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At least 847 records · Page 47Linked to original sources
PubMed1983

Sulphate ion-induced slow transformation of succinate dehydrogenase.

A new type of slow change of succinate dehydrogenase (EC 1.3.99), activity which is induced by sulphate ion is described. After preincubation of submitochondrial particles or soluble succinate dehydrogenase with sulphate both preparations catalyze succinate:phenazine methosulphate reductase reaction with a significant lag. When added to the assay medium sulphate ion induces biphasic time-dependent competitive inhibition of the enzyme. The sulphate-induced inhibition is apparently due to a rapid interaction of the anion with an active site of the enzyme which is followed by a slow pH-dependent (pKa = 7.2) transformation of the enzyme-inhibitor complex. pH profiles of the overall succinate dehydrogenase reaction and of equilibrium between fast and slow enzyme-sulphate complexes suggest that the same protolytic equilibrium step is involved in the formation of an active intermediate and an inactive enzyme-sulphate complex.

Animals↗
PubMed1981

The partially reduced species present in purified cytochrome oxidase from baker's yeast is cytochrome a.

Cytochrome oxidase purified from baker's yeast submitochondrial particles is found to exist in a partially reduced state in the resting enzyme. Studies utilizing optical and EPR spectroscopy indicate that the "inactive" fraction contains a reduced low-spin heme, cytochrome a possibly indicating a block of electron transfer from cytochrome a to cytochrome a 3. There is no apparent reduction of either the EPR-detectable copper or the species associated with the 830 nm band. Oxidative titrations of the resting-state yeast cytochrome oxidase indicate that the reduction potential of the species titrating is higher than that of ferricyanide. This "inactive" cytochrome oxidase is not the result of the isolation procedure, but seems to represent a species which is present in the intact yeast.

Cytochrome a Group↗
PubMed2003

Structural and functional organization of Complex I in the mitochondrial respiratory chain.

Metabolic flux control analysis of NADH oxidation in bovine heart submitochondrial particles revealed high flux control coefficients for both Complex I and Complex III, suggesting that the two enzymes are functionally associated as a single enzyme, with channelling of the common substrate, Coenzyme Q. This is in contrast with the more accepted view of a mobile diffusable Coenzyme Q pool between these enzymes. Dilution with phospholipids of a mitochondrial fraction enriched in Complexes I and III, with consequent increased theoretical distance between complexes, determines adherence to pool behavior for Coenzyme Q, but only at dilution higher than 1:5 (protein:phospholipids), whereas, at lower phospholipid content, the turnover of NADH cytochrome c reductase is higher than expected by the pool equation.

Animals↗
PubMed1980

The prosthetic groups in succinate dehydrogenase. Number and stoichiometry.

I. Succinate:Q oxidoreductase (EC 1.3.99.1) as present in beef-heart submitochondrial particles contains equal amounts of FAD, a [2Fe-2S] cluster and a [4Fe-4S] cluster. Both Fe-S clusters are reducible by succinate. 2. A second type of [2Fe-2S] cluster, called center S-2, that has been proposed to be present in purified preparations of succinate dehydrogenase and isolated Complex II (Ohnishi, T., Winter, D.B., Lim, J. and King, T.E. (1973) Biochem. Biophys. Res. Commun. 53, 231--237) is an artifact introduced by the purification procedure. 3. It is suggested that the 70 000 dalton subunit which is known to bind the flavin, accomodates also the [4Fe-4S] cluster whereas the 28 000 dalton subunit contains the [2Fe-2S] cluster.

Animals↗
PubMed1993

Mechanism of cytotoxicity of paraquat. II. Organ specificity of paraquat-stimulated lipid peroxidation in the inner membrane of mitochondria.

