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1.
Achromobacter denitrificans YD35 is an NO 2−-tolerant bacterium that expresses the aconitase genes acnA3, acnA4, and acnB, of which acnA3 is essential for growth tolerance against 100 m m NO 2−. Atmospheric oxygen inactivated AcnA3 at a rate of 1.6 × 10 −3 min −1, which was 2.7- and 37-fold lower compared with AcnA4 and AcnB, respectively. Stoichiometric titration showed that the [4Fe-4S] 2+ cluster of AcnA3 was more stable against oxidative inactivation by ferricyanide than that of AcnA4. Aconitase activity of AcnA3 persisted against high NO 2− levels that generate reactive nitrogen species with an inactivation rate constant of k = 7.8 × 10 −3 min −1, which was 1.6- and 7.8-fold lower than those for AcnA4 and AcnB, respectively. When exposed to NO 2−, the acnA3 mutant (AcnA3Tn) accumulated higher levels of cellular citrate compared with the other aconitase mutants, indicating that AcnA3 is a major producer of cellular aconitase activity. The extreme resistance of AcnA3 against oxidation and reactive nitrogen species apparently contributes to bacterial NO 2− tolerance. AcnA3Tn accumulated less cellular NADH and ATP compared with YD35 under our culture conditions. The accumulation of more NO by AcnA3Tn suggested that NADH-dependent enzymes detoxify NO for survival in a high NO 2− milieu. This novel aconitase is distributed in Alcaligenaceae bacteria, including pathogens and denitrifiers, and it appears to contribute to a novel NO 2− tolerance mechanism in this strain. 相似文献
2.
Mass spectrometric analysis shows that assimilation of inorganic nitrogen (NH 4+, NO 2−, NO 3−) by N-limited cells of Selenastrum minutum (Naeg.) Collins results in a stimulation of tricarboxylic acid cycle (TCA cycle) CO 2 release in both the light and dark. In a previous study we have shown that TCA cycle reductant generated during NH 4+ assimilation is oxidized via the cytochrome electron transport chain, resulting in an increase in respiratory O 2 consumption during photosynthesis (HG Weger, DG Birch, IR Elrifi, DH Turpin [1988] Plant Physiol 86: 688-692). NO 3− and NO 2− assimilation resulted in a larger stimulation of TCA cycle CO 2 release than did NH 4+, but a much smaller stimulation of mitochondrial O 2 consumption. NH 4+ assimilation was the same in the light and dark and insensitive to DCMU, but was 82% inhibited by anaerobiosis in both the light and dark. NO 3− and NO 2− assimilation rates were maximal in the light, but assimilation could proceed at substantial rates in the light in the presence of DCMU and in the dark. Unlike NH 4+, NO 3− and NO 2− assimilation were relatively insensitive to anaerobiosis. These results indicated that operation of the mitochondrial electron transport chain was not required to maintain TCA cycle activity during NO 3− and NO 2− assimilation, suggesting an alternative sink for TCA cycle generated reductant. Evaluation of changes in gross O 2 consumption during NO 3− and NO 2− assimilation suggest that TCA cycle reductant was exported to the chloroplast during photosynthesis and used to support NO 3− and NO 2− reduction. 相似文献
3.
Short-term changes in pyridine nucleotides and other key metabolites were measured during the onset of NO 3− or NH 4+ assimilation in the dark by the N-limited green alga Selenastrum minutum. When NH 4+ was added to N-limited cells, the NADH/NAD ratio rose immediately and the NADPH/NADP ratio followed more slowly. An immediate decrease in glutamate and 2-oxoglutarate indicates an increased flux through the glutamine synthase/glutamate oxoglutarate aminotransferase. Pyruvate kinase and phospho enolpyruvate carboxylase are rapidly activated to supply carbon skeletons to the tricarboxylic acid cycle for amino acid synthesis. In contrast, NO 3− addition caused an immediate decrease in the NADPH/NADP ratio that was accompanied by an increase in 6-phosphogluconate and decrease in the glucose-6-phosphate/6-phosphogluconate ratio. These changes show increased glucose-6-phosphate dehydrogenase activity, indicating that the oxidative pentose phosphate pathway supplies some reductant for NO 3− assimilation in the dark. A lag of 30 to 60 seconds in the increase of the NADH/NAD ratio during NO 3− assimilation correlates with a slow activation of pyruvate kinase and phospho enolpyruvate carboxylase. Together, these results indicate that during NH 4+ assimilation, the demand for ATP and carbon skeletons to synthesize amino acid signals activation of respiratory carbon flow. In contrast, during NO 3− assimilation, the initial demand on carbon respiration is for reductant and there is a lag before tricarboxylic acid cycle carbon flow is activated in response to the carbon demands of amino acid synthesis. 相似文献
4.
