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1.
The artificial electron donor system, phenazine methosulfate (PMS)-ascorbate, inhibited active transort of solutes in Pseudomonas aeruginosa irrespective of whether the active transport systems were shock sensitive or shock resistant. N,N,N′,N′-tetramethylphenylenediamine could be substituted for PMS but a higher concentration was required. PMS-ascorbate also inhibited active transport in several other bacterial species with the exception of Escherichia coli and of a nonpigmented strain of Serratia marcescens. PMS-ascorbate previously has been shown to energize active transport in isolated membrane vesicles, even those prepared from the same bacterial species in whose intact cells active transport was inhibited. The apparent K m of glucose active transport in untreated cells of P. aeruginosa was 40 μM while the K m of glucose transport in cells incubated with PMS-ascorbate was 25 mM, and PMS-ascorbate had no effect on efflux of accumulated glucose. These results strongly suggested that facilitated diffusion resulted upon exposure of the cells to PMS-ascorbate. Thus, PMS-ascorbate appeared to have an uncoupler-like effect on cells of P. aeruginosa. The experimental data also pointed out that there are fundamental differences between the response of intact cells and membrane vesicles to exogenous electron donors. 相似文献
2.
3-BrPA (3-bromopyruvate) is an alkylating agent with anti-tumoral activity on hepatocellular carcinoma. This compound inhibits cellular ATP production owing to its action on glycolysis and oxidative phosphorylation; however, the specific metabolic steps and mechanisms of 3-BrPA action in human hepatocellular carcinomas, particularly its effects on mitochondrial energetics, are poorly understood. In the present study it was found that incubation of HepG2 cells with a low concentration of 3-BrPA for a short period (150 microM for 30 min) significantly affected both glycolysis and mitochondrial respiratory functions. The activity of mitochondrial hexokinase was not inhibited by 150 microM 3-BrPA, but this concentration caused more than 70% inhibition of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and 3-phosphoglycerate kinase activities. Additionally, 3-BrPA treatment significantly impaired lactate production by HepG2 cells, even when glucose was withdrawn from the incubation medium. Oxygen consumption of HepG2 cells supported by either pyruvate/malate or succinate was inhibited when cells were pre-incubated with 3-BrPA in glucose-free medium. On the other hand, when cells were pre-incubated in glucose-supplemented medium, oxygen consumption was affected only when succinate was used as the oxidizable substrate. An increase in oligomycin-independent respiration was observed in HepG2 cells treated with 3-BrPA only when incubated in glucose-supplemented medium, indicating that 3-BrPA induces mitochondrial proton leakage as well as blocking the electron transport system. The activity of succinate dehydrogenase was inhibited by 70% by 3-BrPA treatment. These results suggest that the combined action of 3-BrPA on succinate dehydrogenase and on glycolysis, inhibiting steps downstream of the phosphorylation of glucose, play an important role in HepG2 cell death. 相似文献
3.
Propionate inhibits citrullinogenesis when succinate (plus rotenone) or glutamate are the oxidizable substrates used. Propionate decreases the intramitochondrial concentration of carbamylphosphate by decreasing the ATP content. When the energy supply for citrullinogenesis is provided by an influx of exogenous ATP, propionate is no longer an inhibitor. Pyruvate inhibits citrullinogenesis with glutamate but not with succinate (plus rotenone) as oxidizable substrates. Propionate and pyruvate deplete mitochondrial ATP but probably by different mechanisms. 相似文献
4.
Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity. 相似文献
5.
Optimal cell yield of Pseudomonas aeruginosa grown under denitrifying conditions was obtained with 100 mM nitrate as the terminal electron acceptor, irrespective of the medium used. Nitrite as the terminal electron acceptor supported poor denitrifying growth when concentrations of less than 15 mM, but not higher, were used, apparently owing to toxicity exerted by nitrite. Nitrite accumulated in the medium during early exponential phase when nitrate was the terminal electron acceptor and then decreased to extinction before midexponential phase. The maximal rate of glucose and gluconate transport was supported by 1 mM nitrate or nitrite as the terminal electron acceptor under anaerobic conditions. The transport rate was greater with nitrate than with nitrite as the terminal electron acceptor, but the greatest transport rate was observed under aerobic conditions with oxygen as the terminal electron acceptor. When P. aeruginosa was inoculated into a denitrifying environment, nitrate reductase was detected after 3 h of incubation, nitrite reductase was detected after another 4 h of incubation, and maximal nitrate and nitrite reductase activities peaked together during midexponential phase. The latter coincided with maximal glucose transport activity. 相似文献
6.