The production of superoxide anion (O2-) statistically increased, stimulated by paraquat (1,1'dimethyl-4,4'-bipyridylium dichloride) in lung, liver, kidney and heart submitochondrial particles (SMP) isolated from rats given paraquat intravenously. Paraquat also stimulated O2- production in bovine liver NADH-ubiquinone oxidoreductase (complex I). The reaction mixture used in these assays turned its color into blue proving the occurrence of paraquat free radicals. The pH optimum for NADH dependent O2- production with paraquat was 9.5. O2- production was stimulated by paraquat even in the presence of rotenone, one of the mitochondrial respiratory chain inhibitors. The lipid peroxidation increased in lung SMP but not in heart SMP of paraquat-treated rats. These results may suggest that paraquat was reduced by complex I, but there was difference in the lipid peroxidation by the paraquat radical between rat lung and heart.

Animals↗
PubMed1999

Molecular characterisation of a NADH ubiquinone oxidoreductase subunit 5 from Schistosoma mansoni and inhibition of mitochondrial respiratory chain function by testosterone.

Complementary DNA, encoding the mitochondrial enzyme NADH-ubiquinone oxidoreductase subunit 5 (SmND5) of the human parasite Schistosoma mansoni was isolated by screening a S. mansoni cDNA library with a human androgen receptor (hAR) cDNA probe. The complete nucleotide and deduced aminoacid sequences of SmND5 were determined. Southern blot analysis revealed the occurrence of a single copy gene for SmND5 and by means of RT-PCR, it was shown that sex- and stage-specific expression of SmND5 occurred. In order to establish a functional relationship between the mitochondrial enzyme and the androgen receptor, the effects of testosterone were compared to those of classical respiratory chain inhibitors, using adult schistosome and beef heart submitochondrial particles. Physiological concentrations of testosterone were able to inhibit the maintenance of proton gradient across the mitochondrial membranes, as well as ATP synthesis. The steroid was found to be cytotoxic to the larvae, but not to adult schistosomes. A model is proposed to explain the observed in vivo testosterone-related differences in worm burdens, in experimental chronic infections.

Amino Acid Sequence↗
PubMed1984

Clathrin-coated vesicles from rat liver: enzymatic profile and characterization of ATP-dependent proton transport.

Clathrin-coated vesicles isolated from rat liver exhibited an enzymatic profile distinct from that of rat liver plasma membranes, lysosomes, microsomes, and mitochondria. The coated vesicles catalyzed ATP-dependent proton transport that acidified the vesicle interior, as measured by the fluorescence quenching of acridine orange. H+ transport by coated vesicles was not inhibited by vanadate (0.1 mM) or ouabain (2 mM) and differed from H+ transport by rat liver submitochondrial particles in its greater resistance to inhibition by oligomycin (10 pM to 10 microM) and N,N'-dicyclohexylcarbodiimide (DCCD) (0.1-100 microM) and its sensitivity to N-ethylmaleimide (0.1-2 mM). H+ transport was stimulated by valinomycin in the presence of K+, exhibited no specific cation requirement, but was dependent upon the presence of a permeant anion, with Cl- and Br- being the most effective of the anions studied. Finally, H+ transport was poorly supported by GTP, UTP, or ADP and exhibited no consistent relationship to the coated vesicle-associated ouabain-insensitive ATPase activity.

Adenosine Triphosphatases↗
PubMed1981

On the site of action of the inhibition of the mitochondrial respiratory chain by lipoxygenase.

Treatment of beef-heart submitochondrial particles with reticulocyte lipoxygenases gives rise to a strong irreversible inhibition of the NADH and succinate oxidase activities. This is not accompanied by any loss of the Fe-S clusters of the respiratory chain as determined by EPR spectroscopy. The inhibitory blockage is located between both the NADH and succinate dehydrogenases and Q-10. The inhibitory action of treatment with lipoxygenase also takes place in the absence of Q-10. The Fe-S clusters of the mitochondrial outer membrane are destroyed by lipoxygenase treatment, without any effect on the rotenone-insensitive NADH: cytochrome c oxidoreductase activity. It is concluded that these clusters are not involved in this enzyme.

Animals↗
PubMed1985

Effect of ischemia on heart submitochondrial superoxide production.