Most studies of bacterial denitrification have used nitrate (NO 3−) as the first electron acceptor, whereas relatively less is understood about nitrite (NO 2−) denitrification. We isolated novel bacteria that proliferated in the presence of high levels of NO 2− (72 mM). Strain YD50.2, among several isolates, was taxonomically positioned within the α subclass of Proteobacteria and identified as Ochrobactrum anthropi YD50.2. This strain denitrified NO 2−, as well as NO 3−. The gene clusters for denitrification ( nar, nir, nor, and nos) were cloned from O. anthropi YD50.2, in which the nir and nor operons were linked. We confirmed that nirK in the nir- nor operon produced a functional NO 2− reductase containing copper that was involved in bacterial NO 2− reduction. The strain denitrified up to 40 mM NO 2− to dinitrogen under anaerobic conditions in which other denitrifiers or NO 3− reducers such as Pseudomonas aeruginosa and Ralstonia eutropha and nitrate-respiring Escherichia coli neither proliferated nor reduced NO 2−. Under nondenitrifying aerobic conditions, O. anthropi YD50.2 and its type strain ATCC 49188 T proliferated even in the presence of higher levels of NO 2− (100 mM), and both were considerably more resistant to acidic NO 2− than were the other strains noted above. These results indicated that O. anthropi YD50.2 is a novel denitrifier that has evolved reactive nitrogen oxide tolerance mechanisms.Environmental bacteria maintain the global nitrogen cycle by metabolizing organic and inorganic nitrogen compounds. Denitrification is critical for maintenance of the global nitrogen cycle, through which nitrate (NO 3−) or nitrite (NO 2−) is reduced to gaseous nitrogen forms such as N 2 and nitrous oxide (N 2O) ( 19, 47). Decades of investigations into denitrifying bacteria have revealed their ecological impact ( 9), their molecular mechanisms of denitrification ( 13, 25, 47), and the industrial importance of removing nitrogenous contaminants from wastewater ( 31, 36). Bacterial denitrification is considered to comprise four successive reduction steps, each of which is catalyzed by NO 3− reductase (Nar), NO 2− reductase (Nir), nitric oxide (NO) reductase (Nor), and N 2O reductase (Nos). The reaction of each enzyme is linked to the electron transport chain on the cellular membrane and accompanies oxidative phosphorylation, implying that bacterial denitrification is of as much physiological significance as anaerobic respiration ( 25, 47). Most denitrifying bacteria are facultative anaerobes and respire with oxygen under aerobic conditions. Because denitrification is induced in the absence of oxygen, it is considered an alternative mechanism of energy conservation that has evolved as an adaptation to anaerobic circumstances ( 13, 47).Nitrite and NO are hazardous to bacteria, since they generate highly reactive nitrogen species (RNS) under physiological conditions and damage cellular DNA, lipid, and proteins ( 28, 37). Denitrifying bacteria are thought to be threatened by RNS since they reduce NO 3− to generate NO 2− and NO as denitrifying intermediates. Furthermore, denitrifying bacteria often inhabit environments where they are exposed to NO 2− and NO and hence high levels of RNS. Recent reports suggest that pathogenic bacteria invading animal tissues are attacked by NO generated by macrophages ( 12). Such bacteria involve denitrifiers, and some of them, for example, Neisseria meningitidis ( 1) and Pseudomonas aeruginosa, acquire resistance to NO by producing Nor ( 44). The utilization (reduction) of NO by Brucella increases the survival of infected mice ( 2). These examples suggest that production of a denitrifying mechanism affects bacterial survival of threats from both endogenous and extracellular RNS. However, the mechanism of RNS tolerance induced by denitrifying bacteria is not fully understood.Ubiquitous gram-negative Ochrobactrum strains are widely distributed in soils and aqueous environments, where they biodegrade aromatic compounds ( 11), organophosphorus pesticides ( 45), and other hydrocarbons ( 38) and remove heavy metal ions such as chromium and cadmium ( 24). Having been isolated from clinical specimens, Ochrobactrum anthropi is currently recognized as an