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PubMed1980

Organization of unc gene cluster of Escherichia coli coding for proton-translocating ATPase of oxidative phosphorylation.

The proton-translocating ATPase (F1-F0) of oxidative phosphorylation (ATP phosphohydrolase, EC 3.6.1.3) is coded for by a set of structural genes comprising the unc operon in Escherichia coli. We have analyzed several new transducing phages and plasmids carrying various lengths of the DNA segments of the unc operon by complementation assay using 14 new unc- mutants and representatives of previously described strains which were made available to us. Transducing phages carrying parts of the unc gene cluster were isolated: lambda uncA-9 and lambda glmS phages converted only some of the unc- mutants to the Unc+, as determined by complementation assays. A new hybrid plasmid (pMCR533) carrying part of the unc operon was constructed by inserting the HindIII fragment of lambda asn-5 DNA (a phage carrying the entire unc operon) into the unique HindIII site of pBR322. This plasmid transformed eight unc- strains to Unc+, including uncB402 and uncA401, but did not complement uncD11 or four other strains. Two minichromosomes which carry the E. coli replication origin were also tested: plasmid pNH05 transformed the uncB402 but not the uncA401 strain to Unc+, whereas plasmid pMCF1 transformed none of the mutants tested. Analysis of the DNAs from these transducing phages and plasmids with restriction endonucleases suggested that all of the structural genes for the F1-F0 complex are localized within a DNA segment of approximately 4.5 megadaltons containing two EcoRI sites. The approximate locations of the unc- mutations were mapped on this DNA segment.

Adenosine Triphosphatases↗
PubMed1998

Energy balance in muscle activity: simulations of ATPase coupled to oxidative phosphorylation and to creatine kinase.

Energy balance refers to the dynamic homeostasis of ATP and related forms of chemical potential within cells. This regulation is accomplished mainly by oxidative metabolism in most mammals. This homeostasis matches dynamically the energy demands of cellular ATPases (net decrease in chemical potential energy) with the energy supply by mitochondrial oxidative phosphorylation (net increase in chemical potential energy). Muscle cells are distinguished from most other cell types in their ability to attain energy balance with more than a 10-fold range of ATPase demand. Creatine kinase maintains a near to equilibrium flux: PCr + ADP<-->ATP + Cr. One important function of creatine kinase is to buffer ATP and ADP concentrations. A system of differential equations describe the coupled operation of cellular ATPase, creatine kinase and oxidative phosphorylation. These equations used experimentally measured concentrations of relevant metabolites and enzyme activities to simulate energy balance in muscle cells. The principle of energy balance is adequately illustrated by simulations with only a three component system.

Adenosine Diphosphate↗
PubMed2003

ATP generation in the Trypanosoma brucei procyclic form: cytosolic substrate level is essential, but not oxidative phosphorylation.

Trypanosoma brucei is a parasitic protist responsible for sleeping sickness in humans. The procyclic form of this parasite, transmitted by tsetse flies, is considered to be dependent on oxidative phosphorylation for ATP production. Indeed, its respiration was 55% inhibited by oligomycin, which is the most specific inhibitor of the mitochondrial F0/F1-ATP synthase. However, a 10-fold excess of this compound did not significantly affect the intracellular ATP concentration and the doubling time of the parasite was only 1.5-fold increased, suggesting that oxidative phosphorylation is not essential for procyclic trypanosomes. To further investigate the sites of ATP production, we studied the role of two ATP producing enzymes, which are involved in the synthesis of pyruvate from phosphoenolpyruvate: the glycosomal pyruvate phosphate dikinase (PPDK) and the cytosolic pyruvate kinase (PYK). The parasite was not affected by PPDK gene knockout. In contrast, inhibition of PYK expression by RNA interference was lethal for these cells. In the absence of PYK activity, the intracellular ATP concentration was reduced by up to 2.3-fold, whereas the intracellular pyruvate concentration was not reduced. Furthermore, we show that this mutant cell line still excreted acetate from d-glucose metabolism, and both the wild type and mutant cell lines consumed pyruvate present in the growth medium with similar high rates, indicating that in the absence of PYK activity pyruvate is still present in the trypanosomes. We conclude that PYK is essential because of its ATP production, which implies that the cytosolic substrate level phosphorylation is essential for the growth of procyclic trypanosomes.