Glucose transport by Pseudomonas aeruginosa was studied. These studies were enhanced by the use of a mutant, strain PAO 57, which was unable to grow on glucose but which formed the inducible glucose transport system when grown in media containing glucose or other inducers such as 2-deoxy-d-glucose. Both PAO 57 and parental strain PAO transported glucose with an apparent K(m) of 7 muM. Free glucose was concentrated intracellularly by P. aeruginosa PAO 57 over 200-fold above the external level. These data constitute direct evidence that glucose is transported via active transport by P. aeruginosa. Various experimental data clearly indicated that P. aeruginosa PAO transported methyl alpha-d-glucose (alpha-MeGlc) via the glucose transport system. The apparent K(m) of alpha-MeGlc transport was 7 mM which indicated a 1,000-fold lower affinity of the glucose transport system for alpha-MeGlc than for glucose. While only unchanged alpha-MeGlc was detected intracellularly in P. aeruginosa, alpha-MeGlc was actually concentrated intracellularly less than 2-fold over the external level. Membrane vesicles of P. aeruginosa PAO retained transport activity for gluconate. This solute was concentrated intravesicularly several-fold over the external level. A component of the glucose transport system is believed to have been lost during vesicle preparation since glucose per se was not transported. Instead; glucose was converted to gluconate by membrane-associated glucose dehydrogenase and gluconate was then transported into the vesicles. Although this may constitute an alternate system for glucose transport, it is not a necessary prerequisite for glucose transport by intact cells since P. aeruginosa PAO 57, which lacks glucose dehydrogenase, was able to transport glucose at a rate equal to the parental strain. 相似文献
7.
19 The effect of pent-4-enoic acid, propionic acid and several other short-chain fatty acids on citrulline synthesis in rat liver mitochondria was studied. 2.Pent-4-enoate at 1 mM inhibited mitochondrial citulline synthesis by about 80-90%. It is concluded that pent-4-enoate inhibits citrulline synthesis by interfering with some aspect of mitochondrial energy metabolism. This results in impairment of mitochondrial ornithine uptake or depletion of mitochondrial ATP, which, in turn, impairs carbamoyl phosphate synthesis or both. Evidence in support of this conclusion includes: pent-4-enoate has no effect on citrulline synthesis supported by succinate or exogenous ATP; pent-4-enoate lowers the medium plus mitochondrial ATP concentration; finally, when glutamate is the oxidizable substrate, pent-4-enoate decreases the carbamoyl phosphate concentration in mitochondria incubated without ornithine to minimize citrulline synthesis and impairs the mitochondrial uptake of ornithine, but it has neither effect when succinate is the oxidizable substrate. 4. Propionate, butyrate and crotonate also inhibit mitochondrial citrulline synthesis, but much less than pent-4-enoate. 5. Acetate, pentanoate, pent-2-enoate, hexanoate, octanoate, isovalerate, tiglylate and alpha-methylbutyrate have little or no effect on mitochondrial citrulline synthesis. 相似文献
8.
Glucose stimulated the rate of incorporation of [3H]leucine into HCLO4-insoluble fraction of cultured rat heart muscle cells under both aerobic and anaerobic conditions. In the aerobic system the incorporation proceeded at a constant rate during 3h of incubation with and without glucose whereas in the anaeorbic system the incorporation ceased after approx. 60 min and could be renewed only by the addition of glucose. No correlation was found to exist between the above effect of glucose on protein synthesis and glucose-dependent changes in the intracellular ATP concentration. The extent of the stimulation of protein synthesis was related to the concentration of glucose. The effect of glucose was suppressed by cycloheximide but was not affected by actinomycin D. Glucose had no effect on the rate of transport of alpha-aminoisobutyric acid. Mannose also stimulated [3H]leucine incorporation. Substances that did not produce lactate were ineffective. Iodoacetate inhibited the stimulatory effect of glucose, but pyruvate, which by itself had no apprecialbe stimulatory action, relieved the inhibition induced by iodoacetate. There was no concomitant change in the concentration of ATP when iodoacetate inhibition was reversed by pyruvate. L-Lactate or other intermediates of energy metabolism could not relieve the inhibitory effect of iodoacetate. 相似文献
9.
