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PubMed1999

Active/de-active state transition of the mitochondrial complex I as revealed by specific sulfhydryl group labeling.

The sensitivities of NADH oxidase and/or NADH-ubiquinone reductase activities of submitochondrial particles and purified complex I towards N-ethylmaleimide (NEM) and other SH-reagents were studied. Only thermally de-activated preparations IA.D. Vinogradov (1998) Biochim. Biophys. Acta 1364, 169-185] were inhibited by SH-reagents whereas the redox-pulsed, activated enzyme was resistant to the inhibitors. The pH profile of the pseudo-first order inhibition rate suggested a pKa of about 10 for the de-activation-dependent, NEM-reactive sulfhydryl group. NADH-ubiquinone reductase of activated particles treated with an excess of NEM followed by removal of the inhibitor was still capable of slow reversible active/de-active transition. When active, NEM-treated particles were de-activated and further inhibited by N-fluorescein maleimide, specific incorporation of the fluorescence label into low molecular mass polypeptide was evident. Comparison of the specific fluorescence labeling of submitochondrial particles, crude and purified complex I showed that the active/de-active state-dependent SH-group is located in a 15 kDa polypeptide (most likely in the 15 kDa IP subunit of the iron-sulfur protein-containing fraction of complex I).

Animals↗
PubMed1988

Fate of nucleotides bound to reconstituted Fo-F1 during adenosine 5'-triphosphate synthesis activation or hydrolysis: role of protein inhibitor and hysteretic inhibition.

The protein ATPase inhibitor entraps about five nucleotides in pig heart mitochondrial F1, one at least being a triphosphate [Di Pietro, A., Penin, F., Julliard, J.H., Godinot, C., & Gautheron, D.C. (1988) Biochem. Biophys. Res. Commun. 152, 1319-1325]. The fate of these nucleotides was studied during ATP synthesis driven by NADH oxidation in reconstituted inverted submitochondrial particles. Iodinated F1, containing 0.7 mol of endogenous nucleotides/mol, was first loaded with tritiated adenine nucleotides in the presence or absence of the protein inhibitor and then reassociated with F1-depleted submitochondrial particles (ASU particles) to reconstitute an efficient NADH-driven ATP synthesis. In the absence of the protein inhibitor, 1.7 mol of labeled nucleotides remained bound per mole of reassociated F1, 0.8-0.9 mol being rapidly exchangeable against medium ADP or ATP, as measured after rapid filtration through nitrocellulose filters. In the presence of the protein inhibitor, as many as 3.25 mol of labeled nucleotides remained bound per mole of reassociated F1. Under hydrolysis conditions where ATPase activity was highly inhibited, no release of tritiated nucleotide occurred. In contrast, under ATP synthesis conditions where the protonmotive force was generated by NADH oxidation, the progressive reversal of inhibition by the protein inhibitor was correlated to a concomitant release of tritiated nucleotide. When ATP synthesis became fully active, about one nucleotide was completely exchanged whereas more than three nucleotides remained tightly bound and did not appear to be directly involved in ATP synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1984

Loose binding of testicular mitochondrial ATPase to the inner membrane.

Rat testis mitochondrial ATPase was not inhibited by oligomycin at pH 7.5. It was inhibited only at higher alkaline pH's, and showed a lower sensitivity both to oligomycin and N,N'-dicyclohexylcarbodiimide and a higher one to efrapeptin. In submitochondrial particles, testis ATPase was only slightly inhibited by oligomycin, ossamycin, and efrapeptin. The possibility of a loose binding of F1 to the membrane was supported by its recovery from the supernatant of the submitochondrial particles. Furthermore, by electron microscopy, after hypoosmotic shock and negative staining of the mitochondrial preparations, most of the inner mitochondrial membranes showed only a few "knobs" or none at all. The capacity of the testis mitochondrial preparation to produce ATP was tested and compared to that from liver. ATP synthetase/ATPase activity ratio was 30/1 in liver mitochondria, whereas in the testis it was 3/1. In spite of this large difference, at least part of the testis ATPase must be firmly bound to the membrane, since it is able to form ATP. The rest seems to be loosely bound and its functional significance is still unknown.

Adenosine Triphosphatases↗
PubMed1994

The effect of sulfite on the ATP hydrolysis and synthesis activity of membrane-bound H(+)-ATP synthase from various species.

The action of sulfite on ATP hydrolysis and synthesis activities is investigated in membrane vesicles prepared from the cyanobacterium Synechococcus 6716, chromatophores from the photosynthetic purple bacterium Rhodospirillum rubrum, membrane vesicles from the related non-photosynthetic bacterium Paracoccus denitrificans, and bovine heart submitochondrial particles. Without any further pretreatment ATP hydrolysis is stimulated by sulfite in all four membrane preparations. Typically ATP synthesis in the cyanobacterial membrane vesicles is inhibited by sulfite, whereas ATP synthesis in chromatophores and the submitochondrial particles is not. These differences in sensitivity of ATP synthesis to sulfite, however, correspond well with the distribution of (photosynthetic) sulfur oxidizing pathways in the remaining three organisms/organelles compared in this study.

