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1.
Protoplasts isolated from the apical segments of Cuscuta reflexa exhibited blue light-sensitive PM-linked NADH oxidase activity and increased rate of Ca2+-uptake in presence of NADH in dark, which was also stimulated by blue light. Contrary to marginal inhibition by Con A treatment, the ATPase inhibitors significantly inhibited the Ca2+ uptake by the protoplasts both in dark and under blue light. The Ca2+-calmodulin antagonists, W-7 and calmidazolium, also inhibited Ca2+-uptake by protoplasts under similar conditions. The state of PM polarization was monitored by the fluorescent dye 9-amino acridine. It was observed that PM-linked NADH oxidation caused hyperpolarization of the membrane, the exposure of which to blue light resulted in membrane depolarization. The presence of Ca2+-calmodulin antagonists or Con A treatment completely abolished the blue light-induced membrane depolarization. It is argued that these actities at the PM, having some glycoproteic components, are functionally closely involved in blue light-induced signal transduction in Cuscuta  相似文献   

2.
Four-day-old corn (Zea mays L. ) seedlings, which had grown in aerated 0.5 x Hoagland solution (pH 5.7) in dark were stressed by 50, 100 and 150 mmol/L NaCI solution for 6, 12 and 24 h respectively. The root segments (0 to 20 mm from the tip) were sampled for study. The results showed that the relative elongation rate (RER), H+-extrusion rate and Fe(CN)3-6 reduction rate of the roots declined with the increase of NaC1 concentration, but H+-extmsion rate restored somewhat with the time course. NADH oxidation rate increased up to 12 h with the increase of NaC1 concentration and then decreased significantly, while under the same concentration of NaC1 NADH oxidation rate decreased with the time course. The relative coeffecient (r) of RER and H+-extrusion rate under NaCI stress was 0.999 8. Therefore, it is suggested that the inhibition of the elongation growth by NaC1 stress is closely related to the inhibition of redox system and H + -ATPase activity of plasma membrane.  相似文献   

3.
L Kato  M Ishaque 《Cytobios》1975,12(45):31-43
Particles from Mycobacterium lepraemurium catalysed the oxidation of NADH with oxygen as the terminal electron acceptor. The preparations contained cytochromes of the a + a3'b and c types, as well as CO-binding pigments. The NADH oxidase activity was sensitive to inhibitors of the flavoprotein system as well as to HQNO and antimycin A. In addition, a cytochrome oxidase sensitive to cyanide was also present. The system was inhibited by the thiol-binding agent, PCMB, and thus indicated the involvement of sulphydryl group in the enzymatic oxidation of NADH. The sensitivity of the NADH oxidase system to all the inhibitors of the respiratory chain and the effect of these inhibitors on the absorption spectra suggested that cytochromes of the b, c, a + a3 types are involved in the transfer of electrons in NADH oxidation.  相似文献   

4.
过量表达NADH氧化酶加速光滑球拟酵母合成丙酮酸   总被引:1,自引:0,他引:1  
[目的]进一步提高光滑球拟酵母(Torulopsis glabrata)发酵生产丙酮酸的生产强度.[方法]将来源于乳酸乳球菌(Lactococcus lactis)中编码形成水的NADH氧化酶noxE基因过量表达于丙酮酸工业生产菌株T. glabrata CCTCC M202019中,获得了一株NADH氧化酶活性为34.8 U/mg蛋白的重组菌T. glabrata-PDnoxE.[结果]与出发菌株T. glabrata CCTCC M202019相比,细胞浓度、葡萄糖消耗速率和丙酮酸生产强度分别提高了168%、44.9%和12%,发酵进行到36 h葡萄糖消耗完毕.补加50 g/L葡萄糖继续发酵20 h,则使丙酮酸浓度提高到67.2 g/L.葡萄糖消耗速度和丙酮酸生产强度增加的原因在于形成水的NADH氧化酶过量表达,导致NADH和ATP含量分别降低了18.1%和15.8%.而NAD<' 增加了11.1%.[结论]增加细胞内NAD<' 含量能有效地提高酵母细胞葡萄糖的代谢速度及目标代谢产物的生产强度.  相似文献   