Adenosine Triphosphate↗
PubMed1997

Interactions of 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene with mitochondrial oxidative phosphorylation.

The effects of DDE (2,2-bis(p-chlorophenyl)-1,1-dichloroethylene), the major metabolite of DDT (2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane), on rat liver mitochondrial bioenergetic activities were examined. The approach developed by M. D. Brand (Biochim Biophys Acta 1018: 128-133, 1990) was used to assess the effects of DDE because it is possible to discriminate the sites of action of compounds having pleiotypic effects on oxidative phosphorylation. Data were further confirmed using a "classical" approach, including measurements of transmembrane potential, respiratory indexes, enzymatic activities and membrane permeability to protons. DDE up to 40 nmol/mg protein affected the proton motive force generating system. In fact, DDE interacted with succinate dehydrogenase (complex II), decreasing respiration and membrane potential. In this concentration range, the permeability of the inner membrane to protons remained intact. Only higher concentrations (> or = 80 nmol/mg) increased permeability to protons, uncoupling oxidation from phosphorylation. The phosphorylative system was not affected because the rate of ATP synthesis was unchanged. In addition, data from carbonyl cyanide m-chlorophenylhydrazone-uncoupled rotenone-inhibited preparations or submitochondrial particles indicated that F0F1 ATPase activity is not affected by DDE. Therefore, DDE inhibition of complex II and putative inhibition of succinate translocation explain the depression of mitochondrial respiration. The use of appropriate substrates and assay conditions indicates that complexes I, III and IV were not affected by DDE. The uncoupling of oxidative phosphorylation at high concentrations (> 80 nmol DDE/mg protein) was probably related to deleterious effects on the integrity of the mitochondrial membrane. We confirmed that the technique originally proposed by Brand is useful for characterizing the effects of xenobiotics on oxidative phosphorylation. In addition, data provided by this technique closely agree with data from classical studies.

Adenosine Triphosphate↗
PubMed1983

Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves.

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 NO(2) (-) concentration in the control leaves was very low in both light and darkness; 98% or more of the NO(2) (-) formed from NO(3) (-) was further assimilated in control leaves. More NO(2) (-) accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO(2) (-), 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 NO(2) (-) 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 NO(2) (-) formed from NO(3) (-) was further assimilated, whereas in light, 95% or more of the NO(2) (-) was further assimilated.

Journal Article↗
PubMed2000

Nuclear genes and oxidative phosphorylation disorders: a review.

UNLABELLED: Knowledge concerning the approximately 70 human nuclear genes creating the essential building-blocks of the five multi-protein subunit complexes of the oxidative phosphorylation (OXPHOS) system has been expanded greatly in the past few years. However, knowledge concerning the numerous human genes involved in the regulation of transcription, translation, post-translational modification, mitochondrial signalling, import, quality control, folding and assembly of the OXPHOS system is still rather scanty. It may be expected that this scenario, by the application of direct (candidate gene identification by comparison between known genes in lower species and the human expressed sequence tag database) and indirect genetic strategies (the chromosome transfer technique, linkage analysis and positional cloning) will rapidly change. By now, a limited number of structural and non-structural nuclear gene defects have been found. CONCLUSION: This review summarises the state of our current knowledge of nuclear gene mutations in oxidative phosphorylation disorders.

DNA↗
PubMed1977

An alternating site sequence for oxidative phosphorylation suggested by measurement of substrate binding patterns and exchange reaction inhibitions.

Catalysis by beef heart submitochondrial particles of the medium Pi in equilibrium HOH, Pi in equilibrium ATP, and the ATP in equilibrium HOH exchanges is strongly inhibited while the ATPase and intermediate Pi in equilibrium HOH exchange are accelerated when medium ADP is removed by pyruvate kinase action. Arsenate readily blocks completely the Pi in equilibrium ATP and medium Pi in equilibrium HOH exchange reactions, but not the ATP in equilibrium HOH exchange reaction. The residual ATP in equilibrium HOH exchange in presence of arsenate is inhibited by 2,4-dinitrophenol. These results and other data are explained by an alternating site model for oxidative phosphorylation. In this model during net oxidative phosphorylation ATP is formed at one site but is transitorily tightly bound and not released until ADP and Pi bind at a second site and the membrane ATPase complex is energized. Under conditions of net ATP hydrolysis, ATP binding at one site is accompanied by hydrolysis of the transitorily tightly bound ATP as a second site. Attractive features are only one site of input for conformational energization of the membrane ATPase, a single conformational transition that accounts for both the promotion of ADP and Pi binding in a competent mode and the release of tightly bound ATP, and a symmetry of catalytic sites. The Pi in equilibrium ATP exchange is not inhibited by increase in MgADP and MgATP at constant ratios, and the energy-linked ADP in equilibrium ATP exchange is not inhibited by increased concentrations of MgATP and Pi at a constant ratio. Such exchange patterns indicate a random binding and release of ADP and Pi.

