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1.
The role of a recently identified K+ATP channel in preventing H2O2 formation was examined in isolated pea stem mitochondria. The succinate-dependent H2O2 formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H2O2 formation was not influenced. The succinate-sustained H2O2 generation was also unaffected by nigericin (a H+/K+ exchanger), but completely prevented by valinomycin (a K+ ionophore). In addition, cyclosporin A (a K+ATP channel opener) inhibited this H2O2 formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K+ channel faces the intermembrane space. Finally, the succinate-dependent H2O2 formation was partially prevented by phenylarsine oxide (a thiol oxidant).  相似文献   

2.
Abstract: We studied the action of H2O2 on the exocytosis of glutamate by cerebrocortical synaptosomes. The treatment of synaptosomes with H2O2 (50–150 µ M ) for a few minutes results in a long-lasting depression of the Ca2+-dependent exocytosis of glutamate, induced by KCl or by the K+-channel inhibitor 4-aminopyridine. The energy state of synaptosomes, as judged by the level of phosphocreatine and the ATP/ADP ratio, was not affected by H2O2, although a transient decrease was observed after the treatment. H2O2 did not promote peroxidation, as judged by the formation of malondialdehyde. In indo-1-loaded synaptosomes, the treatment with H2O2 did not modify significantly the KCl-induced increase of [Ca2+]i. H2O2 inhibited exocytosis also when the latter was induced by increasing [Ca2+]i with the Ca2+ ionophore ionomycin. The effects of H2O2 were unchanged in the presence of superoxide dismutase and the presence of the Fe3+ chelator deferoxamine. These results appear to indicate that H2O2, apparently without damaging the synaptosomes, induces a long-lasting inhibition of the exocytosis of glutamate by acting directly on the exocytotic process.  相似文献   

3.
Abstract Everted vesicles of the methanogenic strain Gö1 synthesized ATP in response to methanogenesis from methyl-coenzyme M and H2. Simultaneously, a transmembrane pH gradient (ΔpH) was generated as evident from fluorescence quenching of acridine orange. Protonophorous uncouplers prevented ΔpH generation and ATP synthesis, but did not affect methanogenesis. The ATP synthase inhibitor diethylstilbestrol (DES) inhibited ATP synthesis but had no effect on methanogenesis and on ΔpH formation, indicating the essential role of the transmembrane proton potential in ATP synthesis. Progress has also been made in assigning specific functions to membrane components in methanogenesis from methyl-CoM and H2. Separation of cell extracts into cytoplasmic and membrane fraction revealed an essential role of membrane-bound components in electron transfer: methanogenesis catalyzed by the cytoplasmic fraction from strain Gö1 was stimulated several fold by membranes from various methanogens. This stimulation was prevented if the membranes had been treated with oxidants (O2, K3[Fe(CN)6]) or SH reagents (Ag+, p -chloromercuribenzoate, iodoacetamide) pointing to the involvement of functional SH groups in methanogenesis from methyl-CoM and H2.  相似文献   

4.
Abstract In cell suspensions of the methanogenic bacterium strain Gö1 or Methanosarcina barkeri H2 formation from methanol in the presence of 2-bromoethanesulfonic acid (BES) was strictly dependent on sodium ions; apparent K S for Na+, 1.3±0.3 mM.H2 formation was inhibited by the uncoupler tetrachlorosalicylanilide (TCS), but this inhibition could be temporarily overcome, when a sodium pulse (100 mM) was given to the cell suspension. On the other hand, H2 formation from formaldehyde in the presence of BES (rate: 300 nmol H2/h·mg protein as compared to 25 nmol H2/h·mg protein from methanol) was not sodium-dependent, not TCS-sensitive and not inhibited by addition of monensin. H2 formation was accompanied by CO2 formation in stoichiometric amounts, 3 H2:1 CO2 for methanol and 2 H2:1 CO2 for formaldehyde oxidation.  相似文献   