emerging opportunistic pathogen, although relatively little is known about its pathogenesis and factors contributing to its virulence ( 7, 30). Manipulation systems have been developed to investigate these issues at the molecular genetic level ( 33). Some O. anthropi strains have been identified as denitrifiers ( 21), although the denitrifying properties of these strains have not been investigated in detail. This study was undertaken to examine the denitrifying properties of O. anthropi in more detail. O. anthropi YD50.2 was selected for this study and was isolated herein. The strain denitrified high levels of NO 2− (up to 40 mM) to dinitrogen under anaerobic conditions. The strain was highly resistant to acidified NO 2− under nondenitrifying aerobic conditions. These results indicate that O. anthropi YD50.2 has mechanisms that produce tolerance to RNS. 相似文献
5.
The nr 1 soybean ( Glycine max [L.] Merr.) mutant does not contain the two constitutive nitrate reductases, one of which is responsible for enzymic conversion of nitrite to NO x (NO + NO 2). It was tested for possible nonenzymic NO x formation and evolution because of known chemical reactions between NO 2− and plant metabolites and the instability of nitrous acid. It did not evolve NO x during the in vivo NR assay, but intact leaves did evolve small amounts of NO x under dark, anaerobic conditions. Experiments were conducted to compare NO 3− reduction, NO 2− accumulation, and the NO x evolution processes of the wild type (cv Williams) and the nr 1 mutant. In vivo NR assays showed that wild-type leaves had three times more NO 3− reducing capacity than the nr 1 mutant. NO x evolution from intact, anerobic nr 1 leaves was approximately 10 to 20% that from wild-type leaves. Nitrite content of the nr 1 mutant leaves was usually higher than wild type due to low NO x evolution. Lag times and threshold NO 2− concentrations for NO x evolution were similar for the two genotypes. While only 1 to 2% of NO x from wild type is NO 2, the nr 1 mutant evolved 15 to 30% NO 2. The kinetic patterns of NO x evolution with time weré completely different for the mutant and wild type. Comparisons of light and heat treatments also gave very different results. It is generally accepted that the NO x evolution by wild type is primarily an enzymic conversion of NO 2− to NO. However, this report concludes that NO x evolution by the nr 1 mutant was due to nonenzymic, chemical reactions between plant metabolites and accumulated NO 2− and/or decomposition of nitrous acid. Nonenzymic NO x evolution probably also occurs in wild type to a degree but could be easily masked by high rates of the enzymic process. 相似文献
6.
We examined nitrate-dependent Fe 2+ oxidation mediated by anaerobic ammonium oxidation (anammox) bacteria. Enrichment cultures of “ Candidatus Brocadia sinica” anaerobically oxidized Fe 2+ and reduced NO 3− to nitrogen gas at rates of 3.7 ± 0.2 and 1.3 ± 0.1 (mean ± standard deviation [SD]) nmol mg protein −1 min −1, respectively (37°C and pH 7.3). This nitrate reduction rate is an order of magnitude lower than the anammox activity of “ Ca. Brocadia sinica” (10 to 75 nmol NH 4+ mg protein −1 min −1). A 15N tracer experiment demonstrated that coupling of nitrate-dependent Fe 2+ oxidation and the anammox reaction was responsible for producing nitrogen gas from NO 3− by “ Ca. Brocadia sinica.” The activities of nitrate-dependent Fe 2+ oxidation were dependent on temperature and pH, and the highest activities were seen at temperatures of 30 to 45°C and pHs ranging from 5.9 to 9.8. The mean half-saturation constant for NO 3− ± SD of “ Ca. Brocadia sinica” was determined to be 51 ± 21 μM. Nitrate-dependent Fe 2+ oxidation was further demonstrated by another anammox bacterium, “ Candidatus Scalindua sp.,” whose rates of Fe 2+ oxidation and NO 3− reduction were 4.7 ± 0.59 and 1.45 ± 0.05 nmol mg protein −1 min −1, respectively (20°C and pH 7.3). Co-occurrence of nitrate-dependent Fe 2+ oxidation and the anammox reaction decreased the molar ratios of consumed NO 2− to consumed NH 4+ (ΔNO 2−/ΔNH 4+) and produced NO 3− to consumed NH 4+ (ΔNO 3−/ΔNH 4+). These reactions are preferable to the application of anammox processes for wastewater treatment. 相似文献
7.
