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PubMed1990

A rapid bioassay for chemicals that induce pro-oxidant states.

A new bioassay has been developed that allows rapid, sensitive detection of chemicals such as paraquat and adriamycin, which manifest their acute toxicity, mutagenicity or carcinogenicity by inducing a pro-oxidant state in vivo. Submitochondrial particles isolated from bovine myocardium are used to catalyze NADH-dependent enzymatic reduction of these chemicals to free radicals. The highly reactive species generated in this system reduce molecular dioxygen to the superoxide anion radical, which is detected spectrophotometrically using the adrenochrome reaction. The anticancer drug adriamycin, the herbicides paraquat and diquat, the analytical dye sulfonazo III, and the experimental carcinogen 4-nitroquinoline-N-oxide have been used to test the sensitivity of this new method. This assay can be used to screen fresh water samples for the presence of pollutants that can generate oxygen-centered free radicals in vivo, or to test newly synthesized chemicals for this activity, and may therefore be valuable for environmental monitoring and preliminary toxicity evaluation of industrial or pharmaceutical products.

4-Nitroquinoline-1-oxide↗
PubMed1994

Binding of malate dehydrogenase and NADH channelling to complex I.

As previously reported, mitochondrial malate dehydrogenase (MDH) binds to purified complex I of the electron transport system. With conditions used in previous reports, MDH binds even more extensively, but probably predominantly non-specifically, to the matrix side of the inner mitochondrial membrane of submitochondrial particles (SMP). Herein we report experimental conditions for highly specific binding of malate dehydrogenase to complex I within SMP. These conditions permit us to demonstrate NADH channelling from malate dehydrogenase to complex I using the competing reaction test. This test, though not ideal for all situations, has several advantages over the enzyme buffering test previously used. These advantages should facilitate further studies elucidating NADH channelling to complex I from MDH and other dehydrogenases. Independent evidence of NADH channelling to the electron transport chain and the potential advantages of substrate channelling in general are also discussed. Substrate channelling from MDH in particular may be especially beneficial because of the unfavourable equilibrium and kinetics of this enzyme reaction.

Animals↗
PubMed1975

Energy conservation and uncoupling in mitochondria.

Energy conservation and uncoupling in mitochondria are examined in the light of three important new findings: (a) Studies with the photoaffinity-labeling uncoupler 2-azido-4-nitrophenol have shown that mitochondria contain a specific uncoupler binding site (apparently a polypeptide of Mr = 30,000 +/- 10%). (b) This site fractionates into an enzyme complex (complex V), which is capable of oligomycin- and uncoupler-sensitive ATP-Pi exchange. It is absent from electron transfer complexes I, III, and IV, which represent segments of the respiratory chain containing coupling sites 1, 2, and 3, respectively. (c) Trinitrophenol is a membrane-impermeable uncoupler (uncouples submitochondrial particles, but not mitochondria) and a poor protonophore. There is an excellent correlation between the uncoupling potencies and the affinities of uncouplers for the mitochondrial uncoupler-binding site. There is no correlation between uncoupling potency and protonophoric activity of uncouplers when a membrane-permeable uncoupler is compared with a membrane-impermeable one.

Adenosine Triphosphate↗
PubMed1975

Exploring sites on mitochondrial ATPase for catalysis, regulation, and inhibition.

Evidence is presented that mitochondrial ATPase has two types of sites that bind adenine nucleotides. The catalytic site, C, binds the substrates ATP, GTP, or ITP and the inhibitor guanylyl imidodiphosphate (GMP-PNP). A second type of site, R, binds ATP, ADP, adenylyl imidodiphosphate (AMP-PNP), and the chromium complexes of ATP or ADP. All of these substances binding to the R site inhibit the hydrolysis of ATP in a competitive manner; their inhibition of hydrolysis of ITP and GTP is noncompetitive. GMP-PNP inhibits oxidative phosphorylation in submitochondrial particles but AMP-PNP does not. The localization on mitochondrial membranes of sites for the binding of various antibiotics that inhibit oxidative phosphorylation is discussed.

Adenosine Diphosphate↗
PubMedDate not supplied

Vanadate inhibition of mitochondrial respiration and H+ ATPase activity in Saccharomyces cerevisiae.