Adenosine Triphosphate↗
PubMed1982

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Partial characterization and changes in activity during exponential growth.

1. The mitochondrial adenosine triphosphatase (ATPase) of Acanthamoeba castellanii is Mg2+-requiring (optimum cation: ATP ratio of 1.5) and has two pH optima of activity (at pH 6.6 and 8.1). 2. ATPase activity of submitochondrial particles is effectively inhibited by twelve different inhibitors of energy conservation suggesting similarities in inhibitor-binding sites to other previously characterized complexes. 3. Gel filtration by passage through Sephadex G-50 increases ATPase activity of submitochondrial particles between 1.5 and 3.5 fold indicating the presence of a low molecular weight inhibitor protein. 4. After removal of the inhibitor protein, sensitivity to inhibitors of energy conservation decreases by between 1.5 and 14 fold. Crude F1-inhibitor preparations from A. castellanii, Schizosaccharomyces pombe, Tetrahymena pyriformis and bovine heart also inhibit ATPase activity. 5. Large variations in ATPase activity, F1-inhibitor protein activity, and amounts of immunologically-determined ATPase protein were observed during exponential growth, and the correlation between changes in these measurements is discussed. 6. The results are also discussed highlighting the similarities between the mitochondrial ATPase of A. castellanii and other mitochondrial ATPases.

Adenosine Triphosphatases↗
PubMed1983

A temperature-dependent structural change of mitochondrial ATPase.

The temperature dependence of the intrinsic tryptophan fluorescence in either bovine heart submitochondrial particles or oligomycin-sensitive ATPase isolated therefrom shows a discontinuity at near 25 degrees C, which coincides with the temperature where a break in the Arrhenius plot of ATPase activity is found. Addition of n-butanol to submitochondrial particles induces a decrease of tryptophan fluorescence in the whole temperature range. The discontinuity is interpreted as a temperature-dependent structural change and related to a viscosity-induced phase separation of the intrinsic mitochondrial proteins.

1-Butanol↗
PubMed2000

The in vitro evaluation of the toxicities of three related herbicide formulations containing ester derivatives of 2,4,5-T and 2,4-D using sub-mitochondrial particles.

The aim of this investigation was to determine the contribution made by the different components of herbicide formulations to the overall toxicity of the formulations. Three related herbicide formulations were chosen. The first, Agent Orange, consisted only of the butyl esters of 2,4,5-T and 2,4-D. The second was Agent Orange diluted with diesel fuel and the third formulation tested was a tree and blackberry killer, which consisted of the butyl ester of 2,4,5-T, the ethyl ester of 2,4-D, diesel fuel and two surfactants. The potential toxic effects of these three formulations were evaluated by determining their inhibitory effects on the oxidative functions of submitochondrial particles prepared from beef heart mitochondria. The effective concentration that caused a 50% inhibition of the activities of the submitochondrial particles was determined for all three formulations. When the toxicity of the individual components of these formulations was evaluated, it was established that the so-called 'inert' components i.e. diesel fuel and surfactants contributed approximately 50% of the overall toxicity of the complete formulations. Hence the results confirm the importance of evaluating the toxicity of complete formulations, rather than only focussing on the active components. While cellular and sub-cellular assays cannot account for pharmacokinetic and pharmacodynamic changes that may affect the toxicity of xenobiotics, the sub-mitochondrial particle test is useful as an initial screening assay.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1995

Translocation of loops regulates transport activity of mitochondrial ADP/ATP carrier deduced from formation of a specific intermolecular disulfide bridge catalyzed by copper-o-phenanthroline.

The cross-linking reagent copper-o-phenanthroline complex (Cu(OP)2) specifically caused a decrease in the amount of the 30-kDa ADP/ATP carrier in bovine submitochondrial particles associated predominantly with formation of a 60-kDa protein consisting of a cross-linked dimer of the carrier. However, Cu(OP)2 had no effect on mitochondria. The transport of ADP via the carrier through submitochondrial particle membranes was found to be inhibited in parallel with the progress of intermolecular cross-linking. Analysis of the cross-linked site showed that a disulfide bridge was formed only between two Cys56 residues in a pair of the first loops facing the matrix space. The transport inhibitor bongkrekic acid, which locks the m-state conformation of the carrier, had no effect on disulfide bridge formation catalyzed by Cu(OP)2, but carboxyatractyloside, which locks the c-state conformation by acting from the cytosolic side, completely inhibited the cross-linking. These results show that the ADP/ATP carrier functions as a dimer form, and a pair of the first loops protrudes into the matrix space in the m-state, but possibly intrudes into the membrane in the c-state. Thus, it is suggested that a pair of the first loops acts as a gate and that its opening and closing are regulated by their translocation.