5.
Human red cells (RBC) respond to moderate Ca2+-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca2+-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase (GAPDH/PGK)-steps. 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca2+-extrusion rate and not to the loaded Ca2+. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca2+-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the GAPDH/PGK equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. NAD/NADH ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.  相似文献   

6.
The particulate fraction of Rhodopseudomonas viridis when supplied with succinate catalyses the reduction of NAD+ by light; this reaction is inhibited by uncouplers of oxidative phosphorylation but not by oligomycin. Formation of NADH takes place in the dark when ATP or PPi is supplied. Both light and dark reactions are inhibited by valinomycin and nigericin, when added together, but not by either separately. NADH formation in R. viridis appears to take place by an energy-dependent reversal of electron flow and energy may be conserved in the form of a membrane potential. The addition of ATP caused the oxidation of both C553 and C558 in chromatophores; carbonylcyanide p-trifluoromethoxyphenylhydrazone and oligomycin abolished this oxidation.

The NAD+ and NADH concentrations at equilibrium in the light-dependent reaction were determined and the oxidation-reduction potential of this couple calculated. From this value it was calculated that under these experimental conditions the energy requirement to form NADH from the succinate/fumarate couple at Eh = o V was 9.4 kcal.

Particles of R. viridis contained an active transhydrogenase, driven by either light or ATP, that was sensitive to uncouplers of oxidative phosphorylation; the light-driven reaction was insensitive to oligomycin and was inhibited by antimycin A and 2-heptyl-4-hydroxyquinone-N-oxide.

R. viridis did not grow aerobically but particles contained NADH oxidase activity that was cyanide sensitive. There was no spectroscopic evidence for cytochromes of the b-type in reduced-minus-oxidised spectra of particles or in pyridine haemochrome spectra of whole cells.  相似文献   


7.
摘要:【目的】为进一步提高光滑球拟酵母(Torulopsis glabrata)葡萄糖代谢速率及丙酮酸生产强度。【方法】将源于荚膜胞浆菌(Histoplasma capsulatum)的编码选择性氧化酶的AOX1基因过量表达于T. glabrata中,获得了一株线粒体内NADH氧化途径发生改变且胞内总NADH 氧化酶活性提高1.8倍的重组菌株AOX。【结果】与出发菌株CON比较,细胞浓度以及发酵周期降低了20.3%和10.7%,而平均比葡萄糖消耗速率和丙酮酸合成速率分别提高了34.7%和54.1%。其原因  相似文献   

8.
It has been reported that nonmitochondrial NAD(P)H oxidases make an important contribution to intracellular O2-* in vascular tissues and, thereby, the regulation of vascular function. Topological analyses have suggested that a well-known membrane-associated NAD(P)H oxidase may not release O2-* into the cytosol. It is imperative to clarify the source of intracellular O2-* associated with this enzyme and its physiological significance in vascular cells. The present study hypothesized that an NAD(P)H oxidase on the sarcoplasmic reticulum (SR) in coronary artery smooth muscle (CASM) regulates SR ryanodine receptor (RyR) activity by producing O2-* locally. Western blot analysis was used to detect NAD(P)H oxidase subunits in purified SR from CASM. Fluorescent spectrometric analysis demonstrated that incubation of SR with NADH time dependently produced O2-*, which could be substantially blocked by the specific NAD(P)H oxidase inhibitors diphenylene iodonium and apocynin and by SOD or its mimetic tiron. This SR NAD(P)H oxidase activity was also confirmed by HPLC analysis of conversion of NADH to NAD+. In experiments of lipid bilayer channel reconstitution, addition of NADH to the cis solution significantly increased the activity of RyR/Ca2+ release channels from these SR preparations from CASM, with a maximal increase in channel open probability from 0.0044 +/- 0.0005 to 0.0213 +/- 0.0018; this effect of NADH was markedly blocked in the presence of SOD or tiron or the NAD(P)H oxidase inhibitors diphenylene iodonium, N-vanillylnonanamide, and apocynin. These results suggest that a local NAD(P)H oxidase system on SR from CASM regulates RyR/Ca2+ channel activity and Ca2+ release from SR by producing O2-*.  相似文献   