Inactivation of TPI1, the Saccharomyces cerevisiae structural gene encoding triose phosphate isomerase, completely eliminates growth on glucose as the sole carbon source. In tpi1-null mutants, intracellular accumulation of dihydroxyacetone phosphate might be prevented if the cytosolic NADH generated in glycolysis by glyceraldehyde-3-phosphate dehydrogenase were quantitatively used to reduce dihydroxyacetone phosphate to glycerol. We hypothesize that the growth defect of tpi1-null mutants is caused by mitochondrial reoxidation of cytosolic NADH, thus rendering it unavailable for dihydroxyacetone-phosphate reduction. To test this hypothesis, a tpi1Δ nde1Δ nde2Δ gut2Δ quadruple mutant was constructed. NDE1 and NDE2 encode isoenzymes of mitochondrial external NADH dehydrogenase; GUT2 encodes a key enzyme of the glycerol-3-phosphate shuttle. It has recently been demonstrated that these two systems are primarily responsible for mitochondrial oxidation of cytosolic NADH in S. cerevisiae. Consistent with the hypothesis, the quadruple mutant grew on glucose as the sole carbon source. The growth on glucose, which was accompanied by glycerol production, was inhibited at high-glucose concentrations. This inhibition was attributed to glucose repression of respiratory enzymes as, in the quadruple mutant, respiratory pyruvate dissimilation is essential for ATP synthesis and growth. Serial transfer of the quadruple mutant on high-glucose media yielded a spontaneous mutant with much higher specific growth rates in high-glucose media (up to 0.10 h −1 at 100 g of glucose·liter −1). In aerated batch cultures grown on 400 g of glucose·liter −1, this engineered S. cerevisiae strain produced over 200 g of glycerol·liter −1, corresponding to a molar yield of glycerol on glucose close to unity. 相似文献
8.
Mg:ATP-dependent H + pumping has been studied in microsomal vesicles from 24-hour-old radish ( Raphanus sativus L.) seedlings by monitoring both intravesicular acidification and the building up of an inside positive membrane potential difference (Δ ψ). ΔpH was measured as the decrease of absorbance of Acridine orange and Δ ψ as the shift of absorbance of bis(3-propyl-5-oxoisoxazol-4-yl)pentamethine oxonol. Both Mg:ATP-dependent Δ pH and Δ ψ generation are completely inhibited by vanadate and insensitive to oligomycin; moreover, Δ pH generation is not inhibited by NO 3−. These findings indicate that this membrane preparation is virtually devoid of mitochondrial and tonoplast H +-ATPases. Both intravesicular acidification and Δ ψ generation are influenced by anions: Δ pH increases and Δ ψ decreases following the sequence SO 42−, Cl −, Br −, NO 3−. ATP-dependent H + pumping strictly requires Mg 2+. It is very specific for ATP (apparent Km 0.76 millimolar) compared to GTP, UTP, CTP, ITP. Δ pH generation is inhibited by CuSO 4 and diethylstilbestrol as well as vanadate. Δ pH generation is specificially stimulated by K + (+ 80%) and to a lesser extent by Na + and choline (+28% and +14%, respectively). The characteristics of H + pumping in these microsomal vesicles closely resemble those described for the plasma membrane ATPase partially purified from several plant materials. 相似文献
9.