Animals↗
PubMed1995

DBHBM (3,5-dibromo-4-hydroxy-benzylidenemalonitrile) is a novel inhibitor of electron transfer through the QN center of the mitochondrial bc1 complex.

DBHBM (3,5-dibromo-4-hydroxy-benzylidenemalonitrile) inhibited the NADH- or succinate-supported rate of O2 consumption in beef heart submitochondrial particles (Ki = 7 x 10(-7) M). Oxygen comsumption was restored with the addition of ascorbate/TMPD, indicating that the inhibitory effect was on the ubiquinol-cytochrome c reductase activity of the respiratory chain. Difference spectra with submitochondrial particles indicated that DBHBM blocked electron transport through the cytochrome bc1 complex, in a mode closely similar to that of antimycin A. The reduction rates of cytochrome b by succinate were strongly inhibited in the presence of DBHBM plus myxothiazol, but not by DBHBM plus antimycin A. These data suggest that DBHBM may bind primarily to the QN center. In the purified bc1 complex, DBHBM and antimycin A induced a red shift from 562 to 566 nm of the alpha peak of cytochrome b, supporting the idea that DBHBM influences predominantly the ligand field of the b562 (bh) heme. Difference spectra in the presence or absence of myxothiazol showed that DBHBM induced the same red shift with a maximum at 565 nm and a minimum at 559 nm. We conclude that DBHBM blocks electron transfer at the QN center and thus may be considered a novel group III inhibitor of the bc1 complex.

Animals↗
PubMed1985

Mechanism of inhibition of mitochondrial adenosine triphosphatase by dicyclohexylcarbodiimide and oligomycin: relationship to ATP synthesis.

Measurement of the rate of [gamma-32P]ATP binding (k1) and release (k-1) from catalytic sites on submitochondrial particles permitted calculation of the affinity constant in catalytic sites (k1 = K1/k1-1) of 10(12) M-1. This value is the same as that determined previously for the solubilized ATPase (F1) from beef heart mitochondria. Treatment of submitochondrial particles with dicyclohexylcarbodiimide or oligomycin so as to cause about 90% inhibition of ATPase activity was accompanied by a decrease in the binding of [gamma-32P]ATP in high-affinity catalytic sites. Under the conditions of the experiment, it is expected that the inhibitors reacted not with the ATPase itself but with other proteins in the oligomycin-sensitive ATPase complex (F0-F1). It is proposed that dicyclohexylcarbodiimide and oligomycin inhibit ATPase activity by causing a conformational change in the F0 portion of the complex that is transmitted to F1, resulting in an impaired binding of substrate in catalytic sites. These observations of apparent conformational interactions between F0 and F1 on the mitochondrial membrane are relevant to the mechanism of the coupling device that links the energy store to ATP formation in oxidative phosphorylation. It is proposed that a change in the state of ionization of one or more charged amino acid residues in F0 results in a conformational change in F0 which, transmitted to F1, reversibly alters the catalytic sites and facilitates the release of product ATP.

Adenosine Triphosphate↗
PubMed1987

Structural and functional differences in H+-ATPases with native and reconstituted inhibitor protein.

Anti F1 antibodies that react with the alpha and beta subunits of the mitochondrial F0-F1 ATPase complex do not interfere with the natural inhibitor protein-ATPase interaction as revealed by inhibitor peptide titration curves. Submitochondrial particles with endogenous or added bound inhibitor protein show differences in immunoprecipitation. Submitochondrial particles which are partially depleted of inhibitor protein gave the same immunoprecipitation curve as the Mg-ATP particle. Anti F1 antibodies induce differential effects in ATP hydrolysis and ATP-Pi exchange. ATP hydrolysis is stimulated in Mg-ATP particles to 200%, while inhibitor depleted and inhibitor reconstituted particles are inhibited by the presence of the antibodies. ATP-Pi exchange is stimulated in inhibitor reconstituted particles and inhibited in Mg-ATP and inhibitor depleted particles. These results suggest that the inhibitor protein when endogenously bound confers a different conformation to the F1-ATPase than that of the F1 ATPase with added bound inhibitor protein.

Adenosine Triphosphate↗
PubMed1983

Comparative cardiac oxygen radical metabolism by anthracycline antibiotics, mitoxantrone, bisantrene, 4'-(9-acridinylamino)-methanesulfon-m-anisidide, and neocarzinostatin.

