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PubMed1988

ATP hydrolysis induces variable porosity to mannitol in the mitochondrial inner membrane.

Osmotic titration of ATPase activity in rat liver mitochondria was consistent with enhanced porosity of the mitochondrial inner membrane to mannitol due to ATP hydrolysis even when endogenous respiration was inhibited by rotenone. The occluded ATPase activity, which exhibits osmotic activation with an optimum near isotonicity, depends both on the ATPase activity per se and on the activity of the ADP/ATP carrier. Purified ADP/ATP carrier incorporated into small, unilamellar liposomes was critically shown to exhibit dependence of its activity on the osmotic pressure differences across the membrane, with maximal activity corresponding to isotonicity, regardless of the actual internal tonicity.

Adenosine Triphosphate↗

PubMed1988

Analysis of mechanisms of free-energy coupling and uncoupling by inhibitor titrations: theory, computer modeling and experiments.

The rates of ATP synthesis and of ATP-driven NAD reduction have been measured in bovine heart submitochondrial particles as a function of the fraction of inhibited redox pumps (in titrations with either antimycin or rotenone) and of the fraction of inhibited ATPases (in titrations with DCCD). The flux control coefficients of the redox and ATPase proton pumps on the rates of ATP synthesis and of ATP-driven NAD reduction have been derived and found to be equal to 1 for both pumps; i.e., both pumps appear to be 'completely rate limiting'. A theoretical analysis of the inhibitor titration approach based on kinetic models of chemiosmotic coupling and on the theory of metabolic control is presented. This analysis (i) shows that the results of the single inhibitor titrations are incompatible with a delocalized chemiosmotic mechanism of energy coupling if the proton conductance of the membrane is sufficiently low with respect to the conductances of the pumps; and (ii) suggests an experimental approach based on the determination of the P/O and the respiratory control ratios at different degrees of inhibition of the proton pumps to establish the origin of the 'loose coupling' of submitochondrial particle preparations. Three independent types of observation show that the 'loose coupling' of the particle preparation is not mainly due to an increased membrane proton conductance. The same and other independent observations are consistent with the view that the loose coupling of submitochondrial particle preparation is due mainly to inhomogeneity, i.e. to the presence of a subpopulation of highly leaky non-phosphorylating vesicles respiring at maximal rate. The results as a whole together with the simulations and analysis presented lead to the conclusion that the mechanism of free-energy coupling in submitochondrial particles is not completely delocalized.

Adenosine Triphosphatases↗

PubMed1993

Characterization of NAD(P)H-dependent ubiquinone reductase activities in rat liver microsomes.

Exogenous ubiquinone-10 was efficiently reduced by rat liver microsomes in the presence of NADH and NADPH under anaerobic conditions. Ubiquinone-10 reduced under anaerobic conditions was rapidly re-oxidized by the re-aeration. The reduction and re-oxidation were not observed when the reactions were carried out with the boiled microsomes or without microsomes, suggesting that the reactions were enzymatically catalyzed by the electron transport system(s) from NAD(P)H to O2 through the ubiquinone. The Km and Vmax of the reductase activity for NADH were 0.4 mM and 1.7 nmol/min per mg of protein, and those for NADPH were 19 microM and 2.1 nmol/min per mg of protein, respectively. The NADH-dependent oxidoreduction system was different from the NADPH-dependent system because of the following observations; (1) rotenone inhibited only the NADH-dependent ubiquinone-10 reductase, (2) dicoumarol inhibited the NADPH-dependent ubiquinone-10 reduction more potently than the NADH-dependent reduction and (3) the activity oxidizing the reduced ubiquinone-10 in the presence of NADH was less than that in the presence of NADPH. Endogenous ubiquinone-9 was also reduced and re-oxidized in essentially the same manner as exogenous ubiquinone-10. Thus, ubiquinone-10 oxidoreductase in rat liver microsomes acts on endogenous ubiquinone-9.

Animals↗

PubMed1976

Monitoring membrane potentials in Ehrlich ascites tumor cells by means of a fluorescent dye.