9.
The plasma membrane NADH oxidase activity partially purified from the surface of HeLa cells exhibited hydroquinone oxidase activity. The preparations completely lacked NADH:ubiquinone reductase activity. However, in the absence of NADH, reduced coenzyme Q10 (Q10H2=ubiquinol) was oxidized at a rate of 15+/-6 nmol min-1 mg protein-1 depending on degree of purification. The apparent Km for Q10H2 oxidation was 33 microM. Activities were inhibited competitively by the cancer cell-specific NADH oxidase inhibitors, capsaicin and the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). With coenzyme Q0, where the preparations were unable to carry out either NADH:quinone reduction or reduced quinone oxidation, quinol oxidation was observed with an equal mixture of the Q0 and Q0H2 forms. With the mixture, a rate of Q0H2 oxidation of 8-17 nmol min-1 mg protein-1 was observed with an apparent Km of 0.22 mM. The rate of Q10H2 oxidation was not stimulated by addition of equal amounts of Q10 and Q10H2. However, addition of Q0 to the Q10H2 did stimulate. The oxidation of Q10H2 proceeded with what appeared to be a two-electron transfer. The oxidation of Q0H2 may involve Q0, but the mechanism was not clear. The findings suggest the potential participation of the plasma membrane NADH oxidase as a terminal oxidase of plasma membrane electron transport from cytosolic NAD(P)H via naturally occurring hydroquinones to acceptors at the cell surface.  相似文献   

10.
Human red cells (RBC) respond to moderate Ca2+-loading with increased ATP consumption and stimulation of glycolytic flux. 1. Ca2+-induced metabolite transitions at different pH-values showed a clearcut crossover at the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase (GAPDHPGK)-steps. 2. The behavior of glycolytic metabolites in iodoacetate-treated, GAPDH-inhibited, and in phosphoenolpyruvate-loaded RBC ruled out activation of hexokinase, phosphofructokinase and pyruvate kinase. 3. Glycolytic stimulation is linked to Ca2+-extrusion rate and not to the loaded Ca2+. 4. Adenine nucleotides and inorganic phosphate could be ruled out as the connecting link between glycolytic activation and Ca2+-extrusion. 5. NADH oxidation was observed at all pH-values studied when the RBC were incubated either at low or high extracellular potassium. NADH is product-inhibitor of GAPDH. The concentration (34 μM) of thermodynamically free NADH calculated from the GAPDHPGK equilibrium reactants was in the inhibitory range: any decrease in NADH is therefore followed by activation of GAPDH. NADNADH ratio seems to be the connecting link between ATP consuming ion transport and ATP generation by glycolysis.  相似文献   

11.
A mathematical model of the glycolytic system with the cytoplasmic coenzymes NAD+ and NADH as essential variables is proposed. It has been shown that any increase in the steady-state concentration of NADH will reduce the range of activity of the "generalized" ATPase, wherein the level of ATP is stabilized. Such a reduction in the range of ATP stabilization may be caused by an increasing rate of the pyruvate loss into non-glycolytic pathways, in particular, into mitochondria. This effect may be compensated by increasing oxidation of NADH by the dehydrogenases of H+-transferring cytosol-mitochondrial shuttles (malate-aspartate or alpha-glycerophosphate). The properties of the complete model were compared with those of its simplified version, which takes account only of the phosphotransferase reactions of glycolysis. The effects of various factors, which do not alter the level of NADH in the system, may be studied within the scope of the simplified model.  相似文献   