The effects of vanadate on mitochondrial respiration and H+ ATPase activity in Saccharomyces cerevisiae were studied. A 50% inhibition of oxygen uptake in isolated mitochondria was produced by 4.4 mM-V2O5. Activity of H+ ATPase in whole mitochondria was inhibited by 50% by 5.5 microM-V2O5, in submitochondrial particles by 55 microM-V2O5; and in the chloroform-released H+ ATPase by 0.5 mM-V2O5. Vanadate was also found to relieve growth inhibition caused by the mitochondrial H+ ATPase inhibitors NN'-decyclohexylcarbodiimide and oligomycin. These results imply that vanadate could affect mitochondrial respiration by interacting with the H+ ATPase in S. cerevisiae.

Adenosine Triphosphatases↗
PubMed1996

Mutations at histidine 211 of the yeast F1-ATPase beta-subunit affect the stability and assembly of the ATPase and the structure of the active site.

The role of H211 of the yeast F1-ATPase beta-subunit was investigated by site-specific mutagenesis and characterization of the resulting enzymes. Five amino acids (N, D, I, K, and A) were substituted for H211 of the ATP2 gene. The mutated genes were expressed in an atp2::LEU2 host, and only yeast expressing H211N respired aerobically. The respiratory phenotypes of the other four mutants were suppressed by a second site mutation (L203F). The ATPases from the single mutant strains were unstable when removed from the mitochondrial inner membrane, preventing purification. Submitochondrial particles were prepared from each strain and the activities were stable under a variety of conditions, allowing determination of Vmax and Km for ATP hydrolysis. Mutations of H211 caused increases in Km of 3.7- to 7.4-fold, while L203F had little effect. The suppressive effect of the L203F mutation was also expressed in the Km values of the double mutant strains. The ATPases from the H211 mutants had diminished sensitivity to oligomycin, and their pH optima were 1.5-2.0 units less than the wild-type optimum. Values of pKa for the groups involved in catalysis were estimated for the wild-type enzyme and three H211 mutants (N, D, and K). Each mutant enzyme showed a substantial decrease in the pKa of the group(s) which serves as a base in acid-base catalysis. The results of this study demonstrate that H211 is important in maintaining the structure of the wild-type enzyme complex and also contributes to the structure of the active site. L203 is also required for the stability of the enzyme complex and may have a structural or functional interaction with H211. Neither H211 nor L203 is required for catalysis by F1.

Binding Sites↗
PubMed1997

Changes of energy metabolism induced by 1-methyl-4-phenylpyridinium (MPP+)-related compounds in rat pheochromocytoma PC12 cells.

We examined effects of three structurally related pyridinium compounds, 1-methyl-4-phenylpyridinium (MPP+), paraquat, and 1-methyl-4-(4'-nitrophenyl) pyridinium (analog 1), on the energy metabolism in pheochromocytoma PC12 cells. MPP+ inhibited the intracellular NADH oxidation by the mitochondrial respiratory chain, judging from the decrease of the cytosolic NAD+/NADH ratio. Paraquat enhanced the oxidation of NADH and decreased intracellular ATP more than MPP+. The inhibition of the mitochondrial respiration by MPP+ was partially compensated by enhanced glycolysis, while paraquat inhibited glycolysis at the level of hexokinase probably due to the intracellular production of oxygen radicals. Analog 1 moderately enhanced glycolysis, moderately increased a cytosolic ratio of NAD+/NADH, and caused only a slight decline of intracellular ATP. Paraquat was the most cytotoxic of the three compounds. Thus, the three structurally related compounds, MPP+, paraquat, and analog 1, showed different effects on the mitochondrial respiratory chain and the glycolytic pathway in PC 12 cells. Their properties found in the cells well reflected those obtained by using bovine heart submitochondrial particles.

Adenosine Triphosphate↗
PubMed1998

3,5,3'-triiodothyronine induces mitochondrial permeability transition mediated by reactive oxygen species and membrane protein thiol oxidation.