1. The fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide was measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potentials under a variety of different ionic and metabolic conditions. 2. In the presence of valinomycin, fluorescent intensity is dependent on log [K+]medium (the fluorescent intensity increased with increasing [K+]medium) where K+ replaced Na+ in the medium. Cellular K+ content also influenced fluorescent intensity in the presence of valinomycin. With lower cellular K+, fluorescent intensity in the presence of valinomycin for any given concentration was increased. 3. In the presence of gramicidin fluorescent intensity was highest in Krebs-Ringer and decreased with the substitution of choline+ for Na+. 4. The observations with ionophores are consistent with the hypothesis that the dye monitors membrane potential in these cells with an increase in fluorescence indicating membrane depolarization (internal becomes more positive). 5. The estimated membrane potentials were influenced by the way in which the cells were treated. Upon dilution of the cells from 1 in 20 to 1 in 300 the initial estimations were between -50 and -60 mV. With incubation at 1 in 300 dilution for 1 h at room temperature or a 37 degrees C, the membrane potentials ranged from -18 to -42 mV. 6. Estimations of membrane potential on the basis of chloride distribution (Cl-cell/Cl-medium) in equilibrated cells ranged from -13 to -32 mV. 7. Addition of glucose to cells equilibrated at 37 degrees C for 30 min in the presence of rotenone led to a decrease in fluorescent intensity indicating hyperpolarization. Addition of ouabain in turn led to a 70 to 100% reversal of fluorescent intensity. This hyperpolarization is therefore probably due to the electrogenic activity of the sodium pump. 8. The addition of amino acids known to require external Na+ for transport increased fluorescent intensity (depolarization) reaching a maximum at higher concentrations of amino acids. Plots of 1/deltafluorescence vs. 1/[glycine] were linear with an apparent Km of 2-3 mM. The increase in fluorescence with amino acids always required external Na+. Plots of 1/fluorescence vs. 1/[Na+]medium were also linear with an apparent Km of 29 mM. These apparent Km values compare favorably with those derived from amino acid transport studies using tracers. These data indicate that the Na+-dependent transport of amino acids in these cells is electrogenic.

Amino Acids↗

PubMed1977

Iron uptake by immature erythroid cells. Mechanism of dependence on metabolic energy.

The mechanism by which the utilization of transferrin-bound iron is linked with cellular metabolism was investigated using rabbit reticulocytes and bone marrow cells. The rate of metabolism was altered by the use of inhibitors which act at different sites in the metabolic pathway (NaF, sodium fluoroacetate, rotenone, 2,4-dinitrophenol, NaCN) and by the addition of metabolic substrates (inosine, sodium pyruvate, sodium lactate). Measurements were made of the rates of iron and transferrin uptake and, in many of the experiments of cellular ATP and NADH concentrations. The results showed that there was a significant correlation between the rate of iron uptake and the ATP concentration of the cells, but no correlation was found with the NADH concentration. The rate of transferrin uptake was inhibited to a lesser degree than that of iron uptake, and only when the ATP concentration had fallen below that necessary to inhibit iron uptake. It is concluded that the rate of uptake of transferrin-bound iron by immature erythroid cells is dependent on the intracellular concentration of ATP but is independent of the NADH concentration.

Adenosine Triphosphate↗

PubMed1981

Control of passive permeability of Chinese hamster ovary cells by external and intracellular ATP.

External ATP causes passive permeability change in several transformed cells, but not in untransformed cells. We studied the effect of external ATP on the passive permeability of CHO-K1 cells, a transformed clone of Chinese hamster ovary cells. Treatment of the cells with external ATP alone did not produce a permeability change, and this was observed only when a mitochondrial inhibitor, such as rotenone or oligomycin, was present together with ATP. These inhibitors reduced the concentration of intracellular ATP and a permeability change by external ATP was observed when intracellular ATP was decreased more than 70%. This requirement for permeability change of CHO-K1 cells was quite unique, since passive permeability change of other transformed cells so far tested was induced by ATP alone. Treatment of CHO-K1 cells with cyclic AMP analogues increased their sensitivity to external ATP about 2-fold. The roles of external and intracellular ATP in controlling passive permeability are discussed.

Adenosine Triphosphate↗

PubMed1981

Effects of orthovanadate on salt and water effluxes from the gills of seawater eels, Anguilla anguilla.