NADH-dependent formation of superoxide anions (O2-) by rabbit cardiac submitochondrial particles (SMP) was stimulated after exposure of the isolated heart to 90 min of ischemic perfusion. This effect was more evident in the rotenone-inhibited region of the respiratory electron chain in comparison to the antimycin-inhibited region. The kinetic study of the NADH-dependent reaction showed that at the level of the rotenone-inhibited region, ischemia reduced Km value for NADH, differently from the antimycin-inhibited region where the kinetic constants remain unchanged. No significant changes of the Vmax values were observed in both SMP-producing O2- sites. The ischemic perfusions also produced a reduction of mitochondrial function, particularly evident when glutamate as substrate was studied.

Animals↗
PubMed1982

Kinetic mechanism of mitochondrial adenosine triphosphatase. ADP-specific inhibition as revealed by the steady-state kinetics.

1. A substantial increase of the initial rate of ATP hydrolysis was observed after preincubation of bovine heart submitochondrial particles with phosphoenolpyruvate and pyruvate kinase. 2. The activation was accompanied by an increase of Vmax, without change of Km for ATP. 3. The activated particles catalysed the biphasic hydrolysis of ATP in the presence of an ATP-regenerating system; the initial rapid phase was followed by a second, slower, phase in a time-dependent fashion. 4. The higher the ATP concentration used as a substrate, the higher is the rate of transition between these two phases. 5. The particles catalysed the hydrolysis of ITP with a lag phase; after preincubation with phosphoenolpyruvate and pyruvate kinase, ITP was hydrolysed at a constant rate. 6. Qualitatively the same phenomena were observed when soluble mitochondrial ATPase (F1-ATPase) prepared by the conventional method in the presence of ATP was used as nucleotide triphosphatase. 7. A kinetic scheme is proposed, in which the intermediate active enzyme-product complex (E.ADP) formed during ATP hydrolysis is in slow equilibrium with the inactive E*.ADP complex forming as a result of dislocation of ADP from the active site of ATPase to the other site, which is not in rapid equilibrium with the surrounding medium.

Adenosine Diphosphate↗
PubMed1986

Extraction, partial purification and functional reconstitution of two mitochondrial carriers transporting keto acids: 2-oxoglutarate and pyruvate.

Bovine heart submitochondrial particles were treated with a medium containing Triton X-114 and cardiolipin. The extract was subjected to hydroxyapatite chromatography. Only a few major polypeptides of similar molecular masses were found in the eluate, as shown by electrophoresis in an SDS-polyacrylamide gel stained with silver. The eluate was reconstituted into liposomes and was shown to catalyse two different transport activities: 2-oxoglutarate-2-oxoglutarate exchange sensitive to phthalonate and phenylsuccinate and pyruvate-pyruvate exchange sensitive to 2-cyano-4-hydroxycinnamate. Since both activities were found to have characteristics similar to those described for intact mitochondria, it was concluded that at least two of the polypeptides found in the hydroxyapatite eluate correspond to the two mitochondrial carriers.

Animals↗
PubMed1981

A new species of bound ubisemiquinone anion in QH2: cytochrome c oxidoreductase.

Using a combination of EPR and low temperature diffuse reflectance spectroscopy, a new species of semiquinone anion has been detected in QH2:cytochrome c oxidoreductase in submitochondrial particles under conditions of oxidant-induced extra reduction of cytochrome b. In contrast to the previously detected semiquinone anion, this new species is insensitive to antimycin but sensitive to treatment with 2,3-dimercaptopropanol and O2. The two species can easily be distinguished on the basis of their respective EPR properties since they differ in g-value, line width, and microwave power saturation behavior. It is concluded that the two species of semiquinone anion are bound to different domains on QH2:cytochrome c oxidoreductase. The existence of two different semiquinone anions in the enzyme strongly supports a mechanism of electron flow as proposed in the Q-cycle.

Animals↗
PubMed1991

Ubiquinone protects against loss of tocopherol in rat liver microsomes and mitochondrial membranes.