5.
Abstract: The effects of 1-methyl-4-phenylpyridinium (MPP+) on the oxygen consumption, ATP production, H2O2 production, and mitochondrial NADH-CoQ1 reductase (complex I) activity of isolated rat brain mitochondria were investigated. Using glutamate and malate as substrates, concentrations of 10–100 µ M MPP+ had no effect on state 4 (−ADP) respiration but decreased state 3 (+ADP) respiration and ATP production. Incubating mitochondria with ADP for 30 min after loading with varying concentrations of MPP+ produced a concentration-dependent decrease in H2O2 production. Incubation of mitochondria with ADP for 60 min after loading with 100 µ M MPP+ caused no loss of complex I activity after washing of MPP+ from the mitochondrial membranes. These data are consistent with MPP+ initially binding specifically to complex I and inhibiting both the flow of reducing equivalents and the production of H2O2 by the mitochondrial respiratory chain, without irreversibly damaging complex I. However, mitochondria incubated with H2O2 in the presence of Cu2+ ions showed decreased complex I activity. This study provides additional evidence that cellular damage initiated by MPP+ is due primarily to energy depletion caused by specific binding to complex I, any increased damage due to free radical production by mitochondria being a secondary effect.  相似文献   

6.
Abstract Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H2 turnover per gram soil. Dilution did not affect the fraction of H2/CO2-dependent methanogenesis which made up 22% of total CH4 production. However, dilution resulted in a ten fold decrease of the H2 steady state partial pressure from approximately 4 to 0.4 Pa indicating that H2/CO2-dependent methanogenesis was more or less independent of the H2 pool. The rates of H2 production calculated from the H2 turnover rate constants and the H2 steady state partial pressures accounted for only < 5% of H2/CO2-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H2/CO2-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H2 transfer within syntrophic bacterial associations and was not significantly affected by the outside H2 pool.  相似文献   

7.
Abstract: Hydrogen peroxide (H2O2) is produced from several sources in brain and may be involved in neurodegeneration and second messenger signaling. Little is known about the effects of H2O2 on transmitter storage in brain synaptic vesicles. Neurotransmitter uptake into synaptic vesicles is driven by an electrochemical proton gradient generated by the vacuolar H+-ATPase (V-ATPase) in the vesicle membrane. We report here that the V-ATPase in bovine brain synaptic vesicles is highly sensitive to inhibition by micromolar concentrations of H2O2. Glutamate uptake by the vesicles is also inhibited, very likely as a secondary consequence of ATPase inactivation. Dithiothreitol or reduced glutathione reverse H2O2-induced inhibition of the V-ATPase, and ATP or GTP partially protect the ATPase from inhibition by H2O2. These and other results suggest that the mechanism of inhibition of the V-ATPase by H2O2 involves oxidation of a reactive cysteine sulfhydryl group in the ATP binding site. Inhibition of V-ATPase activity would decrease the amount of transmitter stored in synaptic vesicles and thus down-regulate transmitter release during episodes of oxidative stress or in response to second messenger signaling.  相似文献   

8.
Washed bacterial suspensions obtained from the pig hindgut were incubated under 13CO2 in a buffer containing NaH13CO3 and carbohydrates. Incorporation of 13C into short chain fatty acids was assayed by quantitative nuclear magnetic resonance. The effects of different levels of H2 added to the gas phase (0, 20 and 80% v/v) and of the specific methanogenesis inhibitor 2-bromoethane-sulphonic acid (BES) were determined. In control incubations increasing the concentration of H2 markedly increased methane production. Single- and double-labelled acetate and butyrate were formed in all incubations. In the absence of BES, increasing H2 significantly increased the incorporation of 13CO2 into butyrate and the proportion of double-labelled acetate in total labelled acetate. The addition of BES proved to be very successful as a methane inhibitor and greatly enhanced the amount of mono- and double-labelled acetate, especially at the highest H2 partial pressure. The results suggest that methanogenesis inhibited both routes of reductive acetogenesis, i.e. the homoacetate fermentation of hexose (represented for the most part by single labelling) and the synthesis of acetate from external CO2 and H2 (represented mostly by double labelling). A highly significant interaction between BES and H2 concentration was observed. At the highest pH2 BES increased the proportion of labelled acetate in total acetate from 17.1% for the control to 50.9%. It was concluded that although acetogenesis and methanogenesis can occur simultaneously in the pig hindgut, reductive acetogenesis may become a significant pathway of acetate formation in the absence of methanogenesis.  相似文献   