Orthovanadate (5 . 10(-7) M) perfused through isolated gills at a constant rate increased the perfusion pressure by 40% but inhibited the effluxes of Na+ and Cl- by 40%. Water efflux was unaltered. Ouabain (10(-4) M) and rotenone (10(-4) M) influenced salt and water effluxes in the same way but did not alter perfusion pressures. Orthovanadate (10(-5) M) perfused at constant rate increased the pressure nearly 2.5-fold; under these conditions effluxes of Na+, Cl- and H2O were all increased approximately 2.5-fold.

Anguilla↗

PubMed1985

Reciprocal effects of phenothiazines and naphthalene sulfonamides on the external ATP-dependent permeability change in Chinese hamster ovary cells.

External ATP causes a great increase in passive permeability to phosphorylated metabolites in several transformed cells, but not in untransformed cells. We have previously demonstrated that the external ATP-dependent permeability change was induced in Chinese hamster ovary cells, CHO-K1, only in the presence of a mitochondrial inhibitor (rotenone, KCN) or a cytoskeleton-attacking agent, vinblastine (Kitagawa, T. and Akamatsu, Y. Biochim. Biophys. Acta 649, 76-82 (1981); 734, 25-32 (1983]. A similar ATP-dependent permeability change was also induced in CHO cells when the cells were treated with 10-30 microM trifluoperazine. This permeability change, like the previously mentioned ones, was found to be reversible and the treated cells remained viable. The permeability change induced by ATP and trifluoperazine was independent of changes in cellular ATP concentration and this property was the same as that of the permeability change with external ATP and vinblastine. Since trifluoperazine is known to interact with calmodulin and to inhibit calmodulin-dependent cellular functions, these results may indicate that calmodulin associated with the cytoskeleton plays an important role in control of the permeability change, although nonspecific perturbation by the drug of the membranes cannot be ruled out. Chlorpromazine and a naphthalene sulfonamide, W-7, also induced an ATP-dependent permeability change. However, these drugs, like mitochondrial inhibitors, reduced the cellular ATP concentration to induce the permeability change. Thus, a clear difference in the action of these drugs in intact cells was also shown in this study. Possible mechanisms for the ATP-dependent permeability change in mammalian cells are discussed.

Adenosine Triphosphate↗

PubMed1991

The use of the potential-sensitive fluorescent probe bisoxonol in mast cells.

The regulation of the plasma membrane potential of rat peritoneal mast cells at the resting state and during activation was investigated using bisoxonol as a potential-sensitive fluorescent dye. Fluorescence microphotography showed that this negatively charged probe was not only present in the plasma membrane, but was also distributed in the cytoplasm. The intracellular localization of bisoxonol was confirmed by conducting experiments which showed that bisoxonol fluorescence was not enhanced in ATP-permeabilized mast cells. Rotenone (10(-7) M) and oligomycin (10(-6) M) did not change the fluorescence of bisoxonol showing, therefore, mitochondrial depolarization was not recorded with bisoxonol and suggesting that bisoxonol may represent a useful probe to study plasma membrane potential changes in the absence of exocytosis. We showed that, in non-stimulated mast cells, the blockade of the sodium pump enhanced the fluorescence of bisoxonol as did gramicidin a non selective ionophore used to fully depolarize the cells. High concentration of potassium (30 mM) as well as different ionic channel blockers did not significantly change the fluorescence intensity of bisoxonol, suggesting that ionic channel permeabilities were not involved in maintaining the resting plasma membrane potential of mast cells. Mast cells stimulated by compound 48/80 completely lost the fluorescence, shown by fluorescence microphotography, suggesting that exocytotic phenomena might induce a dye redistribution which is not only due to changes in the plasma membrane potential. In mast cells pretreated with pertussis toxin, which blocks mast cell-exocytosis, compound 48/80 induced a delayed (2 min) decrease of bisoxonol fluorescence which was shown to be dependent on the activity of the sodium pump. Considering that bisoxonol is a useful potential-sensitive probe in exocytosis-deprived mast cells, our results suggest that the sodium pump is mainly involved in the changes of plasma membrane potential of mast cells.

Adenosine Triphosphate↗

PubMed1993

Intracellular pH regulation in cecal epithelial cells from the chick.