12.
Cell-free extracts from aerobically grown Streptococcus agalactiae cells possess a reduced nicotinamide adenine dinucleotide (NADH) oxidase which is linked to oxygen. It is inhibited by cyanide, although cytochromes evidently are not involved. Adenosine triphosphate (ATP) formation occurs during the reaction, but 66 to 75% of the total ATP is formed nonoxidatively. The remaining 25 to 35% of the ATP formation is related to the oxidation of NADH. The formation of ATP in the oxidative reaction can be prevented by excluding oxygen or adding cyanide to prevent NADH oxidation. It can also be prevented by adding methylene blue or pyruvate, which bypasses electron transport to oxygen, but does not interfere with NADH oxidation. Potential sources of ATP, such as glycolysis, the pyruvate oxidase reaction, or the oxidative pentose cycle, are not present, and the high nonoxidative ATP formation is ascribed to the adenylate kinase reaction. The reaction requires adenosine diphosphate (ADP) as a phosphate acceptor. NADH oxidation is independent of ADP. Antimycin A, amytal, and 2,4-dinitrophenol decreased, but did not prevent, oxidative formation of ATP. P:O ratios ranged from 0.15 to 0.25. All of the oxidative activity was in the soluble portion of the cell-free extracts.  相似文献   

13.
《BBA》1987,893(3):386-397
Three NAD(P)H dehydrogenases were found and purified from a soluble fraction of cells of the purple non-sulfur bacterium Rhodobacter capsulatus, strain B10. Molecular mass of NAD(P)H, NADPH and NADH dehydrogenases are 67 000 (4 · 18 000), 35 000 and 39 000, and the isoelectric points are 4.6, 4.3 and 4.5, respectively. NAD(P)H dehydrogenase is characterized by a higher sensitivity to quinacrine, NADPH dehydrogenase by its sensitivity to p-chloromercuribenzoate and NADH dehydrogenase by its sensitivity to sodium arsenite. In contrast to the other two enzymes, NAD(P)H dehydrogenase is capable of oxidizing NADPH as well as NADH, but the ratio of their oxidation rates depends on the pH. All NAD(P)H dehydrogenases reacted with ferricyanide, 2,6-dichlorophenolindophenol, benzoquinone and naphthoquinone, but did not exhibit transhydrogenase, reductase or oxidase activity. Moreover, NADH dehydrogenase was also capable of reducing FAD and FMN. NAD(P)H and NADH dehydrogenases possessed cytochrome-c reductase activity, which was stimulated by menadione and ubiquinone Q1. The activity of NAD(P)H and NADH dehydrogenases depended on culture-growth conditions. The activity of NAD(P)H dehydrogenase from cells grown under chemoheterotrophic aerobic conditions was the lowest and it increased notably under photoheterotrophic anaerobic conditions upon lactate or malate growth limitation. The activity of NADH dehydrogenase was higher from the cells grown under photoheterotrophic anaerobic conditions upon nitrate growth limitation and under chemoheterotrophic aerobic conditions. NADPH dehydrogenase synthesis dependence on R. capsulatus growth conditions was insignificant.  相似文献   

14.
SYNOPSIS. Diphenylamine was shown to be a potent inhibitor of cyanide insensitive respiration in both bloodstream and newly established culture forms of the same isolate of Trypanosoma brucei, with the L-α-glycerophosphate oxidase system having the greatest sensitivity to the inhibitor. The NADH oxidase activity of bloodstream forms was at least twice as sensitive to diphenylamine as the corresponding activity in culture forms, suggesting different routes of NADH oxidation in the 2 forms. The oxidation of L-α-glycerophosphate was inhibited to a similar degree in both culture and bloodstream forms. L-α-glycerophosphate oxidation in bloodstream forms differed from that found in culture forms in that the bloodstream system, unlike that in the culture form, was unable to donate electrons to cytochrome c. In culture form trypanosomes there was a distinct difference in the degree of diphenylamine inhibition on the oxidation of L-α-glycerophosphate, NADH, and succinate, suggesting the participation of separate flavoproteins in the oxidation of these substrates.  相似文献   