9.
Abstract Washed whole cells of Methanospirillum hungatei incubated in TES buffer retained methanogenic activity in the absence of any reducing agents. Washed cells grown with 80% H2-20% CO2 and acetate produced methane from H2/CO2 and 50 mM formate at 1.1 to 1.8 and 15 μmol methane · h−1· mg−1 protein, respectively. Cadmium at a concentration of 15 μM and 50 μM mercury, copper or zinc completely inhibited methane production from H2/CO2 by M. hungatei . The chelating agent, EDTA, protected the cells from inhibition by cadmium but acetate and citrate did not. The activity of formate dehydrogenase and hydrogenase remaining in cells after incubation with copper, mercury, zinc or cadmium was reduced with formate dehydrogenase being the more sensitive.  相似文献   

10.
Abstract The effect of cadmium (Cd) on methane formation from methanol and/or H2–CO2 by Methanosarcina barkeri was examined in a defined growth medium and in a simplified buffer system containing 50 mM Tes with or without 2 mM dithiothreitol (DTT). No inhibition of methanogenesis by high concentrations of cadmium was observed in growth medium. Similarly, little inhibition of methanogenesis by whole cells in the Tes buffer system was observed in the presence of 430 μM Cd or 370 μM mercury (Hg) with 2 mM DTT. When the concentration of DTT was reduced to 0.4 mM, almost complete inhibition of methanogenesis from H2–CO2 and methanol by 600 μM Cd was observed. In the absence of DTT, 150 μM Cd inhibited methanogenesis from H2–CO2 completely and from methanol by 97%. Methanogenesis from H2–CO2 was more sensitive to Cd than that from methanol.  相似文献   

11.
SUMMARY: Sterilized raw sewage sludge enriched with sulphate and inoculated with pure strains of Desulphovibrio desulphuricans produced negligible sulphide. Unsterilized sludge supplemented with 7% (w/v) CaSO4.2H2O and inoculated with crude cultures of sulphate-reducing bacteria obtained from sewage yielded 1·0% S2- (wt S2- produced as H2S/vol. of raw sludge) in 6 months at 30°. By repeated subculture more active cultures developed which produced 1% S2- in 7 days and 1·2–1·9% in 28 days. Digested sludge yielded only 0·1% S2-. In semicontinuous fermentations at 30°, raw sludge without added sulphate produced 20 times its own volume of gas containing 70% CH4 and 30% CO2. When 5% CaSO4.2H2O and an active crude culture of sulphate reducers were added, gas production decreased steadily to zero. There were no differences in pH, temperature and redox potential in sludges producing methane or sulphide. The chief cause of inhibition appeared to be the action of sulphide: 0·02% soluble sulphide (S2-) totally inhibited methane formation; 0·01% S2- initially decreased gas production by one-quarter but there was a slow recovery to normal, suggesting acclimatization of the methane-producing organisms to sulphide.
Linked fermentations, in which gas from a methane fermentation swept H2S from a sulphide fermentation, gave a final gas mixture of about 60% CH4, 30% CO2 and 5–10% H2S. The yield of sulphide depended on the rate of sweeping.  相似文献   