Liver microsomes and submitochondrial particles (SMP) were isolated from rats fed on a vitamin E or coenzyme Q10/vitamin E enriched diet in order to clarify the antioxidant interactions between coenzyme Q10 and vitamin E. Electron spin resonance spectrometry shows that the decay of vitamin E radicals (tocopheroxy radicals) generated by the arachidonic acid/lipoxygenase oxidation system proceeded at a higher rate in vitamin E enriched microsomes and SMP than in those enriched with coenzyme Q10/vitamin E. Vitamin E levels determined by high performance liquid chromatography revealed that when subjected to enzymatic oxidation, membranes enriched with vitamin E alone were depleted of vitamin E earlier than those enriched with both coenzyme Q10 and vitamin E. These results show that coenzyme Q10 conserves vitamin E, which would help prolong membrane resistance against oxidative damage.

Animals↗
PubMed1995

ATP synthesis catalyzed by the mitochondrial F1-F0 ATP synthase is not a reversal of its ATPase activity.

The ADP(Mg2+)-deactivated oligomycin-sensitive F1-F0 ATPase of coupled submitochondrial particles treated with the substoichiometric amount of oligomycin was studied to test whether ATP synthesis and hydrolysis proceed in either direction through the same intermediates. The initial rates of ATP hydrolysis, oxidative phosphorylation, ATP-dependent, succinate-supported NAD+ reduction, and ATP-induced delta microH+ generation were measured using deactivated ATPase trapped by azide [Biochem. J. (1982) 202, 15-23]. Three ATP consuming reactions were strongly inhibited when azide was present in the assay mixtures, whereas ATP synthesis was not altered by azide. The unidirectional effect of azide is not consistent with three alternating binding sites mechanism operating in ATP synthesis and support our hypothesis on the existence of nucleotide(Mg2+)-controlled 'synthase' and 'hydrolase' states of the mitochondrial F1-F0 ATPase.

Adenosine Triphosphate↗
PubMed2006

Topography and chemical reactivity of the active-inactive transition-sensitive SH-group in the mitochondrial NADH:ubiquinone oxidoreductase (Complex I).

The spatial arrangement and chemical reactivity of the activation-dependent thiol in the mitochondrial Complex I was studied using the membrane penetrating N-ethylmaleimide (NEM) and non-penetrating anionic 5,5'-dithiobis-(2-nitrobenzoate) (DTNB) as the specific inhibitors of the enzyme in mitochondria and inside-out submitochondrial particles (SMP). Both NEM and DTNB rapidly inhibited the de-activated Complex I in SMP. In mitochondria NEM caused rapid inhibition of Complex I, whereas the enzyme activity was insensitive to DTNB. In the presence of the channel-forming antibiotic alamethicin, mitochondrial Complex I became sensitive to DTNB. Neither active nor de-activated Complex I in SMP was inhibited by oxidized glutathione (10 mM, pH 8.0, 75 min). The data suggest that the active/de-active transition sulfhydryl group of Complex I which is sensitive to inhibition by NEM is located at the inner membrane-matrix interface. These data include the sidedness dependency of inhibition, effect of pH, ionic strength, and membrane bilayer modification on enzyme reactivity towards DTNB and its neutral analogue.

Animals↗
PubMed1997

Pro- and anti-oxidant activities of the mitochondrial respiratory chain: factors influencing NAD(P)H-induced lipid peroxidation.