15.
Previously, we showed the presence in radish (Raphanus sativus L.) plasmalemma vesicles of an NAD(P)H oxidase, active at pH 4.5-5.0, which elicits the formation of anion superoxide (Vianello and Macrí (1989) Biochim. Biophys. Acta 980, 202-208). In this work, we studied the role of hydrogen peroxide and iron ions upon this oxidase activity. NADH oxidation was stimulated by ferrous ions and, to a lesser extent, by ferric ions. Salicylate and benzoate, two known hydroxyl radical scavengers, inhibited both basal and iron-stimulated NADH oxidase activity. The iron chelators EDTA (ethylenediaminetetraacetic acid) and DFA (deferoxamine melysate) at high concentrations (2 mM) inhibited the NADH oxidation, whereas they were ineffective at lower concentrations (80 microM); the subsequent addition of ferrous ions caused a rapid and limited increase of oxygen consumption which later ceased. Hydrogen peroxide was not detected during NADH oxidation but, in the presence of salicylate, its formation was found in significant amounts. NADH oxidase activity was also associated to a Fe2+ oxidation which was only partially inhibited by salicylate. Ferrous ion oxidation was partially inhibited by catalase and prevented by superoxide dismutase, while ferric ion reduction was abolished by catalase and unaffected by superoxide dismutase. These results show that during NADH oxidation iron ions undergo oxidoreduction and that hydrogen peroxide is produced and rapidly consumed. As previously suggested, this oxidation appears linked to the univalent oxidoreduction of iron ions by a reduced flavoprotein of radish plasmalemma which is then converted to a radical form. The latter, reacting with oxygen generates the superoxide anion which dismutases to H2O2. Hydrogen peroxide, through a Fenton's reaction, may react with Fe2+ to produce hydroxyl radicals, or with Fe3+ to generate the superoxide anion.  相似文献   

16.
Inhibition of NADH oxidation by pyridine derivatives   总被引:1,自引:0,他引:1  
The neurotoxicity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, an impurity in an illicit drug, is expressed after its oxidation to 1-methyl-4-phenylpyridinium by monoamine oxidase. The pyridinium is concentrated by carrier-mediated transport into the mitochondria where it inhibits NADH dehydrogenase and, hence, ATP synthesis. Some structurally related compounds have been tested for their effect on the oxidation of NAD+-linked substrates in intact mitochondria, and for the inhibition of the accumulation of the pyridinium into mitochondria and of NADH dehydrogenase activity in a membrane preparation. Some pyridine derivatives are more inhibitory to NADH dehydrogenase than is 1-methyl-4-phenylpyridinium but these are not concentrated into mitochondria by the uptake system. 4-Phenylpyridine, one of the most effective inhibitors, both occurs naturally and is an environmental pollutant.  相似文献   

17.
The membrane fraction of Bacterionema matruchotii contains an electron transport chain with oxidizing activity for NADH and succinate. Respiration was inhibited by KCN, 2-heptyl-4-hydroxyquinoline-N-oxide, UV light irradiation and CO. UV light irradiation, analysis of membrane extracts, and reconstitution of respiration in UV light treated membranes suggested that respiration is mediated by a menaquinone derivative. The membranes contained cytochromes a, b, and c. Inhibition studies and the effect of KCN and CO on the cytochrome spectrum indicated the presence of an a+a3 cytochrome oxidase and cytochrome o. The membrane fraction from cells grown under O2-limiting conditions contained nitrate reductase activity. In B. matruchotii, electron transport is coupled to oxidative phosphorylation as judged by the effects of substrates and inhibitors on the intracellular ATP concentration.  相似文献   