We examined nitrate assimilation and root gas fluxes in a wild-type barley ( Hordeum vulgare L. cv Steptoe), a mutant ( nar1a) deficient in NADH nitrate reductase, and a mutant ( nar1a; nar7w) deficient in both NADH and NAD(P)H nitrate reductases. Estimates of in vivo nitrate assimilation from excised roots and whole plants indicated that the nar1a mutation influences assimilation only in the shoot and that exposure to NO 3− induced shoot nitrate reduction more slowly than root nitrate reduction in all three genotypes. When plants that had been deprived of nitrogen for several days were exposed to ammonium, root carbon dioxide evolution and oxygen consumption increased markedly, but respiratory quotient—the ratio of carbon dioxide evolved to oxygen consumed—did not change. A shift from ammonium to nitrate nutrition stimulated root carbon dioxide evolution slightly and inhibited oxygen consumption in the wild type and nar1a mutant, but had negligible effects on root gas fluxes in the nar1a; nar7w mutant. These results indicate that, under NH 4+ nutrition, 14% of root carbon catabolism is coupled to NH 4+ absorption and assimilation and that, under NO 3− nutrition, 5% of root carbon catabolism is coupled to NO 3− absorption, 15% to NO 3− assimilation, and 3% to NH 4+ assimilation. The additional energy requirements of NO 3− assimilation appear to diminish root mitochondrial electron transport. Thus, the energy requirements of NH 4+ and NO 3− absorption and assimilation constitute a significant portion of root respiration. 相似文献
10.
The aim of this work was to determine which of the two reactions ( i.e. phosphorylation or dephosphorylation) involved in the establishment of the phosphorylated status of the wheat leaf phospho enolpyruvate carboxylase and sucrose phosphate synthase protein responds in vivo to NO 3− uptake and assimilation. Detached mature leaves of wheat ( Triticum aestivum L. cv Fidel) were fed with N-free (low-NO 3− leaves) or 40 m m NO 3− solution (high-NO 3− leaves). The specific inhibition of the enzyme-protein kinase or phosphatase activities was obtained in vivo by addition of mannose or okadaic acid, respectively, in the uptake solution. Mannose at 50 m m, by blocking the kinase reaction, inhibited the processes of NO 3−-dependent phospho enolpyruvate carboxylase activation and sucrose phosphate synthase deactivation. Following the addition of mannose, the deactivation of phospho enolpyruvate carboxylase and the activation of sucrose phosphate synthase, both due to the enzyme-protein dephosphorylation, were at the same rate in low-NO 3− and high-NO 3− leaves, indicating that NO 3− had no effect per se on the enzyme-protein phosphatase activity. Upon treatment with okadaic acid, the higher increase of phospho enolpyruvate carboxylase and decrease of sucrose phosphate synthase activities observed in high NO 3− compared with low NO 3− leaves showed evidence that NO 3− enhanced the protein kinase activity. These results support the concept that NO 3−, or a product of its metabolism, favors the activation of phospho enolpyruvate carboxylase and deactivation of sucrose phosphate synthase in wheat leaves by promoting the light activation of the enzyme-protein kinase(s) without affecting the phosphatase(s). 相似文献
11.
Nitrite oxidation is the second step of nitrification. It is the primary source of oceanic nitrate, the predominant form of bioavailable nitrogen in the ocean. Despite its obvious importance, nitrite oxidation has rarely been investigated in marine settings. We determined nitrite oxidation rates directly in 15N-incubation experiments and compared the rates with those of nitrate reduction to nitrite, ammonia oxidation, anammox, denitrification, as well as dissimilatory nitrate/nitrite reduction to ammonium in the Namibian oxygen minimum zone (OMZ). Nitrite oxidation (⩽372 n M NO 2− d −1) was detected throughout the OMZ even when in situ oxygen concentrations were low to non-detectable. Nitrite oxidation rates often exceeded ammonia oxidation rates, whereas nitrate reduction served as an alternative and significant source of nitrite. Nitrite oxidation and anammox co-occurred in these oxygen-deficient waters, suggesting that nitrite-oxidizing bacteria (NOB) likely compete with anammox bacteria for nitrite when substrate availability became low. Among all of the known NOB genera targeted via catalyzed reporter deposition fluorescence in situ hybridization, only Nitrospina and Nitrococcus were detectable in the Namibian OMZ samples investigated. These NOB were abundant throughout the OMZ and contributed up to ∼9% of total microbial community. Our combined results reveal that a considerable fraction of the recently recycled nitrogen or reduced NO 3− was re-oxidized back to NO 3− via nitrite oxidation, instead of being lost from the system through the anammox or denitrification pathways. 相似文献
12.
