Inhibition of xanthine oxidase and xanthine dehydrogenase by nitric oxide. Nitric oxide converts reduced xanthine-oxidizing enzymes into the desulfo-type inactive form.
Xanthine oxidase (XO) and xanthine dehydrogenase (XDH) were inactivated by incubation with nitric oxide under anaerobic conditions in the presence of xanthine or allopurinol. The inactivation was not pronounced in the absence of an electron donor, indicating that only the reduced enzyme form was inactivated by nitric oxide. The second-order rate constant of the reaction between reduced XO and nitric oxide was determined to be 14.8 +/- 1.4 M-1 s-1 at 25 degrees C. The inactivated enzymes lacked xanthine-dichlorophenolindophenol activity, and the oxypurinol-bound form of XO was partly protected from the inactivation. The absorption spectrum of the inactivated enzyme was not markedly different from that of the normal enzyme. The flavin and iron-sulfur centers of inactivated XO were reduced by dithionite and reoxidized readily with oxygen, and inactivated XDH retained electron transfer activities from NADH to electron acceptors, consistent with the conclusion that the flavin and iron-sulfur centers of the inactivated enzyme both remained intact. Inactivated XO reduced with 6-methylpurine showed no "very rapid" spectra, indicating that the molybdopterin moiety was damaged. Furthermore, inactivated XO reduced by dithionite showed the same slow Mo(V) spectrum as that derived from the desulfo-type enzyme. On the other hand, inactivated XO reduced by dithionite exhibited the same signals for iron-sulfur centers as the normal enzyme. Inactivated XO recovered its activity in the presence of a sulfide-generating system. It is concluded that nitric oxide reacts with an essential sulfur of the reduced molybdenum center of XO and XDH to produce desulfo-type inactive enzymes. (+info)
The superoxide dismutase activity of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774.
Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli [Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol. 178, 6736-6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues-Pousada, C. (1999) Eur. J. Biochem. 259, 235-243]. Thus, the SOD activity of Dfx isolated from the sulphate-reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U x mg-1, which increases approximately 2.5-fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low-spin iron species with g-values at 2.27 (g perpendicular) and 1.96 (g parallel). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs. Evidence for another SOD in D. desulfuricans is also presented - this enzyme is inhibited by cyanide, and N-terminal sequence data strongly indicates that it is an analogue to Cu,Zn-SODs isolated from other sources. This is the first indication that a Cu-containing protein may be present in a sulphate-reducing bacterium. (+info)
Cytosolic Ca2+ movements of endothelial cells exposed to reactive oxygen intermediates: role of hydroxyl radical-mediated redox alteration of cell-membrane Ca2+ channels.
1. The mode of action of reactive oxygen intermediates in cysosolic Ca2+ movements of cultured porcine aortic endothelial cells exposed to xanthine/xanthine oxidase (X/XO) was investigated. 2. Cytosolic Ca2+ movements provoked by X/XO consisted of an initial Ca2+ release from thapsigargin-sensitive intracellular Ca2+ stores and a sustained Ca2+ influx through cell-membrane Ca2+ channels. The Ca2+ movements from both sources were inhibited by catalase, cell-membrane permeable iron chelators (o-phenanthroline and deferoxamine), a *OH scavenger (5,5-dimethyl-1-pyrroline-N-oxide), or an anion channel blocker (disodium 4, 4'-diisothiocyano-2, 2'-stilbenedisulphonic acid), suggesting that *O2- influx through anion channels was responsible for the Ca2+ movements, in which *OH generation catalyzed by intracellular transition metals (i.e., Haber-Weiss cycle) was involved. 3. After an initial Ca2+ elevation provoked by X/XO, cytosolic Ca2+ concentration decreased to a level higher than basal levels. Removal of X/XO slightly enhanced the Ca2+ decrease. Extracellular addition of sulphydryl (SH)-reducing agents, dithiothreitol or glutathione, after the removal of X/XO accelerated the decrement. A Ca2+ channel blocker, Ni2+, abolished the sustained increase in Ca2+, suggesting that Ca2+ influx through cell-membrane Ca2+ channels was extracellularly regulated by the redox state of SH-groups. 4. The X/XO-provoked change in cellular respiration was inhibited by Ni2+ or dithiothreitol as well as inhibitors of Haber-Weiss cycle, suggesting that Ca2+ influx was responsible for *OH-mediated cytotoxicity. We concluded that intracellular *OH generation was involved in the Ca2+ movements in endothelial cells exposed to X/XO. Cytosolic Ca2+ elevation was partly responsible for the oxidants-mediated cytotoxicity. (+info)
Isolation and characterization of the Saccharomyces cerevisiae XPT1 gene encoding xanthine phosphoribosyl transferase.
A new Saccharomyces cerevisiae gene, XPT1, was isolated as a multicopy suppressor of a hypoxanthine phosphoribosyl transferase (HPRT) defect. Disruption of XPT1 affects xanthine utilization in vivo and results in a severe reduction of xanthine phosphoribosyl transferase (XPRT) activity while HPRT is unaffected. We conclude that XPT1 encodes XPRT in yeast. (+info)
Reactive oxygen species activate a Ca2+-dependent cell death pathway in the unicellular organism Trypanosoma brucei brucei.