18.
Cells of Vibrio costicola at pH 8.5 generate both membrane potential (inside negative) and delta pH (inside acidic) in the presence of a proton conductor, carbonyl cyanide m-chlorophenylhydrazone (CCCP). The generation of CCCP-resistant membrane potential was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide that is known to inhibit the Na+-motive NADH oxidase of Vibrio alginolyticus. NADH oxidase, but not lactate oxidase, of inverted membrane vesicles prepared from V. costicola required Na+ for a maximum activity and was inhibited by 2-heptyl-4-hydroxyquinoline-N-oxide. By the oxidation of NADH, inverted membrane vesicles generated concentration gradients of Na+ across the membrane, whose magnitude was always larger than that of delta pH by about 50 mV. In contrast, magnitudes of delta pH and Na+ concentration gradients generated by the oxidation of lactate were similar. Na+ translocation in the presence of lactate was inhibited by CCCP but little affected by valinomycin. On the other hand, Na+ translocation in the presence of NADH was resistant to CCCP and stimulated by valinomycin. Amiloride, an inhibitor for a eucaryotic Na+/H+ antiport system, inhibited the lactate-dependent Na+ translocation but had little effect on the NADH-dependent Na+ translocation. These results indicate that a primary event of lactate oxidation is the translocation of H+, which then causes the generation of Na+ concentration gradients via the secondary Na+/H+ antiport system. We conclude that the NADH oxidase of V. costicola translocates Na+ as an immediate result of respiration, leading to the generation of Na+ electrochemical potential.  相似文献   

19.
To investigate the effects of chronic exposure to ketone bodies on glucose-induced insulin secretion, we evaluated insulin release, intracellular Ca2+ and metabolism, and Ca2+ efficacy of the exocytotic system in rat pancreatic islets. Fifteen-hour exposure to 5 mM d-beta-hydroxybutyrate (HB) reduced high glucose-induced insulin secretion and augmented basal insulin secretion. Augmentation of basal release was derived from promoting the Ca2+-independent and ATP-independent component of insulin release, which was suppressed by the GDP analog. Chronic exposure to HB affected mostly the second phase of glucose-induced biphasic secretion. Dynamic experiments showed that insulin release and NAD(P)H fluorescence were lower, although the intracellular Ca2+ concentration ([Ca2+](i)) was not affected 10 min after exposure to high glucose. Additionally, [Ca2+](i) efficacy in exocytotic system at clamped concentrations of ATP was not affected. NADH content, ATP content, and ATP-to-ADP ratio in the HB-cultured islets in the presence of high glucose were lower, whereas glucose utilization and oxidation were not affected. Mitochondrial ATP production shows that the respiratory chain downstream of complex II is not affected by chronic exposure to HB, and that the decrease in ATP production is due to decreased NADH content in the mitochondrial matrix. Chronic exposure to HB suppresses glucose-induced insulin secretion by lowering the ATP level, at least partly by inhibiting ATP production by reducing the supply of NADH to the respiratory chain. Glucose-induced insulin release in the presence of aminooxyacetate was not reduced, which implies that chronic exposure to HB affects the malate/aspartate shuttle and thus reduces NADH supply to mitochondria.  相似文献   

20.
Inverted membrane vesicles prepared from Vibrio alginolyticus generated a membrane potential (positive inside) and accumulated Na+ by the oxidation of NADH. Generation of the membrane potential required Na+ and was inhibited by 2-heptyl-4-hydroxyquinoline N-oxide, a specific inhibitor of the Na+-dependent NADH oxidase. Collapse of the membrane potential by valinomycin stimulated the uptake of Na+. In contrast, accumulation of H+ was not detected under all the conditions tested. These results suggest that only Na+ is translocated by the Na+-dependent NADH oxidase of V. alginolyticus.  相似文献   

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