Adenosine Triphosphate↗
PubMed1984

Thiols in oxidative phosphorylation: inhibition and energy-potentiated uncoupling by monothiol and dithiol modifiers.

Three apparently different modifications of submitochondrial particles (SMP) or ATP synthase preparations (complex V) inhibit oxidative phosphorylation and ATP-32Pi exchange activities, all of which are reversible by addition of mono- or dithiols. (a) Triphenyltin chloride inhibits ATP synthesis and hydrolysis without uncoupling. The inhibition by triphenyltin chloride is reversible by addition of beta-mercaptoethanol, dithiothreitol, or dihydrolipoamide. (b) Factor B is a water-soluble protein of Mr (11-12) X 10(3), contains a vicinal dithiol, and is required for energy transfer to and from F1-ATPase when tested with SMP-rendered factor B deficient by extraction with ammonia-ethylenediaminetetraacetic acid (EDTA) (AE-SMP). Treatment of factor B with mono- and dithiol modifiers, such as p-(chloromercuri)benzenesulfonate (PCMPS), Cd2+, or diazenedicarboxylic acid bis(dimethylamide) (diamide), inhibits factor B. This inhibition is reversed by addition to modified factor B of appropriate mono- and dithiol compounds. Preparations of AE-SMP are partially F1 deficient and partially uncoupled. The uncoupling can be repaired completely by addition of factor B or low levels of oligomycin, or to a large extent by addition of F1-ATPase + oligomycin sensitivity conferring protein. (c) SMP, AE-SMP, and complex V can be completely uncoupled by treatment at 30 degrees C with phenylarsine oxide, Cd2+, diamide, PCMPS, monobromobimane, and mono- and bifunctional maleimides. The uncoupling by these reagents is potentiated by membrane energization. Uncoupling by diamide is greater than or equal to 80% reversed by dihydrolipoamide or beta-mercaptoethanol, the former being much more potent. Dithiothreitol and dithioerythritol are poorly effective.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2002

Multiple effects of 2,2',5,5'-tetrachlorobiphenyl on oxidative phosphorylation in rat liver mitochondria.

An experimental investigation of the response of the multicomponent oxidative phosphorylation system to the environmental pollutant 2,2',5,5'-tetrachlorobiphenyl (2,2',5,5'-TCB) was performed by modular kinetic analysis in rat liver mitochondria oxidizing succinate (+ rotenone) and glutamate + malate. This approach facilitates the analysis of a complex process by dividing it into a small number of modules, each comprising multiple enzymatic steps, and allows evaluation of changes in the kinetics of individual blocks of the complex system induced by multisite effectors. Kinetic dependencies of the respiratory subsystem, the phosphorylation subsystem, and the proton permeability of the inner membrane on the membrane potential Delta Psi were determined in the control and in the presence of 20 microM 2,2',5,5'-TCB. The toxin inhibited the rate of respiration with both substrates to a similar extent (by 23-26%). We showed that 2,2',5,5'-TCB affected the all three modules of the oxidative phosphorylation system: it inhibited both the respiratory and the phosphorylation subsystems, and increased the membrane leak. As a result, the value of Delta Psi in State 3 of mitochondria oxidizing glutamate + malate remained the same or slightly increased with succinate, indicating that in the former case the respiratory subsystem was more sensitive to 2,2',5,5'-TCB. We explain this by the 2,2',5,5'-TCB-induced inhibition of Complex I. Moreover, 2,2',5,5'-TCB decreased the number of oligomycin-binding sites by 20%, caused a significant drop in the membrane potential generated by ATP hydrolysis, and inhibited activity of ATP hydrolysis in uncoupled mitochondria. Thus, we obtained evidence that at least one of the targets of 2,2',5,5'-TCB action within the phosphorylation module was ATP synthase.