Phospo enolpyruvate carboxylase (PEPC) is absent from humans but encoded in the Plasmodium falciparum genome, suggesting that PEPC has a parasite-specific function. To investigate its importance in P. falciparum, we generated a pepc null mutant (D10 Δpepc), which was only achievable when malate, a reduction product of oxaloacetate, was added to the growth medium. D10 Δpepc had a severe growth defect in vitro, which was partially reversed by addition of malate or fumarate, suggesting that pepc may be essential in vivo. Targeted metabolomics using 13C-U-D-glucose and 13C-bicarbonate showed that the conversion of glycolytically-derived PEP into malate, fumarate, aspartate and citrate was abolished in D10 Δpepc and that pentose phosphate pathway metabolites and glycerol 3-phosphate were present at increased levels. In contrast, metabolism of the carbon skeleton of 13C, 15N-U-glutamine was similar in both parasite lines, although the flux was lower in D10 Δpepc; it also confirmed the operation of a complete forward TCA cycle in the wild type parasite. Overall, these data confirm the CO 2 fixing activity of PEPC and suggest that it provides metabolites essential for TCA cycle anaplerosis and the maintenance of cytosolic and mitochondrial redox balance. Moreover, these findings imply that PEPC may be an exploitable target for future drug discovery. 相似文献
13.
The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO 3− and NO 2− assimilation were studied using detached barley ( Hordeum vulgare L. cv Numar) leaves in which only endogenous NO 3− or NO 2− were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO 3− reduction in both light and darkness, while photosynthetic inhibitors did not. The NO2− concentration in the control leaves was very low in both light and darkness; 98% or more of the NO2− formed from NO3− was further assimilated in control leaves. More NO2− accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO2−, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO2− reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors. Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO2− formed from NO3− was further assimilated, whereas in light, 95% or more of the NO2− was further assimilated. 相似文献
14.
Resistance to thyroid hormone (RTH), a human syndrome, is characterized by high thyroid hormone (TH) and thyroid-stimulating hormone (TSH) levels. Mice with mutations in the thyroid hormone receptor beta (TRβ) gene that cannot bind steroid receptor coactivator 1 (SRC-1) and Src-1−/− mice both have phenotypes similar to that of RTH. Conversely, mice expressing a mutant nuclear corepressor 1 ( Ncor1) allele that cannot interact with TRβ, termed NCoRΔID, have low TH levels and normal TSH. We hypothesized that Src-1−/− mice have RTH due to unopposed corepressor action. To test this, we crossed NCoRΔID and Src-1−/− mice to create mice deficient for coregulator action in all cell types. Remarkably, NCoR ΔID/ΔID
Src-1−/− mice have normal TH and TSH levels and are triiodothryonine (T 3) sensitive at the level of the pituitary. Although absence of SRC-1 prevented T 3 activation of key hepatic gene targets, NCoR ΔID/ΔID
Src-1−/− mice reacquired hepatic T 3 sensitivity. Using in vivo chromatin immunoprecipitation assays (ChIP) for the related coactivator SRC-2, we found enhanced SRC-2 recruitment to TR-binding regions of genes in NCoR ΔID/ΔID
Src-1−/− mice, suggesting that SRC-2 is responsible for T 3 sensitivity in the absence of NCoR1 and SRC-1. Thus, T 3 targets require a critical balance between NCoR1 and SRC-1. Furthermore, replacement of NCoR1 with NCoRΔID corrects RTH in Src-1−/− mice through increased SRC-2 recruitment to T 3 target genes. 相似文献
15.