The adenylate energy charges (EC) of Escherichia coli 25922, Pseudomonas aeruginosa 27853, and Streptococcus lactis 7962 rapidly fell in nutrient-rich media from values in excess of 0.9 to below 0.1 when the organisms were exposed to lethal levels of HOCl. The same cells maintained in energy-depleted states were incapable of attaining normal EC values necessary for biosynthesis and growth when challenged with nutrient energy sources after HOCl exposure. These changes correlated quantitatively with loss of replicative capabilities. Initial rates of transport of glucose, succinate, and various amino acids that act as respiratory substrates and the ATP hydrolase activity of the F1 complex from the ATP synthase of E. coli 25922 also declined in parallel with or preceded loss of viability. These results establish that cellular death is accompanied by complete disruption of bacterial ATP production by both oxidative and fermentative pathways as a consequence of inhibition of inner membrane bound systems responsible for these processes. 相似文献
10.
Energy metabolism of Leishmania donovani parasite has been investigated under conditions imitating intralysosomal-like environment in the host organism. Trans-plasma
membrane electron transport and oxygen uptake were inhibited progressively when promastigote cells were exposed to pH 5.5
and 37°C. A special feature of the respiratory chain in amastigote was the absence of complex I, II, and IV. When L. donovani was grown at pH 5.5 and 37°C, the acid excretory product succinate was increased in comparison to cells grown at pH 7.5 and
24°C. The findings of this study showed that the amastigote form catabolized fatty acid to excrete succinic acid when oxidative
phosphorylation was impaired. Amastigote mitochondria failed to generate membrane potential by oxidizable substrates. On the
other hand, the amastigote cell showed absorbance change of safranine O when fatty acid was the oxidizable substrate. The
safranine signal was completely reversed by valinomycin, carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone, malonate, and
oxaloacetate. Our data suggest that the generation of metabolic energy from succinate/H + efflux will contribute to energy requiring process of amastigote significantly. On the basis of these results, we conclude
that due to absence of oxidative phosphorylation in amastigotes, energy linked functions in amastigotes might occur through
fumarate reduction leading to ΔpH generation by succinate excretion. 相似文献
11.
1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K + and gained Na +, Ca 2+ and water. 2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K+ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca2+ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose. 3. When respiration was inhibited in the presence of glucose, K+ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions. 4. In the absence of glucose, the transport of Na+ and K+ was completely stopped by respiratory inhibition. However, more than 50% of the O2 uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport. 5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose. 6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K+ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport. 相似文献
12.
Abstract: The effects of nitroprusside and S -nitrosocysteine, compounds that generate nitric oxide (NO), on synaptosomal energy-producing pathways and energy level were investigated. The decrease in respiration was much faster and more pronounced with S -nitrosocysteine than with nitroprusside. S -Nitrosocysteine, at 10 µ M , inhibited by 80% respiration with glucose and succinate (plus rotenone) in intact synaptosomes and with ascorbate/cytochrome c in broken preparations. Oxygenated hemoglobin reversed and/or prevented the inhibition, whereas glutathione (GSH) prolonged it. Under aerobic conditions, the synaptosomal energy level (creatine phosphate/creatine and ATP/ADP ratios) was reduced by the presence of S -nitrosocysteine, whereas lactate generation was enhanced. The effects on energy parameters were greater at 5 min than at 15 min of incubation and were more pronounced in the presence of GSH. Under strictly anaerobic conditions, lactate production was reduced by the NO-generating compounds in a concentration-dependent manner. It is concluded that (a) inhibition of oxidative phosphorylation by NO leads to a fall in the synaptosomal energy level, which in turn stimulates glycolysis; (b) glycolysis can be inhibited by higher concentrations of the radical; and (c) inhibitory effects on the energy-generating pathway and ATP level could contribute to NO toxicity under some in vivo situations. 相似文献
13.