Animals↗
PubMedDate not supplied

[Acetylcholine and effectiveness of oxidative phosphorylation in isolated rat hepatocytes].

It is shown that ADP and DNP does not intensify the respiration rate in hepatocytes of rats obtained by means of trypsin or EDTA. The same cells obtained using collagenase, phosphorylate added ADP and increase the respiration rate after DNP addition. Acetylcholine added to the cell suspension in a dose of 5 x 4 x 10(-8) M) increases the efficiency of oxidative phosphorylation. The scheme of neurotransmitter regulation of intensity of respiration and efficiency of oxidative phosphorylation on the cell level is suggested.

Acetylcholine↗
PubMed1963

Participation of an intermediate of oxidative phosphorylation in ion accumulation by mitochondria.

Heart mitochondria accumulate massive amounts of Mg(++) and phosphate or Ca(++) and phosphate when incubated under appropriate conditions. Studies with inhibitors of respiration, oligomycin, and uncouplers of oxidative phosphorylation indicate that the energy necessary for these reactions may be provided either in the form of ATP in the absence of electron transport, or in the form of a high-energy intermediate of oxidative phosphorylation, which operates even though ATP is not produced.

Adenosine Triphosphate↗
PubMed1997

Impairment of respiration and oxidative phosphorylation by redox cyclers 2-nitrosofluorene and menadione.

The present study was designed to investigate the effects of 2-nitrosofluorene (NOF), a metabolite of carcinogenic 2-acetylaminofluorene, on mitochondrial respiration and oxidative phosphorylation. NOF reacts with the NADH:ubiquinone oxidoreductase (complex I) and consumes oxygen in a rotenone-insensitive manner. Unlike menadione, which is able to bypass the rotenone-block and to restore ATP-formation, NOF-induced electron flow was almost completely uncoupled. In normal respiration both redox-cyclers decreased the respiratory control and P/O ratios at low concentrations (2-20 nmol/mg) in NADH-dependent oxidation. With succinate as substrate, only NOF was significantly active. In contrast to NOF, the hydroxamic acid N-hydroxy-2-acetylaminofluorene (N-OH-AAF) impaired mitochondrial energy conversion only at much higher concentrations (80 nmol/mg). At concentrations > 10 nmol/mg, NOF inhibited electron flow through the respiratory chain in NADH- and succinate-dependent oxidation, as determined by dinitrophenolate-uncoupled respiration. The small protective effect of L-cysteine indicates that covalent binding of the nitroso-compound to SH-groups may not explain sufficiently the inhibitory effect of NOF. The results support the notion that redox cyclers impair oxidative phosphorylation by establishing alternative pathways for electron transport in the respiratory chain.

2,4-Dinitrophenol↗
PubMedDate not supplied

[The effect of GABA-ergic agents on oxidative phosphorylation in the brain mitochondria in traumatic edema].

The condition of oxidative phosphorylation in the rat brain mitochondria was studied polarographically in dynamics of the brain traumatic edema. It was found that 24 hours after the trauma the rate of oxidation in mitochondria decreased and the degree of conjugation in the respiratory chain compensatorily increased. Piracetam (1 g/kg), phenibut (50 mg/kg) and to a lesser degree sodium oxybutyrate (0.2 g/kg) prevented the 'changes and enhanced the compensatory capacities of mitochondria during the development of traumatic edema of the brain.

Animals↗
PubMedDate not supplied

Regulation of cytochrome c oxidase by adenylic nucleotides. Is oxidative phosphorylation feedback regulated by its end-products?

Cytochrome c oxidase, which catalyzes an irreversible step of the respiratory chain, is one of the rate-controlling steps of oxidative phosphorylation on isolated mitochondria. The rate of electron transfer through the complex is primarily controlled by the associated thermodynamic forces, i.e., the span in redox potential between oxygen and cytochrome c and the protonmotive force. However, the electron flux also depends on the various kinetic effectors, including adenylic nucleotides. Although the number of binding sites for ATP and ADP on cytochrome oxidase is still a matter of debate, experiments performed on the solubilized and reconstituted enzyme provide strong functional evidence that the mammalian cytochrome c oxidase binds adenylic nucleotides on both sides of the inner membrane. These effects include modification in cytochrome c affinity, allosteric inhibition and changes in proton pumping efficiency. Immunological studies have pointed out the role of subunit IV and that of an ATP-binding protein, subunit VIa, in these kinetic regulations. In yeast, the role of the nuclear-encoded subunits in assembly and regulation of the cytochrome c oxidase has been further substantiated by using gene-disruption analysis. Using a subunit VIa-null mutant, the consequences of the ATP regulation on oxidative phosphorylation have been further investigated on isolated mitochondria. Taken together, the data demonstrate that there are multiple regulating sites for ATP on the yeast cytochrome oxidase with respect to the location (matrix versus cytosolic side), kinetic effect (activation versus inhibition) and consequence on the flow-force relationships. The question is therefore raised as to the physiological meaning of such feedback regulation of the respiratory chain by ATP in the control and regulation of cellular energy metabolism.