Intracellular pH (pHi) regulation has been investigated in cells isolated from the proximal ceca of the chicken. pHi was measured with the pH-sensitive dye, 2',7'-bis(carboxyethyl)-5 (6)-carboxyfluorescein in nominally HCO(3-)-free solutions. Under resting conditions the pHi was 7.08. Removal of extracellular Na+ decreased pHi by approx. 0.24 pH units and the subsequent addition of Na+ increased pHi towards the control value. This Na(+)-dependent pHi recovery was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (EIPA). Following an intracellular acidification, by abrupt withdrawal of NH4Cl, pHi alkalinized in the nominally absence of Na+. Rotenone, N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, 4-chloro-7-nitrobenz-2-oxa-1,3-diazole, iodoacetic acid and SCH 28080 inhibited the Na(+)-independent pHi recovery rate by 82, 82, 67, 74, 77 and 50% respectively. Bafilomycin A1 was without effect. Na(+)-independent cell alkalization was stimulated by external K+. In the presence of N-ethylmaleimide addition of Na+ induced a rapid pHi recovery. The initial rate of this recovery exhibited first-order dependence on Na+ concentration and it was inhibited by EIPA. The initial rate of Na(+)-dependent cell alkalization increased with a Hill coefficient greater than one when pHi was reduced from 7.2 to 6.2. The 'set point' for the exchanger is approx. 7.5. These studies demonstrate that in cecal epithelial cells exist at least two mechanisms for proton secretion: a Na(+)-H+ exchanger and a Na(+)-independent proton transport system.

4-Chloro-7-nitrobenzofurazan↗

PubMed1988

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues.

Acyl-CoA oxidase, the first enzyme of the peroxisomal beta-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H2O2-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal beta-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of 14C-labeled acid-soluble products from 1-14C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal beta-oxidation than the acyl-CoA oxidase activity on base of H2O2 production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 microM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.

Acyl-CoA Oxidase↗

PubMed1984

2-Methylbutyryl CoA dehydrogenase from mitochondria of Ascaris suum and its relationship to NADH-dependent 2-methylcrotonyl CoA reduction.

Acyl CoA dehydrogenase and electron-transfer flavoprotein have been isolated and partially purified from mitochondria of the anaerobic nematode, Ascaris suum. Dehydrogenase activity was greatest with 2-methylbutyryl CoA and the relative substrate specificities of the ascarid dehydrogenase(s) differ greatly from their mammalian counterparts. It appears that the ascarid dehydrogenase functions physiologically as a reductase, catalyzing the final step in the synthesis of branched-chain fatty acids. In fact, incubations of A. suum mitochondrial membranes with electron-transfer flavoprotein, 2-methylbutyryl CoA dehydrogenase, 2-methylcrotonyl CoA and NADH resulted in a substantial, rotenone-sensitive, 2-methylbutyrate synthesis. These results suggest that the ascarid electron-transport chain and at least two soluble mitochondrial proteins are involved in the NADH-dependent reduction of 2-methylcrotonyl CoA.

Acyl Coenzyme A↗

PubMed1990

Mitochondrial oxidation of phytanic acid in human and monkey liver: implication that Refsum's disease is not a peroxisomal disorder.

The subcellular site of oxidation of [1-14C]phytanic acid to 14CO2 was investigated in human and monkey liver. In both species, this activity was associated with fractions enriched in mitochondria. Fractions enriched in peroxisomes had no detectable phytanic acid oxidase activity. The mitochondrial inhibitors antimycin A and rotenone significantly decreased 14CO2 production in mitochondria-rich fractions from human and monkey liver. These inhibitors also blocked phytanic acid oxidation in cultured human skin fibroblasts. These data suggest that alpha-oxidation of phytanic acid is a mitochondrial rather than a peroxisomal process in primates.

Animals↗

PubMed1991

Quinone mediated electron transport system in the filarial parasite Setaria digitata.

