scholarly journals Intersubstrate competitions and evidence for compartmentation in mitochondria

1969 ◽  
Vol 113 (4) ◽  
pp. 617-628 ◽  
Author(s):  
E J Harris ◽  
J R Manger
Keyword(s):  
Electrostatic Interaction ◽  
Potassium Salt ◽  
Small Volume ◽  
Dicarboxylic Acids ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Net Charge ◽  
Rate Of Reaction ◽  
Citrate Accumulation

1. The respiration of rat liver mitochondria was compared with different substrates, and with sucrose and saline media. The maximum rates of oxidation obtainable from glutamate, oxoglutarate, glutamate+malate, or succinate were higher in the saline (120mm)–tris (20mm) media than in sucrose (250mm)–tris (20mm) mixtures, but the rate with β-hydroxybutyrate was unchanged. Addition of valinomycin to a medium with sucrose and 5mm-potassium chloride led to rates similar to those measured in saline media; β-hydroxybutyrate oxidation was unaffected. 2. Some pairs of substrates together provided a rate of oxidation greater than the sum of the separate rates. This is accountable if removal of inhibitory products, such as oxaloacetate, compensates for any mutual competition between the substrates. Other pairs showed rates less than the sum of the separate rates, which is accountable by mutual competition. β-Hydroxybutyrate and other substrates, except succinate, provided strictly additive rates; with succinate there was evidence for competition. In the presence of rotenone, succinate oxidation was slowed down by citrate, oxoglutarate (+arsenite) and by β-hydroxybutyrate. 3. The accumulation of substrates in the mitochondria was measured as a function of the concentration and in the presence of possible competitors, or with a potassium salt and valinomycin to induce uptake of K+. The quantities of oxoglutarate, glutamate and pyruvate increased with the mitochondrial K+, but the quantities of β-hydroxybutyrate did not. Most substrates competed between themselves, although citrate accumulation was somewhat increased by oxoglutarate. β-Hydroxybutyrate competed for accumulation only with succinate, and was unaffected by other substrates. β-Hydroxybutyrate accumulation was almost linearly related to applied concentration (up to 5mm), and its rate of reaction was linearly dependent on concentration up to the highest value tested (0·75mm). Hence it differed from other substrates, which are accumulated and oxidized in a manner that follows a saturation law, with Km values about 1–10mm. 4. It is concluded that β-hydroxybutyrate is stored in a compartment operationally distinct from the space containing K+ and the NAD-linked substrates. It seems likely that succinate enters both compartments. 5. The degree of accumulation and the effectiveness of an anion as a competitor (as judged by low Ki) increases with the net charge. This is indicative of an electrostatic interaction with positive sites. It is suggested that the facilitating influence of dicarboxylic acids on the permeation of tricarboxylic acids may be due to the assembling of pairs of the positive carriers by the former, so favouring the chance of there being three or more carriers in a small volume of space near the boundary to interact with the tricarboxylic anion.

scholarly journals The dependence on dicarboxylic acids and energy of citrate accumulation in depleted rat liver mitochondria

1968 ◽  
Vol 109 (2) ◽  
pp. 247-251 ◽  
Author(s):  
E J Harris
Keyword(s):  
Dicarboxylic Acid ◽  
Internal Energy ◽  
Rat Liver ◽  
Dicarboxylic Acids ◽  
Liver Mitochondria ◽  
Organic Anions ◽  
Rat Liver Mitochondria ◽  
Positive Potential ◽  
Citrate Accumulation ◽  
Endogenous Reserves

The accumulation of some organic anions in the space inaccessible to sucrose of rat liver mitochondria was measured. In untreated mitochondria anions were apparently concentrated from 1mm applied concentration by between five- and 22-fold, depending on their charge. After depletion of endogenous reserves either with uncoupling agent or with oligomycin uptakes were decreased. The accumulation of citrate was restored by combinations of a dicarboxylic acid (malate, succinate, maleate or meso-tartrate) and energy. The energy could either be provided by oxidation of a suitable dicarboxylic acid or from ascorbate in the presence of tetramethylphenylenediamine, or from ATP. The restoration of citrate uptake is not necessarily accompanied by a gain of K+, but a cation- and energy-linked citrate uptake can be induced with valinomycin. When citrate is added to mitochondria in the presence of malate the latter is competitively displaced. The anion accumulation could arise from an internal energy-linked positive potential.