This paper is a study of factors influencing the rate of lipid peroxidation in beef heart submitochondrial particles induced by NAD(P)H via the NADH-ubiquinone oxidoreductase (Complex I) of the respiratory chain. In accordance with earlier observations, both NADH and NADPH initiated lipid peroxidation in the presence of ADP-Fe3+. The rate of the reaction, measured as oxygen consumption and formation of thiobarbituric acid reactive substances, was biphasic as a function of NADH concentration, reaching a maximum at low NADH concentrations and then declining. In contrast, the NADPH-initiated lipid peroxidation showed a monophasic concentration profile of hyperbolic character. Rotenone did not eliminate the biphasicity of the NADH-induced reaction, indicating that this was not due to an antioxidant effect of reduced ubiquinone at high NADH concentrations. This conclusion was further supported by the demonstration that extraction of ubiquinone from the particles did not relieve the inhibition of lipid peroxidation by high NADH concentrations. However rhein, another inhibitor of Complex I, eliminated the biphasicity, and even caused a substantial stimulation of the NADH-induced lipid peroxidation in the particles upon extraction of ubiquinone by pentane. No similar effect occurred in the case of NADPH-induced lipid peroxidation. Furthermore, rhein facilitated both NADH- and NADPH-induced lipid peroxidation even in the absence of added ADP-Fe3+, in a fashion similar to that earlier reported with succinate in the presence of theonyltrifluoroacetone. Based on these findings and measurements of the redox states of ubiquinone and cytochromes in the presence of KCN and NADH or NADPH, it is concluded that Complex I may distinguish between electron input from NADH and NADPH by differences in the site(s) of substrate binding and in the pathways and rates of NADH and NADPH oxidation.

Animals↗
PubMed1982

Effects of 2-mercaptoacetate in isolated liver mitochondria in vitro. Competitive inhibition of 3-hydroxybutyrate dehydrogenase and depression of the beta-oxidation pathway.

The effects of 2-mercaptoacetate on the respiration rates induced by different substrates were studied in vitro in isolated liver mitochondria. With palmitoyl-L-carnitine or 2-oxoglutarate as the substrate, the ADP-stimulated respiration (State 3) was dose-dependently inhibited by 2-mercaptoacetate. with glutamate or succinate as the substrate. State-3 respiration was only slightly inhibited by 2-mercaptoacetate. In contrast, the oxidation rate of 3-hydroxybutyrate was competitively inhibited by 2-mercaptoacetate in both isolated mitochondria and submitochondrial particles. In uncoupled mitochondria and in mitochondria in which ATP- and GTP-dependent acyl-CoA biosynthesis was inhibited, the inhibitory effect of 2-mercaptoacetate on palmitoyl-L-carnitine oxidation was abolished; under the same conditions, however, inhibition of 3-hydroxybutyrate oxidation by 2-mercaptoacetate still persisted. These results led to the following conclusions: 2-mercaptoacetate itself enters the mitochondrial matrix, inhibits fatty acid oxidation through a mechanism requiring an energy-dependent activation of 2-mercaptoacetate and itself inhibits 3-hydroxybutyrate oxidation through a competitive inhibition of the membrane-bound 3-hydroxybutyrate dehydrogenase. This study also strongly suggests that the compound responsible for the inhibition of fatty acid oxidation is 2-mercaptoacetyl-CoA.

3-Hydroxybutyric Acid↗
PubMed1980

The biogenesis of rat-liver mitochondrial ATPase. Evidence that the N, N'-dicyclohexyl carbodiimide-binding protein is synthesized outside the mitochondria.

1. Radioactive N,N'-dicyclohexyl carbodiimide (DCCD) is bound as effectively to the N, N'-dicyclohexyl carbodiimide- and oligomycin-sensitive ATPase complex in submitochondrial particles of normal rat liver as to the similar but partially N,N'-dicyclohexyl carbodiimide- and oligomycin-insensitive complex of thiamphenicol-treated rats. The latter complex is deficient in 3 subunits (subunit 6, 7 and 10). 2. Radioactive N,N'-dicyclohexyl carbodiimide is exclusively bound to the subunits present in the bands 8 and 11 of SDS-PAA gels of the purified ATPase complex. These subunits, most likely the dimer and monomer of the N,N'-dicyclohexyl carbodiimide-binding protein, are products of the cytoplasmic protein synthesis. 3. The results together indicate that the N,N'-dicyclohexyl carbodiimide-insensitivity of the ATPase complex formed during in vitro inhibition of mitochondrial protein synthesis, is not caused by a lack of inhibitor binding protein. The same holds for the oligomycin-insensitivity.

Adenosine Triphosphatases↗