% O2; 5% CO2) for 0-48 h caused no change in cell protein or DNA when compared to normoxic controls. Cell XDH+XO activity decreased 98% after 48 h of 95% O2 exposure and decreased 68% after 48 h normoxia. During hyperoxia, the percentage of cell XDH+XO in the XO form increased to 100%, but was unchanged in air controls. Cell catalase activity was unaffected by hyperoxia and lactate dehydrogenase activity was minimally elevated. Hyperoxia resulted in enhanced cell detachment from monolayers, which increased 112% compared to controls. Release of DNA and preincorporated [8-14C]adenine was also used to assess hyperoxic cell injury and did not significantly change in exposed cells. Pretreatment of cells with allopurinol for 1 h inhibited XDH+XO activity 100%, which could be reversed after oxidation of cell lysates with potassium ferricyanide (K3Fe(CN)6). After 48 h of culture in air with allopurinol, cell XDH+XO activity was enhanced when assayed after reversal of inhibition with K3Fe(CN)6, and cell detachment was decreased. In contrast, allopurinol treatment of cells 1 h prior to and during 48 h of hyperoxic exposure did not reduce cell damage. After K3Fe(CN)6 oxidation, XDH+XO activity was undetectable in hyperoxic cell lysates. Thus, XO-derived PROS did not contribute to cell injury or inactivation of XDH+XO during hyperoxia. It is concluded that endogenous cell XO was not a significant source of reactive oxygen species during hyperoxia and contributes only minimally to net cell production of O2- and H2O2 during normoxia.

Animals↗

PubMed1991

Detection of catalase in rat heart mitochondria.

The presence of heme-containing catalase in rat heart mitochondria (20 +/- 5 units/mg) was demonstrated by biochemical and immunocytochemical analysis. Intact rat heart mitochondria efficiently consumed exogenously added H2O2. The rate of H2O2 consumption was not influenced by succinate, glutamate/malate, or N-ethylmaleimide but was significantly inhibited by cyanide. Hydrogen peroxide decomposition by mitochondria yielded molecular oxygen in a 2:1 stoichiometry, consistent with a catalytic mechanism. Mitochondrial fractionation studies and quantitative electron microscopic immunocytochemistry revealed that most catalase was matrix-associated. Electrophoretic analysis and Western blotting of the mitochondrial matrix fraction indicated the presence of a protein with similar electrophoretic mobility to bovine and rat liver catalase and immunoreactive to anti-catalase antibody. Myocardial tissue has a lower catalase-specific activity and a greater mitochondrial H2O2 production/g of tissue than most organs. Thus catalase, representing 0.025% of heart mitochondrial protein, is important for detoxifying mitochondrial derived H2O2 and represents a key antioxidant defense mechanism for myocardial tissue.

Animals↗

PubMed1991

Substrate inhibition of xanthine oxidase and its influence on superoxide radical production.

The influence of substrate inhibition on xanthine oxidase-intramolecular electron transport was studied by steady-state kinetic analysis. Experiments with hypoxanthine and xanthine up to 900 microM indicated an inhibition pattern which fitted an equation of the general form nu 0 = nu max . [S]/(Km + a[S] + b[S]2/Ki). Univalent electron flux to oxygen was favored at substrate concentrations above 50 microM. This augmentation of univalent flux percentage that appeared at a high substrate concentration was greater for hypoxanthine that xanthine and at pH 8.3 than at 9.5. Our results support a mechanism of inhibition in which a substrate-reduced enzyme, non-productive Michaelis complex was formed. It is possible that this non-productive complex favored the univalent pathway of enzyme reoxidation (superoxide production) by increasing the midpoint redox potential of the molybdenum active site.

Animals↗