Potential-dependent anion movement into tonoplast vesicles from oat roots ( Avena sativa L. var Lang) was monitored as dissipation of membrane potentials (Δψ) using the fluorescence probe Oxonol V. The potentials (positive inside) were generated with the H +-pumping pyrophosphatase, which is K + stimulated and anion insensitive. The relative rate of ΔΨ dissipation by anions was used to estimate the relative permeabilities of the anions. In decreasing order they were: SCN − (100) > NO 3− (72) = Cl − (70) > Br − (62) > SO 42− (5) = H 2PO 4− (5) > malate (3) = acetate (3) > iminodiacetate (2). Kinetic studies showed that the rate of Δψ dissipation by Cl − and NO 3−, but not by SCN −, was saturable. The Km values for Cl − and NO 3− uptake were about 2.3 and 5 millimolar, respectively, suggesting these anions move into the vacuole through proteinaceous porters. In contrast to a H +-coupled Cl − transporter on the same vesicles, the potential-dependent Cl − transport was insensitive to 4,4′-diisothiocyano-2,2′-stilbene disulfonate. These results suggest the existence of at least two different mechanisms for Cl − transport in these vesicles. The potentials generated by the H +-translocating ATPase and H +-pyrophosphatase were nonadditive, giving support to the model that both pumps are on tonoplast vesicles. No evidence for a putative Cl − conductance on the anion-sensitive H +-ATPase was found. 相似文献
16.
Growth chamber studies with soybeans ( Glycine max [L.] Merr.) were designed to determine the relative limitations of NO 3−, NADH, and nitrate reductase (NR) per se on nitrate metabolism as affected by light and temperature. Three NR enzyme assays (+NO 3−in vivo, −NO 3−in vivo, and in vitro) were compared. NR activity decreased with all assays when plants were exposed to dark. Addition of NO 3− to the in vivo NR assay medium increased activity (over that of the −NO 3−in vivo assay) at all sampling periods of a normal day-night sequence (14 hr-30 C day; 10 hr-20 C night), indicating that NO 3− was rate-limiting. The stimulation of in vivo NR activity by NO 3− was not seen in plants exposed to extended dark periods at elevated temperatures (16 hr-30 C), indicating that under those conditions, NO 3− was not the limiting factor. Under the latter condition, in vitro NR activity was appreciable (19 μmol NO 2− [g fresh weight, hr] −1) suggesting that enzyme level per se was not the limiting factor and that reductant energy might be limiting. 相似文献
17.
Electrogenic ATPase activity on the peribacteroid membrane from soybean ( Glycine max L. cv Bragg) root nodules is demonstrated. Membrane energization was monitored using suspensions of intact peribacteroid membrane-enclosed bacteroids (peribacteroid units; PBUs) and the fluorescent probe for membrane potential (ΔΨ), bis-(3-phenyl-5-oxoisoxazol-4yl) pentamethine oxonol. Generation of a positive ΔΨ across the peribacteroid membrane was dependent upon ATP, inhibited by N,N′-dicyclohexyl-carbodiimide and vanadate, but insensitive to N-ethylmaleimide, azide, cyanide, oligomycin, and ouabain. The results suggest the presence of a single, plasma membrane-like, electrogenic ATPase on the peribacteroid membrane. The protonophore, carbonyl-cyanide m-chlorophenyl hydrazone, completely dissipated the established membrane potential. The extent of reduction in the steady state membrane potential upon addition of ions was used to estimate the relative permeability of the peribacteroid membrane to anions. By this criterion, the relative rates of anion transport across the peribacteroid membrane were: NO 3− > NO 2− > Cl − > acetate − > malate −. The observation that 10 millimolar NO 3− completely dissipated the membrane potential was of particular interest in view of the fact that NO 3− inhibits symbiotic nitrogen fixation. The possible function of the ATPase in symbiotic nitrogen fixation is discussed. 相似文献
18.