Extracts of Pseudomonas aeruginosa (ATCC 7700) cells grown on glucose, gluconate, or glycerol had enzyme activities related to the Entner-Doudoroff pathway. These activities were present in no more than trace amounts when the bacteria were grown on succinate. Fructose-1,6-diphosphate aldolase could not be detected in extracts of the bacteria grown on any of the above carbon sources. Therefore, it appears that P. aeruginosa degrades glucose via an inducible Entner-Doudoroff pathway. The apparent absence of fructose-1,6-diphosphate aldolase in cells growing on succinate suggests that the bacteria can form hexose and pentose phosphates from succinate by an alternate route. d-Glucose-6-phosphate dehydrogenase, a branch-point enzyme of the Entner-Doudoroff pathway, was purified 50-fold from glucose-grown cells. Its molecular weight, estimated by sucrose density gradient centrifugation, was found to be approximately 190,000. The enzyme was strongly inhibited by adenosine triphosphate, guanosine triphosphate, and deoxyguanosine triphosphate, which decreased the apparent binding of glucose-6-phosphate to the enzyme. It is suggested that adenine nucleotide-linked control of glucose-6-phosphate dehydrogenase may regulate the overall catabolism of hexose phosphates and prevent their wasteful degradation under certain conditions requiring gluconeogenesis. 相似文献
14.
Ca 2+ uptake by rat brain mitochondria was studied under different experimental conditions. The most rapid uptake of Ca 2+ occurred in the presence of ATP, succinate and P i. ATP alone also supported Ca 2+ uptake. In contrast, no Ca 2+ uptake occurred with succinate and P i when no ATP was added. Oligomycin and atractylate completely inhibited ATP-supported Ca 2+ uptake but produced only a partial inhibition of Ca 2+ transport in the presence of ATP, succinate and P i. ATP plays a dual role in its action on brain mitochondria; it can support Ca 2+ uptake by itself and it serves a function in allowing respiration-dependent Ca 2+ uptake to proceed. The latter role of ATP does not involve transfer of energy from the nucleotide. 相似文献
15.
In addition to their critical function in energy metabolism, mitochondria contain a permeability transition pore, which is regulated by adenine nucleotides. We investigated conditions required for ATP to induce a permeability transition in mammalian mitochondria. Mitochondrial swelling associated with mitochondria permeability transition (MPT) was initiated by adding succinate to a rat liver mitochondrial suspension containing alloxan, a diabetogenic agent. If alloxan was added immediately with or 5 min after adding succinate, MPT was strikingly decreased. MPT induced by alloxan was inhibited by EGTA and several agents causing thiol oxidation, suggesting that alloxan leads to permeability transition through a mechanism dependent on Ca(2+) uptake and sulfhydryl oxidation. Antimycin A and cyanide, inhibitors of electron transfer, carbonyl cyanide m-chlorophenylhydrazone, and oligomycin all inhibited MPT. During incubation with succinate, alloxan depleted ATP in mitochondria after an initial transient increase. However, in a mitochondrial suspension containing EGTA, ATP significantly increased in the presence of alloxan to a level greater than that of the control. These results suggest the involvement of energized transport of Ca(2+) in the MPT initiation. Addition of exogenous ATP, however, did not trigger MPT in the presence of alloxan and had no effect on MPT induced by alloxan. We conclude that alloxan-induced MPT requires mitochondrial energization, oxidation of protein thiols, and matrix ATP to promote energized uptake of Ca(2+). 相似文献
16.
To investigate the operation of a succinate transport system in Escherichia coli, mutants defective in succinate metabolism were isolated. Although the metabolic blocks in the mutant cells were not complete, the succinate transport assays became possible.Pyruvate, lactate or many other carbon sources stimulated succinate uptake, and the uptake was strongly inhibited by some electron transport inhibitors, uncouplers of oxidative phosphorylation and sulfhydryl reagents. The mutant strains accumulated succinate into the cells against a concentration gradient when suitable energy sources were supplied.Presence of glucose in the medium strongly repressed the formation of the succinate transport system. The optimum pH for the succinate uptake was between 7.8 and 8.0. 相似文献
17.
Chemotaxis by Pseudomonas aeruginosa RM46 has been studied, and conditions required for chemotaxis have been defined, by using the Adler capillary assay technique. Several amino acids, organic acids, and glucose were shown to be attractants of varying effectiveness for this organism. Ethylenediaminetetraacetic acid was absolutely required for chemotaxis, and magnesium was also necessary for a maximum response. Serine taxis was greatest when the chemotaxis medium contained 1.5 X 10(-5) M ethylenediaminetetraacetic acid and 0.005 M magnesium chloride. It was not necessary to include methionine in the chemotaxis medium. The strength of the chemotactic responses to glucose and to citrate was dependent on prior growth of the bacteria on glucose and citrate, respectively. Accumulation in response to serine was inhibited by the addition of succinate, citrate, malate, glucose, pyruvate, or methionine to the chemotaxis medium. Inhibition by succinate was not dependent on the concentration of attractant in the capillary. However, the degree to which glucose and citrate inhibited serine taxis was dependent on the carbon source utilized for growth. Further investigation of this inhibition may provide information about the mechanisms of chemotaxis in P. aeruginosa. 相似文献
18.