Ca(2+)-loaded rat liver mitochondria treated with 3,5,3'-triiodothyronine (T3) undergo nonspecific inner membrane permeabilization, as evidenced by mitochondrial swelling, a decrease in membrane potential (delta psi), and an increase in the rate of oxygen uptake. T3 analogues thyroxine (T4), 3',5'-diiodothyronine (T2), and 3,5',3'-triiodothyronine (reverse T3), in decreasing order of potency, resulted in a similar but less extensive effect. Permeabilization induced by T3 is dependent on Ca2+ (1 microM) and T3 (0.5-25 microM) concentrations and is inhibited by cyclosporin A, a known inhibitor of mitochondrial permeability transition. Catalase or dithiothreitol also prevents membrane permeabilization, suggesting the participation of membrane protein thiol group oxidation induced by reactive oxygen species. The determination of the mitochondrial membrane protein thiol group content after treatment with Ca2+ and T3 shows a significant decrease, due to thiol oxidation. When mitochondria are incubated in the presence of inorganic phosphate and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, mitochondrial swelling still occurs after treatment with T3 and high Ca2+ concentrations, suggesting that mitochondrial permeabilization is not dependent on T3-induced delta psi or matrix pH alterations. Under these experimental conditions, when no oxygen is present in the incubation medium, no permeabilization occurs, suggesting that the permeabilization is dependent on mitochondrial-generated reactive oxygen species. Confirming this hypothesis, superoxide generation in a suspension of submitochondrial particles is increased when T3 is present. Our results lead to the conclusion that T3 induces a situation of oxidative stress in isolated liver mitochondria, with Ca(2+)-mediated membrane protein thiol oxidation and nonspecific inner membrane permeabilization.

Animals↗
PubMed1999

Mitochondrial superoxide decreases yeast survival in stationary phase.

Yeast lacking mitochondrial superoxide dismutase (MnSOD) display shortened stationary-phase survival and provide a good model system for studying mitochondrial oxidative damage. We observed a marked decrease in respiratory function preceding stationary-phase death of yeast lacking MnSOD (sod2Delta). Agents (mitochondrial inhibitors) that are known to increase or decrease superoxide production in submitochondrial particles affected stationary-phase survival in a manner inversely correlated with their effects on superoxide production, implicating superoxide in this mitochondrial disfunction. Similar but less-dramatic effects were observed in wild-type yeast. The activities of certain mitochondrial enzymes were particularly affected. In sod2Delta yeast the activity of aconitase, a 4Fe-4S-cluster-containing enzyme located in the matrix, was greatly and progressively decreased as the cells established stationary phase. Succinate dehydrogenase activity also decreased in MnSOD mutants; cytochrome oxidase and ATPase activities did not. Aconitase could be reactivated by addition of materials required for cluster assembly (Fe3+ and a sulfur source), both in extracts and in vivo, indicating that inactivation of the enzyme was by disassembly of the cluster. Our results support the conclusion that superoxide is generated in the mitochondria in vivo and under physiological conditions and that MnSOD is the primary defense against this toxicity. When the balance between superoxide generation and MnSOD activity is disrupted, superoxide mediates iron release from mitochondrial iron-sulfur clusters, leading first to loss of mitochondrial function and then to death, independently of mtDNA damage. These results raise the possibility that similar processes may occur in higher eukaryotes.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗
PubMed1994

Estimation of redox properties of chemical compounds by their reactions with free radicals.

The electron-donor and electron-acceptor properties of biologically active compounds were estimated on the basis of their reactions with free radical intermediates of the excited dye eosin. After dye excitation by a short flash of light, the kinetics of dye anion radicals in the pure dye solution were compared with those in the presence of the natural reducing agent NADH or a number of substances which are known as local anesthetics (LA). LA individually slowed the decay of dye anion radicals and enhanced their concentration to a lesser extent than NADH, thus acting as electron donors toward dye radicals. The electron-donor properties of LA studied correlate with their ability to block sodium channels. In contrast, the sodium channel agonist veratridine decreased the concentration of dye anion radicals, thus showing electron-acceptor properties. The opposite redox features of the channel antagonist and the agonist were revealed also by a modified method suitable for steady-light spectroscopy. Promoted by NADH, eosin photobleaching was slowed by the electron acceptor ferricyanide as well as by veratridine and some phenols. The electron-acceptor properties of the latter compounds correlated with their ability to inhibit ATP synthesis by submitochondrial particles. Thus, these methods may be used to evaluate the redox properties of compounds not characterized by electrochemical methods and to predict their biological activity.

Biological Factors↗
PubMed1997

The mitochondrial sulfonylurea receptor: identification and characterization.

Biochemical identification of mitochondrial sulfonylurea receptors has been carried out through binding studies performed with [3H]glibenclamide. The presence of a single class of low affinity binding sites for glibenclamide in the inner mitochondrial membrane has been found, with a KD of 360 +/- 48 nM and BMAX of 48 +/- 7 pmoles/mg in beef heart mitochondria. Glibenclamide binding was affected by other sulfonylureas (glipizide, glisoxepide) but not by potassium channel openers (diazoxide, pinacidil, RP66471). In both rat liver and beef heart mitochondria adenine nucleotides (ATP, ADP, AMP) and nucleotide analogs (triazine dyes) produced large inhibition (from 60 to 80%) of [3H]glibenclamide binding. Photoaffinity labeling of submitochondrial particles with [125I]-glibenclamide revealed a single specifically labeled polypeptide band of 28 kDa by SDS-PAGE that is postulated to be (or to form a part of) the mitochondrial sulfonylurea receptor.