Substantial rates of nitrate reduction could be achieved with a reconstituted system from spinach leaves containing supernatant, mitochondria, NAD +, oxaloacetate (OAA), and an oxidizable substrate. Appropriate substrates were glycine, pyruvate, citrate, isocitrate, fumarate, or glutamate. The reduction of NO 3− with any of the substrates could be inhibited by n-butyl malonate, showing that the transfer of reducing power from the mitochondria to the supernatant involved the malate exchange carrier. The addition of ADP to the reconstituted system decreased NO 3− reduction and this decrease could be reversed by the addition of rotenone or antimycin A. The operation of the OAA/malate shuttle was achieved most quickly in the system when low concentrations (≤0.1 millimolar) of OAA were added. A corresponding increase in the lag time for the operation of the OAA/malate shuttle was observed when the OAA concentration was increased. Concentrations for half-maximal activity of OAA, glycine, NAD +, and NO 3− in the reconstituted system were 42 micromolar, 0.5 millimolar, 0.25 millimolar, and 26 micromolar, respectively. The transfer of reducing power from the mitochondria to the soluble phase via the OAA/malate shuttle can not only provide NADH for cytoplasmic reduction but can also sustain oxidation of tricarboxylic cycle acids and the generation of α-ketoglutarate independently of the respiratory electron transport chain. 相似文献
19.
Oscillatory behavior of mitochondrial inner membrane potential (ΔΨ m) is commonly observed in cells subjected to oxidative or metabolic stress. In cardiac myocytes, the activation of inner membrane pores by reactive oxygen species (ROS) is a major factor mediating intermitochondrial coupling, and ROS-induced ROS release has been shown to underlie propagated waves of ΔΨ m depolarization as well as synchronized limit cycle oscillations of ΔΨ m in the network. The functional impact of ΔΨ m instability on cardiac electrophysiology, Ca 2+ handling, and even cell survival, is strongly affected by the extent of such intermitochondrial coupling. Here, we employ a recently developed wavelet-based analytical approach to examine how different substrates affect mitochondrial coupling in cardiac cells, and we also determine the oscillatory coupling properties of mitochondria in ventricular cells in intact perfused hearts. The results show that the frequency of ΔΨ m oscillations varies inversely with the size of the oscillating mitochondrial cluster, and depends on the strength of local intermitochondrial coupling. Time-varying coupling constants could be quantitatively determined by applying a stochastic phase model based on extension of the well-known Kuramoto model for networks of coupled oscillators. Cluster size-frequency relationships varied with different substrates, as did mitochondrial coupling constants, which were significantly larger for glucose (7.78 × 10 −2 ± 0.98 × 10 −2 s −1) and pyruvate (7.49 × 10 −2 ± 1.65 × 10 −2 s −1) than lactate (4.83 × 10 −2 ± 1.25 × 10 −2 s −1) or β-hydroxybutyrate (4.11 × 10 −2 ± 0.62 × 10 −2 s −1). The findings indicate that mitochondrial spatiotemporal coupling and oscillatory behavior is influenced by substrate selection, perhaps through differing effects on ROS/redox balance. In particular, glucose-perfusion generates strong intermitochondrial coupling and temporal oscillatory stability. Pathological changes in specific catabolic pathways, which are known to occur during the progression of cardiovascular disease, could therefore contribute to altered sensitivity of the mitochondrial network to oxidative stress and emergent ΔΨ m instability, ultimately scaling to produce organ level dysfunction. 相似文献
20.
Nitrate (NO 3−) and nitrite (NO 2−) are the physiological sources of nitric oxide (NO), a key biological messenger molecule. NO 3−/NO 2− exerts a beneficial impact on NO homeostasis and its related cardiovascular functions. To visualize the physiological dynamics of NO 3−/NO 2− for assessing the precise roles of these anions, we developed a genetically encoded intermolecular fluorescence resonance energy transfer (FRET)-based indicator, named sNOOOpy (sensor for NO 3−/NO 2− in physiology), by employing NO 3−/NO 2−-induced dissociation of NasST involved in the denitrification system of rhizobia. The in vitro use of sNOOOpy shows high specificity for NO 3− and NO 2−, and its FRET signal is changed in response to NO 3−/NO 2− in the micromolar range. Furthermore, both an increase and decrease in cellular NO 3− concentration can be detected. sNOOOpy is very simple and potentially applicable to a wide variety of living cells and is expected to provide insights into NO 3−/NO 2− dynamics in various organisms, including plants and animals. 相似文献
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