The function of mitochondria-bound hexokinase, the enzymatic form peculiar to the brain, in utilization of ATP generated inside the organelles, was examined by incubating rat brain mitochondrial fraction with [14C]glucose under various conditions. Addition of succinate and ADP to the incubation medium increased glucose 6-phosphate formation by the mitochondrial hexokinase and caused a smaller increase in ATP concentration in the mitochondria. The glucose phosphorylation was markedly inhibited by the addition of dinitrophenol, potassium cyanide, and oligomycin, and the ATP concentration was decreased. On the other hand, addition of atractyloside suppressed the glucose phosphorylation without affecting the mitochondrial hexokinase activity, whereas addition of antiserum against the mitochondrial hexokinase inhibited both glucose 6-phosphate formation and hexokinase activity. A part of both the glucose phosphorylation and hexokinase activities, however, remained even in the presence of the maximum dose of the anti-hexokinase serum and atractyloside. These results indicate the active utilization of intrinsically generated ATP by the mitochondria-bound hexokinase, a part of which may be located away from the surface of the mitochondrial membrane. 相似文献
19.
It has been proposed that hexokinase bound to mitochondria occupies a preferred site to which ATP from oxidative phosphorylation is channeled directly (Bessman, S. (1966) Am. J. Medicine 40, 740-749). We have investigated this problem in isolated Zajdela hepatoma mitochondria. Addition of ADP to well-coupled mitochondria in the presence of an oxidizable substrate initiates the synthesis of glucose 6-phosphate via bound hexokinase. This reaction is only partially inhibited by oligomycin, carboxyatractyloside, carbonyl cyanide m-chlorophenylhydrazone (CCCP) or any combination of these, suggesting a source of ATP in addition to oxidative phosPhorylation. This source appears to be adenylate kinase, since Ado2P5, an inhibitor of the enzyme, suppresses hexokinase activity by about 50% when added alone or suppresses activity completely when added together with any of the inhibitors of oxidative phosphorylation. Ado2P5 does not uncouple oxidative phosphorylation nor does it inhibit ADP transport (state 3 respiration) or hexokinase. The relative amount of ATP contributed by adenylate kinase is dependent upon the ADP concentration. At low ADP concentrations, glucose phosphorylation is supported by oxidative phosphorylation, but as the adenine nucleotide translocator becomes saturated the ATP contributed by adenylate kinase increases due to the higher apparent Km of the enzyme. Under conditions of our standard experiment ([ADP] = 0.5 mM), adenylate kinase provides about 50% of the ATP used by hexokinase in well-coupled mitochondria. In spite of this, externally added ATP supported higher initial rates of hexokinase activity than ADP. Our findings demonstrate that oxidative phosphorylation is not a specific or preferential source of ATP for hexokinase bound to hepatoma mitochondria. The apparent lack of a channeling mechanism for ATP to hexokinase in these mitochondria is discussed. 相似文献
20.
The effect of various nucleotides on the last step of aldosterone biosynthesis, the so-called "18 oxidation" (transformation of 18-hydroxycorticosterone to aldosterone), was studied by incubation of tritiated 18-hydroxycorticosterone with untreated duck adrenal mitochondria in vitro. The study was carried out in the absence or in the presence of antimycin A which blocks the respiratory chain. Results show that, when oxidative phosphorylation chain functions normally, GTP and CTP had no effect, UTP stimulated this reaction but ADP and ATP inhibited the transformation of 18-hydroxycorticosterone into aldosterone to the same extent. For this reason ATP is included in all controls for experiments studying the effect of ATP when "18 oxidation" is inhibited by antimycin A. When oxidative phosphorylation chain is inhibited by antimycin A, ATP is able to reverse the inhibition of "18 oxidation" induced by antimycin A, in the presence of succinate. Under these conditions UTP is not able to reverse the inhibition induced by antimycin A; GTP and CTP had no effect. Effects of ATP and UTP on the last step of aldosterone biosynthesis are related to different mechanisms. ATP clearly acts as an energy source for "18 oxidation" in the presence of succinate. The role of UTP must still be determined. 相似文献
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