12.
Abstract The intestinal tract of invertebrate and vertebrate animals, including man, is an anoxic habitat wherein microbial formation of acetate from H2+ CO2 is often a major H2-consuming reaction. This paper will discuss the magnitude and microbiology of H2/CO2 acetogenesis in animal guts, its impact on host animal nutrition, competition for H2 between anaerobic microbes, and the global significance of intestinal H2/CO2 acetogenesis.  相似文献   

13.
The effect of hydrogen peroxide on spores of Clostridium perfringens   总被引:3,自引:2,他引:1  
Dithiothreitol (DTT)-treated spores of Clostridium perfringens were much more sensitive to lysis by H2O2 in the presence of Cu2+ than untreated spores. Lysis was greatly inhibited by hydroxyl radical (.OH) scavengers such as thiourea, dimethylthiourea and dimethylsulfoxide, suggesting that lysis of spores by H2O2 involves formation of OH by Cu2+-catalysed decomposition of the peroxide. DTT-treated spores took up Cu2+ at almost the same rate and extent as did isolated cortical fragments. Hydrogen peroxide caused both the decrease in optical density and the hexosamine solubilization of cortical fragments which bound Cu2+.  相似文献   

14.
Abstract The formation of H2 by chemolithoautrophically growing Oligotropha carboxidovorans has been identified as the result of the oxidation of CO mediated by the cytoplasmic species of the molybdenum-containing CO dehydrogenase multienzyme complex as follows: CO + H 2 O → CO 2+ H 2. Purified CO dehydrogenase was shown to carry hydrogen uptake and formation activities in addition to its catabolic function which is the oxidation of CO. Among the electron donors supporting H2 formation were CO, NADH, reduced flavins and reduced viologen dyes. The reduction of protons to H2 by cytoplasmic CO dehydrogenase is interpreted as a detoxification reaction for electrons to prevent cell damage in O. carboxidovorans .  相似文献   

15.
Clostridium pasteurianum fermented glucose to acetate, butyrate, CO2 and H2. In batch cultures the fermentation pattern was only slightly affected by culture pH over the range 8·0 to 5·5. The acetate/butyrate ratio was always higher than or equal to one. Between 2·14 and 2·33 mol H2 was produced per mol glucose fermented. At unregulated pH, more butanol and less butyrate was formed. In a carbon-limited chemostat, the steady-state acetate/butyrate ratio was always lower than one. H2 production was approximately 1·70 mol per mol glucose consumed. Substantial amounts of extracellular protein were formed. With decreasing pH, acetate and formate production decreased, while H2 production was highest at pH 6.0. With increasing dilution rate ( D ), the product spectrum hardly changed, but more biomass was formed. Y glucosemax and Y ATPmax were 55·97 and 31·48 g dry weight per mol glucose or ATP respectively. With increasing glucose input the formation of fatty acids and H2 slightly decreased.
Continuous cultures fermented mannitol to acetate, butyrate, butanol, CO2 and H2. With acetate as co-substrate, butanol production and molar growth yields, Y mannitol and Y ATP, markedly decreased, while the butyrate and H2 production increased. The latter reached a value of 2·21 mol H2 per mol mannitol consumed.  相似文献   

16.
Abstract: Inorganic phosphate (Pi) plays a vital role in intracellular energy metabolism. Its many effects include stimulation of glucose use, enhancement of high-energy phosphate concentrations, and modulation of cytosolic free [Ca2+]. Cultured fetal rat cortical neurons constitutively import Pi, and cytosolic levels positively correlate with [ATP], [NADPH], and energy charge. In the present study, we demonstrate that the concentration of intracellular Pi is an important determinant of acute neuronal survival after an excitotoxic or oxidative insult to cultured fetal rat cortical neurons. Extracellular Pi dose-dependently enhanced survival of cortical neurons after exposure to NMDA at early (≤6 h) time points after termination of the insult. Pi similarly increased neuronal survival after exposure to kainic acid or H2O2. Pi-exposed neurons had higher basal intracellular [Pi], [ATP], and [GSH], and slightly lower cytosolic free [Ca2+], compared with Pi-deprived neurons. Pi-exposed neurons maintained increased [ATP] after exposure to NMDA and displayed reduced formation of reactive oxygen species after exposure to kainic acid or H2O2, compared with Pi-deprived neurons. These findings demonstrate that changes in extracellular and intracellular Pi can affect neuronal survival after excitotoxic or oxidative insults.  相似文献   