Oxidative energy metabolism in germ-free and conventional rat liver mitochondria

1975 ◽  
Vol 228 (2) ◽  
pp. 526-529 ◽  
Author(s):  
DL Sewell ◽  
BS Wostmann ◽  
C Gairola ◽  
MI Aleem
Keyword(s):  
Wistar Rats ◽  
Liver Mitochondria ◽  
The State ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Germ Free ◽  
Adult Rat ◽  
Respiration Rates ◽  
Oxidative Energy Metabolism ◽  
Beta Hydroxybutyrate

The ADP:O ratios and State 3 (ADP-stimulated) and State 4 (controlled) rates of succinate, beta-hydroxybutyrate, isocitrate, glutamate, pyruvate + malate, alpha-ketoglutarate, and ascorbate + N,N,N',N'-tetramethylphenylenediamine (TMPD) oxidation were examined in liver mitochondria from germ-free and conventional rats of both Lobund Wistar (100-day-old) and Fisher (120-day-old) strains. The State 3 respiration rates of isolated mitochondria from germ-free and conventional rats were comparable except for the rate of succinate oxidation in the Wistar rats, which was significantly lower (approx. 10%). The State 4 respiration rates were generally lower in mitochondria isolated from germ-free Fisher rats (approx. 8%) and significantly lower (approx. 18%) in germ-free Wistar rats. The ADP:O ratios were similar in germ-free and conventional rats. Serum thyroxine concentrations indicated delayed maturation of thyroid function in young germ-free rats, but adult animals had concentrations similar to those found in conventional rats. The results indicate that, although absence of a microflora results in a 20-30% reduction in metabolic rate, the germ-free state has little influence on the functional respiration or oxidative phosphorylation of mitochondria isolated from the liver of the adult rat.

Inhibition of Succinic Dehydrogenase and F0F1-ATP Synthase by 4,4'-Diisothiocyanatostilbene-2,2'-disulfonic Acid (DIDS)

1997 ◽  
Vol 52 (11-12) ◽  
pp. 799-806 ◽  
Author(s):  
Celene F. Bernardes ◽  
Jose R. Meyer-Fernandes ◽  
Orlando B. Martins ◽  
Anibal E. Vercesi
Keyword(s):  
Succinic Dehydrogenase ◽  
Atp Synthesis ◽  
Liver Mitochondria ◽  
Disulfonic Acid ◽  
Rat Liver Mitochondria ◽  
Dependent Manner ◽  
Submitochondrial Particles ◽  
Succinate Oxidation ◽  
Hydrolytic Activities ◽  
Dose Dependent

Abstract This study shows that incubation of rat liver mitochondria in the presence of the thiol/ amino reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DID S) is followed by inhibition of both succinate supported respiration and oxidative phosphorylation. Half-maximal inhibition of succinic dehydrogenase activity and succinate oxidation by mitochondria was attained at 55.3 and 60.8 μm DIDS, respectively. DIDS did inhibit the net ATP synthesis and ATP ⇔ [32P]Pi exchange reaction catalyzed by submitochondrial particles in a dose-dependent manner (Ki= 31.7 μm and Ki = 32.7 μm), respectively. The hydrolytic activities of uncoupled heart submitochondrial particles and purified F 1 -ATPase were also inhibited 50% by 31.9 and 20.9 μm DIDS, respectively.

scholarly journals Peroxidative damage of mitochondrial respiration is substrate-dependent

2009 ◽  
pp. 685-692
Author(s):  
R Endlicher ◽  
P Křiváková ◽  
H Rauchová ◽  
H Nůsková ◽  
Z Červinková ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Peroxidative Damage ◽  
Palmitoyl Carnitine ◽  
Low Concentrations ◽  
Tert Butyl Hydroperoxide ◽  
Strong Inhibitory Effect

The concentration-dependence of tert-butyl hydroperoxide (BHP) inhibitory effect on oxygen consumption in isolated rat liver mitochondria was measured in the presence of various respiratory substrates. Strong inhibitory effect at low concentrations of BHP (15-30 µM) was found for oxoglutarate and palmitoyl carnitine oxidation. Pyruvate and glutamate oxidation was inhibited at higher concentrations of BHP (100-200 µM). Succinate oxidation was not affected even at 3.3 mM BHP. Determination of mitochondrial membrane potential has shown that in the presence of NADH-dependent substrates the membrane potential was dissipated by BHP but was completely restored after addition of succinate. Our data thus indicate that beside peroxidative damage of complex I also various mitochondrial NADH-dependent dehydrogenases are inhibited, but to a different extent and with different kinetics. Our data also show that succinate could be an important nutritional substrate protecting hepatocytes during peroxidative damage.