Setaria digitata, a cattle filarial parasite, is known to have peculiarities such as hydrogen peroxide (H2O2) production, cyanide insensitivity, absence of cytochromes and presence of quinones. Estimation of mitochondrial H2O2 with different substrates and inhibitors showed that salicylhydroxamic acid (SHAM), the alternative oxidase inhibitor, inhibited the H2O2 production maximally. Based on the inhibitory studies with rotenone, antimycin A, o-hydroxydiphenyl, SHAM and 2 thenoyltrifluoroacetone, a mechanism for the electron transport is proposed. Quinone Q8 seems to have a central role, hence inhibitors at the level of quinones might prove to be effective in designing drugs for filariasis.

Animals↗

PubMed1991

Conclusive evidence that very-long-chain fatty acids are oxidized exclusively in peroxisomes in human skin fibroblasts.

We have investigated the contribution of peroxisomes and mitochondria to the beta-oxidation of palmitate (C16:0) and cerotate (C26:0) in intact human skin fibroblasts. The oxidation of both fatty acids was found to be inhibited by rotenone plus antimycin and cyanide, respectively, although to a different extent. When 2-[5-(4-chlorophenyl)pentyl]-oxirane-2-carboxylate (POCA) was used to specifically block carnitine palmitoyltransferase I, it was found that palmitate beta-oxidation was inhibited almost completely whereas cerotate beta-oxidation was not affected. Since carnitine palmitoyltransferase is essential for the oxidation of fatty acids in mitochondria this result provides conclusive evidence that oxidation of very-long-chain fatty acids is initiated in peroxisomes and not in mitochondria.

Antimycin A↗

PubMed1992

Quinone dependent NADH dehydrogenation in mitochondria-like particles from Setaria digitata, a filarial parasite.

In the cattle filarial parasite, Setaria digitata, the mitochondria-like particles have been shown to possess site I associated oxidative phosphorylation and rotenone sensitive and insensitive pathways for the dehydrogenation of NADH. Quinone depleted mitochondria-like particles show a loss of activity of these NADH dehydrogenases and also a complete loss of fumarate reductase activity. Reconstitution with quinone restores both NADH linked oxygen uptake and fumarate reductase activity. Thus activities of complex I and fumarate reductase are linked to quinone. Hence an inhibitor at the level of quinone can simultaneously block both aerobic and anaerobic pathways which drive ATP production and may prove useful in the effective control of filariasis.

Animals↗

PubMed1992

Fumarate reductase system of filarial parasite Setaria digitata.

In the cattle filarial parasite Setaria digitata the mitochondria like particles have been shown to possess NADH dependent fumarate reduction coupled with site I electron transport associated phosphorylation. This reduction is catalysed by the fumarate reductase system. The Km for fumarate is 1.47 mM and that for NADH is 0.33 mM. This activity is sensitive to rotenone, antimycin A and o-Hydroxy diphenyl. One ATP is produced for each pair of electrons transferred to fumarate. The fumarate reductase system consisting of NADH-coenzyme Q reductase, cytochrome b like component(s) and succinate dehydrogenase/fumarate reductase is thus very important and hence specific inhibitors of the system may prove useful in the effective control of filariasis.

Animals↗

PubMed1986

The reduction of dithiobis(2-nitrobenzoate) by rat liver mitochondria.

5,5'-Dithiobis(2-nitrobenzoate) (DTNB) is reduced in mitochondrial suspensions to 5-mercapto-2-nitrobenzoate (MNB) by 3-hydroxybutyrate and isocitrate. Although most of the MNB produced is found in the suspension medium, there is also some within the particles. The amount of MNB found in these fraction varies with the DTNB concentration used and is much lower if mitochondrial glutathione (GSH) is depleted with 1-chloro-2,4-dinitrobenzene. If hydroxybutyrate is present, the reduction of DTNB is increased by ATP and oligomycin. The pellet contains only a little MNB and GSH but these are considerably elevated by antimycin and rotenone as well as by ATP and oligomycin. If isocitrate is present, the reduction of DTNB is greatly stimulated by valinomycin, triethyltin and, to a lesser extent, oligomycin. MNB in the pellet falls and GSH concentrations are unchanged. The results suggest that with hydroxybutyrate (an NAD reducing substrate), the rate of reduction of DTNB is limited by the rate of regeneration of GSH while with isocitrate (an NADP reducing substrate) it is limited by the rate of export of MNB from the matrix.

3-Hydroxybutyric Acid↗