Here we examine a cell death process induced by reactive oxygen species (ROS) in the haemoflagellate Trypanosoma brucei brucei. Ca2+ distribution in cellular compartments was measured with stable transformants expressing aequorin targeted to the cytosol, nucleus or mitochondrion. Within 1.5 h of ROS production, mitochondrial Ca2+ transport was impaired and the Ca2+ barrier between the nuclear envelope and cytosol was disrupted. Consequently the mitochondrion did not accumulate Ca2+ efficiently in response to an extracellular stimulus, and excess Ca2+ accumulated in the nucleus. The terminal transferase deoxytidyl uridine end labelling assay revealed that, 5 h after treatment with ROS, extensive fragmentation of nuclear DNA occurred in over 90% of the cells. Permeability changes in the plasma membrane did not occur until an additional 2 h had elapsed. The intracellular Ca2+ buffer, EGTA acetoxymethyl ester, prevented DNA fragmentation and prolonged the onset of changes in cell permeability. Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase 3, caspase 1, calpain, serine protease, cysteine protease or proteasome activity. Moreover, trypanosomes expressing mouse Bcl-2 were not protected from ROS even though protection from mitochondrial dysfunction and ROS have been reported for mammalian cells. Overall, these results demonstrate that Ca2+ pathways can induce pathology in trypanosomes, although the specific proteins involved might be distinct from those in metazoans. (+info)
Formation of the aldehydic choline glycerophospholipids in human red blood cell membrane peroxidized with an azo initiator.
The production of phospholipid hydroperoxide and aldehydic phospholipid was examined in human red blood cell (RBC) membranes after peroxidation with 2,2-azobis(2-amidinopropane)dihydrochloride (AAPH) or xanthine/xanthine oxidase (XO/XOD/Fe3+). Both radical-generation systems caused a profound decrease in the amount of polyunsaturated fatty acid (PUFA) in choline glycerophospholipid (CGP) and induced formation of peroxidized CGP in RBC membranes to different extents. No consistent generation of peroxidized lipids from CGP was evident after peroxidation with XO/XOD/Fe3+, which caused the apparent decomposition of phospholipids and the formation of large amounts of thiobarbituric acid-reactive substance (TBARS). On the other hand, CGP hydroperoxide was formed as a primary product of peroxidation with AAPH. Aldehydic CGP was also detected as a secondary product of hydroperoxide decomposition in AAPH-peroxidized RBC membranes. Aldehydic CGP was preferentially generated from arachidonoyl CGP rather than from linoleoyl CGP in AAPH-peroxidized membranes. AAPH mainly oxidized CGP to hydroperoxide and aldehydic phospholipids. The sum of hydroperoxide and aldehyde of CGP corresponded to the loss of CGP due to peroxidation by AAPH. This result indicates that CGP was mainly converted into these two oxidized phospholipids in AAPH-peroxidized RBC membranes. (+info)
Effects of reactive oxygen and nitrogen metabolites on RANTES- and IL-5-induced eosinophil chemotactic activity in vitro.
Eosinophils and increased production of nitric oxide (NO) and superoxide, components of peroxynitrite, have been implicated in the pathogenesis of a number of allergic disorders including asthma. Peroxynitrite induced protein nitration may compromise enzyme and protein function. We hypothesized that peroxynitrite may modulate eosinophil migration by modulating chemotactic cytokines. To test this hypothesis, the eosinophil chemotactic responses of regulated on activation, normal T cell expressed and secreted (RANTES) and interleukin (IL)-5 incubated with and without peroxynitrite were evaluated. Peroxynitrite-attenuated RANTES and IL-5 induced eosinophil chemotactic activity (ECA) in a dose-dependent manner (P < 0.05) but did not attenuate leukotriene B4 or complement-activated serum ECA. The reducing agents deferoxamine and dithiothreitol reversed the ECA inhibition by peroxynitrite, and exogenous L-tyrosine abrogated the inhibition by peroxynitrite. PAPA-NONOate, a NO donor, or superoxide generated by lumazine or xanthine and xanthine oxidase, did not show an inhibitory effect on ECA. The peroxynitrite generator, 3-morpholinosydnonimine, caused a concentration-dependent inhibition of ECA. Peroxynitrite reduced RANTES and IL-5 binding to eosinophils and resulted in nitrotyrosine formation. These findings are consistent with nitration of tyrosine by peroxynitrite with subsequent inhibition of RANTES and IL-5 binding to eosinophils and suggest that peroxynitrite may play a role in regulation of eosinophil chemotaxis. (+info)
Effects of superoxide anion on intracellular Ca2+ concentration in rabbit pulmonary arterial smooth muscle cells.
AIM: To study the effect of superoxide anion on the Ca2+ homeostasis in smooth muscle cells isolated from the rabbit pulmonary artery. METHODS: Intracellular Ca2+ concentration ([Ca2+]i) was investigated using cell suspension of freshly isolated smooth muscle cells from rabbit pulmonary artery (PASMC). Fura-2 fluorescent ratio obtained at 340 nm and 380 nm wave lengths was measured as an indicator of [Ca2+]i. RESULTS: ATP 30 mumol.L-1 induced a transient increase in the ratio (Ca2+ transient). Thapsigargin, an inhibitor of sarcoplasmic Ca2+ ATPase, induced a phasic increase in the ratio due to Ca2+ leak from intracellular store sites, but not the sustained increase, thereby suggesting the absence of Ca2+ release-activated Ca2+ entry (CRAC) mechanism in PASMC. When PASMC were exposed to superoxide anion by the pretreatment with xanthine and xanthine oxidase (X/XO) for 30 min, sustained component of ATP-induced Ca2+ transient was elevated. The ratios at 5 and 10 min after ATP application (delta ratio5 min and delta ratio10 min) were increased from 0.091 +/- 0.022 to 0.149 +/- 0.048 (P < 0.05) and from 0.021 +/- 0.020 to 0.117 +/- 0.047 (P < 0.01), respectively. But, thapsigargin-induced [Ca2+]i transient was not affected by X/XO. CONCLUSION: Superoxide anion makes ATP-induced Ca2+ transient sluggish, and does not affect Ca2+ leak pathway in PASMC. (+info)