THE INHIBITION OF OXIDATIVE AND PHOSPHORYLATIVE ENZYMES IN RAT LIVER MITOCHONDRIA BY AMINOAZOBENZENE DERIVATIVES

1960 ◽  
Vol 38 (1) ◽  
pp. 1-11 ◽  
Author(s):  
W. C. McMurray
Keyword(s):  
Cytochrome C ◽  
Rat Liver ◽  
Pyridine Nucleotide ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Metabolic Block ◽  
Liver Carcinogen ◽  
Mitochondrial Dehydrogenase Activity

The liver carcinogen, dimethylaminoazobenzene, inhibited in vitro the oxidation of a variety of pyridine nucleotide linked substrates of rat liver mitochondria without affecting the process of oxidative phosphorylation. Cytochrome c oxidase activity was not inhibited by the carcinogen, nor was the succinoxidase activity, but the phosphorylation accompanying succinate oxidation was uncoupled. Similar effects were noted with other aminoazobenzene derivatives, but did not appear to be correlated with the ability of the compounds to evoke tumors.The site of the respiratory inhibition by dimethylaminoazobenzene appears to be at the level between reduced pyridine nucleotide and cytochrome c in the respiratory chain. Mitochondrial dehydrogenase activity was not inhibited, while the oxidation of reduced diphosphopyridine nucleotide was markedly decreased. The reduction of the electron acceptor, ferricyanide, by pyridine nucleotide linked substrates was also strongly inhibited but the reduction of tetrazolium compounds was not affected. The latter observations suggest that dimethylaminoazobenzene produces a metabolic block between reduced flavin and cytochrome c in the mitochondrial electron transport system.

scholarly journals The inhibition of pyruvate and ls(+)-isocitrate oxidation by succinate oxidation in rat liver mitochondria

1969 ◽  
Vol 114 (3) ◽  
pp. 589-596 ◽  
Author(s):  
T. König ◽  
D. G. Nicholls ◽  
P. B. Garland
Keyword(s):  
Cytochrome B ◽  
Rat Liver ◽  
Rat Heart ◽  
Fold Increase ◽  
Liver Mitochondria ◽  
Inhibitory Effect ◽  
The Other ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Rapid Flow

1. The effects of succinate oxidation on pyruvate and also isocitrate oxidation by rat liver mitochondria were studied. 2. Succinate oxidation was without effect on pyruvate and isocitrate oxidation when respiration was maximally activated with ADP. 3. When respiration was partially inhibited by atractylate, succinate oxidation severely inhibited the oxidation of pyruvate and isocitrate. 4. This inhibitory effect of succinate was associated with a two- to three-fold increase in the reduction of mitochondrial NAD+ but no change in the reduction of cytochrome b. 5. It is concluded that, in the partially energy-controlled state, respiration is more severely inhibited at the first phosphorylating site than at the other two. 6. The effects of succinate oxidation are compared with those of palmitoylcarnitine oxidation. It is concluded that a rapid flow of electrons directly into the respiratory chain at the level of cytochrome b is in itself inadequate to inhibit the oxidation of intramitochondrial NADH. 7. The effects of succinate oxidation on pyruvate oxidation were similar in rat heart and liver mitochondria.

scholarly journals The nature of the stimulation of the respiratory chain of rat liver mitochondria by glucagon pretreatment of animals

1982 ◽  
Vol 204 (1) ◽  
pp. 37-47 ◽  
Author(s):  
A P Halestrap
Keyword(s):  
Respiratory Chain ◽  
Electron Flow ◽  
Liver Mitochondria ◽  
Complex Iii ◽  
Secondary Site ◽  
Rat Liver Mitochondria ◽  
Succinate Oxidation ◽  
Q Cycle ◽  
Cytochromes C ◽  
Stimulation Of

1. Studies on the cytochrome spectra of liver mitochondria from control and glucagon-treated rats in State 4, State 3 and in the presence of uncoupler are reported. 2. The stimulation of electron flow between cytochromes c1 and c observed previously [Halestrap (1978) Biochem. J. 172, 399-405] was shown to be an artefact of Ca2+-induced swelling of mitochondria. 3. When precautions were taken to prevent such swelling, glucagon treatment was shown to enhance the reduction of cytochromes c, c1 and b558 in both State 3 and uncoupled conditions with either succinate or glutamate + malate as substrate. An increase in the reduction of cytochromes b562 and b566 was also seen in some, but not all, experiments. 4. In State 4 with succinate but not glutamate + malate as substrate, cytochromes c, c1, b558, b562 and b566 showed increased reduction. 5. Glucagon stimulated oxidation of duroquinol and palmitoylcarnitine by intact mitochondria and of NADH by disrupted mitochondria. 6. No effect of glucagon on succinate dehydrogenase activity or the temperature-dependence of succinate oxidation could be detected. 7. Glucagon enhanced the inhibition of the respiratory chain by colletotrichin, but not antimycin or 8-heptyl-4-hydroxyquinoline N-oxide. 8. These results are interpreted in terms of a primary stimulation by glucagon of the ‘Q cycle’ [Mitchell (1976) J. Theor. Biol. 62, 827-367] within Complex III (ubiquinol:cytochrome c oxidoreductase) and a secondary site of action involving stimulation of electron flow into Complex III from the ubiquinone pool. 9. Ageing of mitochondria, hyperosmotic treatment or addition of 20 mM-benzyl alcohol opposed the effects of glucagon treatment on cytochrome spectra and colletotrichin inhibition of respiration. 10. These results support the hypothesis that glucagon exerts its effects on the mitochondria by perturbing the membrane structure.

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