ATP-Binding Cassette Transporters↗
PubMed1998

Two subunits of the F0F1-ATPase are phosphorylated in the inner mitochondrial membrane.

Inside-out submitochondrial particles from potato tuber mitochondria were incubated with [gamma-32P]ATP. More than 16 phosphorylated polypeptides were detected by autoradiography on an SDS-gel. Two phosphoproteins, migrating at 22 and 28 kDa, were excised from the SDS-gel, electroeluted, and purified further by anion chromatography. The phosphoproteins were N-terminally sequenced. Over the regions sequenced, the 22 and 28 kDa phosphoproteins had 100% sequence identity with potato proteins identified as the delta'-subunit of the F1-ATPase and the b-subunit of the F0-ATPase, respectively. We suggest that phosphorylation of these proteins may control the interaction between F1 and F0 and regulate energy coupling in oxidative phosphorylation.

Adenosine Triphosphate↗
PubMed1999

Biochemical characterization of lucigenin (Bis-N-methylacridinium) as a chemiluminescent probe for detecting intramitochondrial superoxide anion radical production.

Direct detection of intramitochondrial superoxide anion radical (O(-*)(2)) production is of critical importance for investigating the pathophysiological consequences resulting from altered cellular reactive oxygen homeostasis. The purpose of this study with isolated mitochondria was to characterize the biochemical basis for lucigenin as a chemiluminescent probe to detect intramitochondrial O(-*)(2) production. Incubation of isolated mitochondria with lucigenin at non-redox cycling concentration produced lucigenin-derived chemiluminescence (LDCL), which was increased markedly by mitochondrial substrates, pyruvate/malate or succinate. The LDCL was reduced greatly by the membrane permeable superoxide dismutase (SOD) mimetics, 2,2,6,6-tetramethylpiperidine-N-oxyl and Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin, but not by Cu,Zn-SOD. With an ion-pair HPLC method, a concentration-dependent accumulation of lucigenin was detected within mitochondria. The accumulation of lucigenin by mitochondria was reduced markedly in the presence of carbonyl cyanide p-(trifluoromethoxy)phenyhyldrazone, an uncoupler known to dissipate the mitochondrial membrane potential. With submitochondrial particles, we observed that both complexes I and III of the mitochondrial electron transport chain appear to be able to catalyze the one electron reduction of lucigenin, a critical step involved in LDCL. After incubation of mitochondria with lucigenin at non-redox cycling concentrations, formation of N-methylacridone, the proposed end product of the reaction pathway leading to LDCL, within the mitochondrial fraction was also detected. In addition, a significant linear correlation was observed between the LDCL and either the lucigenin accumulation or the N-methylacridone formation within the mitochondria. Taken together, our results conclusively demonstrate that when properly used LDCL can reliably detect intramitochondrial O(-*)(2) production.

Acridines↗
PubMed1999

Two separate transhydrogenase activities are present in plant mitochondria.

Inside-out submitochondrial particles from both potato tubers and pea leaves catalyze the transfer of hydride equivalents from NADPH to NAD(+) as monitored with a substrate-regenerating system. The NAD(+) analogue acetylpyridine adenine dinucleotide is also reduced by NADPH and incomplete inhibition by the complex I inhibitor diphenyleneiodonium (DPI) indicates that two enzymes are involved in this reaction. Gel-filtration chromatography of solubilized mitochondrial membrane complexes confirms that the DPI-sensitive TH activity is due to NADH-ubiquinone oxidoreductase (EC 1.6.5.3, complex I), whereas the DPI-insensitive activity is due to a separate enzyme eluting around 220 kDa. The DPI-insensitive TH activity is specific for the 4B proton on NADH, whereas there is no indication of a 4A-specific activity characteristic of a mammalian-type energy-linked TH. The DPI-insensitive TH may be similar to the soluble type of transhydrogenase found in, e.g., Pseudomonas. The presence of non-energy-linked TH activities directly coupling the matrix NAD(H) and NADP(H) pools will have important consequences for the regulation of NADP-linked processes in plant mitochondria.