Adenine Nucleotides↗
PubMed1996

ADP delivery from adenylate kinase in the mitochondrial intermembrane space to oxidative phosphorylation increases in the presence of macromolecules.

Macromolecules were added to isolated rat liver mitochondria to mimic cytosolic macromolecules and tested for their effects on the ADP delivery from adenylate kinase in the intermembrane space to oxidative phosphorylation. In the presence of 10% (w/v) dextran M20 or bovine serum albumin, approximately 60% of the maximal ADP flux from adenylate kinase to oxidative phosphorylation was not accessible to an extramitochondrial ADP scavenger. In the absence of macromolecules this was 34%. ADP determinations from incubations with macromolecules demonstrated the existence of flux-dependent ADP concentration gradients across the outer membrane which can be as high as 12 microM.

Adenosine Diphosphate↗
PubMed1998

Control of oxidative phosphorylation, gluconeogenesis, ureagenesis and ATP turnover in isolated perfused rat liver analyzed by top-down metabolic control analysis.

We have analyzed the control exerted by the pathways of oxidative phosphorylation, gluconeogenesis, ureagenesis, and maintenance ATP consumption over each other's rates in isolated, perfused rat liver using top-down metabolic control analysis. The livers from fasted rats were perfused with 3-hydroxybutyrate as respiratory substrate, lactate as substrate for gluconeogenesis, and ammonium as substrate for urea synthesis, in conditions where these pathways were only linked by their common intermediates: ATP, ADP, and Pi. The rates of oxygen consumption, glucose and urea synthesis were measured continuously. The pathways were perturbed either by adding specific inhibitors or by adding new pathways that consumed ATP, and the relative changes in pathway rates were used to calculate the flux control coefficients of each pathway over all pathway rates. When the liver was in a relatively inactive metabolic state, where ATP was only being used by the maintenance ATP-consuming pathways, then essentially all the control over ATP production and consumption was located in the maintenance ATP consumers with ATP production having no control. Whereas, when the liver was in a highly active state using extra ATP for both glucose and urea synthesis, then ATP production (from oxidative phosphorylation) had strong control over its own rate and the rates of glucose and urea synthesis, but gluconeogenesis and ureagenesis still had strong control over their own rates and negative control over each others rates, i.e. they competed for the limited ATP supply. The rate of the maintenance ATP consumers is remarkably insensitive to changes in ATP production and consumption, but exerts considerable control over all other pathways. These results indicate that the general assumption that the rates of ATP production and consumption are controlled exclusively by ATP consumers is false under conditions where a significant amount of ATP is used for biosynthetic processes, such as glucose and urea synthesis, and indicate that the latter processes may be partly controlled by regulators of ATP production and by other ATP-consuming pathways.

3-Hydroxybutyric Acid↗
PubMed1980

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+.

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.

Adenosine Diphosphate↗
PubMed1976

Salicylate- and aspirin-induced uncoupling of oxidative phosphorylation in mitochondria isolated from the mucosal membrane of the stomach.

The hypothesis that the damaging effect on the stomach mucosa of salicylic acid and its derivatives is ascribable to an uncoupling of oxidative phosphorylation has been investigated by testing of mitochondria isolated from the corpus gland area of mini-pig gastric mucosa. Mitochondria, influenced by salicylate or acetylsalicylate (0.7-5.6 mmol/l), demonstrated increased respiration rate, decreased respiratory control ratio, and decreased P/O ratio when tested in vitro. Uncoupling of oxidative phosphorylation occurred at a salicylate concentration between 3.5 and 5.6 mmol/l.

Adenosine Diphosphate↗