17.
Soils contain two different activities for oxidation of hydrogen   总被引:1,自引:0,他引:1  
Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H2 mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K m values for H2, one at about 10–40 nM dissolved H2, the other at about 1.2–1.4 μM H2. The low- K m activity was less sensitive to chloroform fumigation than the high- K m activity. If sterile soil was amended with Paracoccus denitrificans or a H2-oxidizing strain isolated from compost soil, it exhibited only a high- K m (0.7–0.9 μM) activity. It also failed to utilize H2 mixing ratios below a threshold of 1.6–3.0 ppmv H2 (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H2 oxidation was determined from the decrease of dissolved H2. However, H2 was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H2: (1) a high- K m, high-threshold activity which apparently is due to aerobic H2-oxidizing bacteria, and (2) a low- K m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.  相似文献   

18.
Abstract: Mitochondrial complexes I, II, and III were studied in isolated brain mitochondrial preparations with the goal of determining their relative abilities to reduce O2 to hydrogen peroxide (H2O2) or to reduce the alternative electron acceptors nitroblue tetrazolium (NBT) and diphenyliodonium (DPI). Complex I and II stimulation caused H2O2 formation and reduced NBT and DPI as indicated by dichlorodihydrofluorescein oxidation, nitroformazan precipitation, and DPI-mediated enzyme inactivation. The O2 consumption rate was more rapid under complex II (succinate) stimulation than under complex I (NADH) stimulation. In contrast, H2O2 generation and NBT and DPI reduction kinetics were favored by NADH addition but were virtually unobservable during succinate-linked respiration. NADH oxidation was strongly suppressed by rotenone, but NADH-coupled H2O2 flux was accelerated by rotenone. α-Phenyl- N-tert -butyl nitrone (PBN), a compound documented to inhibit oxidative stress in models of stroke, sepsis, and parkinsonism, partially inhibited complex I-stimulated H2O2 flux and NBT reduction and also protected complex I from DPI-mediated inactivation while trapping the phenyl radical product of DPI reduction. The results suggest that complex I may be the principal source of brain mitochondrial H2O2 synthesis, possessing an "electron leak" site upstream from the rotenone binding site (i.e., on the NADH side of the enzyme). The inhibition of H2O2 production by PBN suggests a novel explanation for the broad-spectrum antioxidant and antiinflammatory activity of this nitrone spin trap.  相似文献   

19.
Abstract: We investigated the regulation by intracellular Ca2+ of agonist-induced sequestration of Gq protein-coupled histamine H1 receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H1 receptors from the cell surface membrane was detected as the loss of [3H]mepyramine binding sites on intact cells accessible to the hydrophilic H1-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [3H]mepyramine were mirrored by changes in the subcellular distribution of H1 receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H1 receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca2+ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca2+/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H1-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of Gq protein-coupled receptors is transiently inhibited by Ca2+/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.  相似文献   

20.
Abstract Methanosphaera stadtmanae , a member of the Methanobacteriales reduces methanol, but not CO2 with H2 or 2-propanol to produce methane. In cell-free extracts of M. stadtmanae the activities of several enzymes involved in electron transfer were measured. The activities of an F420-nonreactive hydrogenase, NADP+: F420 oxidoreductase, NADP+-dependent 2-propanol dehydrogenase, and a methyl viologen dependent F420 dehydrogenase were observed. Based on the presence of these particular enzyme activities, their cofactor requirements and the absence of F420-dependent hydrogenase activity, a model of the electron transport pathway through the coenzyme F420 to provide electrons for biosynthesis, was formulated.  相似文献   

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