Chromatography, Gel↗
PubMed2002

Antioxidant and prooxidant activities of alpha-lipoic acid and dihydrolipoic acid.

Reactive oxygen (ROS) and nitrogen oxide (RNOS) species are produced as by-products of oxidative metabolism. A major function for ROS and RNOS is immunological host defense. Recent evidence indicate that ROS and RNOS may also function as signaling molecules. However, high levels of ROS and RNOS have been considered to potentially damage cellular macromolecules and have been implicated in the pathogenesis and progression of various chronic diseases. alpha-Lipoic acid and dihydrolipoic acid exhibit direct free radical scavenging properties and as a redox couple, with a low redox potential of -0.32 V, is a strong reductant. Several studies provided evidence that alpha-lipoic acid supplementation decreases oxidative stress and restores reduced levels of other antioxidants in vivo. However, there is also evidence indicating that alpha-lipoic acid and dihydrolipoic acid may exert prooxidant properties in vitro. alpha-Lipoic acid and dihydrolipoic acid were shown to promote the mitochondrial permeability transition in permeabilized hepatocytes and isolated rat liver mitochondria. Dihydrolipoic acid also stimulated superoxide anion production in rat liver mitochondria and submitochondrial particles. alpha-Lipoic acid was recently shown to stimulate glucose uptake into 3T3-L1 adipocytes by increasing intracellular oxidant levels and/or facilitating insulin receptor autophosphorylation presumably by oxidation of critical thiol groups present in the insulin receptor beta-subunit. Whether alpha-lipoic acid and/or dihydrolipoic acid-induced oxidative protein modifications contribute to their versatile effects observed in vivo warrants further investigation.

Animals↗
PubMed1996

Heart FoF1-ATPase changes during the acute phase of Trypanosoma cruzi infection in rats.

The kinetic properties of ATP hydrolysis and synthesis by FoF1-ATPase of heart mitochondria were evaluated during the acute phase of T. cruzi infection in rats. Mitochondria and submitochondrial particles were isolated 7 days (early stage) and 25 days (late stage) following infection of rats with 2 x 10(5) trypomastigote forms of the Y strain of T. cruzi. The kinetic properties for ATP hydrolysis were altered for the early but not the late stage, showing a changed pH profile, increased K0.5 values, and a decreased total Vmax. The Arrhenius' plot for membrane-associated enzyme showed a higher transition temperature with a lower value for the activation energy in body temperature. For the Triton X-100-solubilized enzyme, the plot was similar to the control. A decrease in the efficiency of ADP phosphorylation by mitochondria, measured by the firefly-luciferase luminescence, was observed only during the late stage and appeared to be correlated with a decrease in the affinity of the FoF1-ATPase for ADP. It is proposed that in the early stage, during the acute phase of T. cruzi infection in rats, heart FoF1-ATPase undergoes a membrane-dependent conformational change in order to maintain the phosphorylation potential of mitochondria, which would compensate for the uncoupling of mitochondrial function. Also, during both the early and late stages, the enzyme seems to be under the regulation of the endogenous inhibitor protein for the preservation of cellular ATP levels.

Acute Disease↗
PubMed1983

Binding of 99mTc-gluconate to heart mitochondria.

99mTc-gluconate has previously been shown to bind to isolated rat heart mitochondria. In the presence of potassium cyanide this binding is enhanced several fold, an effect which has been shown to be correlated to the efficiency of the in vivo binding of the isotope complex in ischemic dog hearts. The present investigation shows that the potassium cyanide-induced binding was mainly localized to the mitochondrial inner membrane; in the presence of cyanide other organelles, e.g., nuclei and liver microsomes also showed some binding. Boiling, lipid extraction, or addition of N-ethylmaleimide caused a variable inhibition of the binding of 99mTc-gluconate to rat heart mitochondria, which also was markedly influenced by temperature, pH, time, and concentrations of protein and isotope complex, but not by conditions affecting energy levels or calcium transport. Fractionation of submitochondrial particles exposed to 99mTc-gluconate in the presence of potassium cyanide indicated that the 99mTc-gluconate-binding component cofractionated with cytochrome oxidase. It is postulated that a protein component localized in the mitochondrial inner membrane, possibly cytochrome oxidase, is responsible for the binding of 99mTc-gluconate.

Animals↗
PubMed1984

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase.

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimyristoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18-28 degrees C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots of Km also show breaks in the same temperature interval, with the exception of the dioleoyl-enzyme vesicles, where Km is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24 degrees C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3-18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.

Animals↗