This study examined the effects of various anthracycline antibiotics and mitoxantrone, bisantrene, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and neocarzinostatin on oxygen radical formation by cardiac sarcoplasmic reticulum and submitochondrial particles. Doxorubicin, daunorubicin, rubidazone, and aclacinomycin A stimulated superoxide production by both heart fractions in a dose-dependent fashion that appeared to follow saturation kinetics. The anthracycline drugs also significantly increased hydrogen peroxide production by heart sarcosomes and submitochondrial particles. On the other hand, mitoxantrone, bisantrene, m-AMSA, and neocarzinostatin did not significantly enhance cardiac reactive oxygen metabolism. Thus, it is unlikely that the mechanism of the cardiac toxicity produced by mitoxantrone and m-AMSA in patients previously treated with anthracycline drugs can be directly related to oxidation-reduction cycling catalyzed by cardiac flavin dehydrogenases.

Aminoacridines↗
PubMed1979

The use of arylazido-beta-alanyl-ATP as a photoaffinity label for the isolated and membrane-bound mitochondrial ATPase complex.

The effects of a photoaffinity derivate of ATP, arylazido-beta-alanyl-ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl) adenosine 5'-triphosphate, on submitochondrial particles and the partially purified ATPase complex of beef heart mitochondria have been investigated. In the absence of light the ATP analogue has been found to be a substrate for the E132PA1P1-ATP exchange reaction of submitochondrial particles. When photoirradiated in the presence of arylazido-beta-alanyl-ATP, the ATPase activity and the the the [32P]Pi-ATP exchange reaction are inhibited maximally 80%. Arylazido-beta-alanyl-ATP following photolysis is a noncompetitive inhibitor with respect to ATP while arylazido-beta-alanine, the azido-containing adjunct of the ATP analogue, has no inhibitory effect under the same conditions. The inactivating effect of arylazido-beta-alanyl-ATP is prevented in part by the presence of ATP, or ADP and pyrophosphate. Photolabeling produces a covalent binding of the derivative with the F1ATPase being the major protein labeled. The binding of 0.22 mumol of arylazido-beta-alanyl-ATP/mg of mitochondrial protein is associated with a maximal inhibitory effect. The ATPase activity of the partially purified ATPase complex is also sensitive to photoirradiation in the presence of arylazido-beta-alanyl-ATP. When the ATPase complex is associated with liposomes there is an increase in the specific ATPase activity with a 10-fold increase in Vmax and a 4-fold decrease in KmATP associated with a parallel increase in the apparent affinity and maximal inhibitory effect of the arylazido-beta-alanyl-ATP. The photoinhibition of the ATPase complex in the presence of arylazido-beta-alanyl-ATP results in covalent binding of 1.6 mumol of arylazido-beta-alanyl-ATP/mg of protein. The alpha and beta subunits are the only components of the ATPase complex labeled by the [3H]arylazido-beta-alanyl-ATP. The relationship between the arylazido-beta-alanyl-ATP-labeled sites and the nucleotide binding sites on the mitochondrial ATPase is discussed.

Adenosine Triphosphatases↗
PubMed1980

Stimulation of the alternative oxidase of Neurospora crassa by Nucleoside phosphates.

The alternative-oxidase-mediated succinate oxidase activity of Neurospora crassa decreases drastically when mitochondria are fractionated into submitochondrial particles or treated with deoxycholate. The activity, however, can be completely restored in the presence of nucleoside 5'-monophosphates. The purine nucleoside 5'-monophosphates are more effective than the pyrimidine homologues. 5'-GMP gives a 10-fold stimulation of the alternative-oxidase-mediated succinate oxidase activity in submitochondrial particles. A comparison is made with the results obtained earlier with Moniliella tomentosa [Hanssens & Verachtert (1976) J. Bacteriol. 125, 825--835; Vanderleyden, Van Den Eynde & Verachtert (1980) Biochem. J. 186, 309--316].

Deoxycholic Acid↗
PubMed1980

[Effect of 2,3-dimercaptopropanol on electron transfer in the energy coupling site 2 of the respiratory chain: evidence for the Q-cycle hypothesis].

Incubation of submitochondrial particles from bovine heart with 2,3-dimercaptopropanol (British antilewisite, BAL) results in inhibition of the respiratory chain between cytochromes b and c. BAL exerts no pronounced effect on the spectral and redox properties of cytochromes b566 and b562. In the BAL-treated submitochondrial particles antimycin brings about the bathochromic shift of the cytochrome b562 absorption alpha-band, but does not induce "extra-reduction" of cytochromes b. Upon a combined effect of antimycin and BAL the reduction of cytochromes b by succinate is inhibited. Whereas neither antimycin nor BAL alone prevents this reaction. The data obtained suggest the existence of two pathways of electron transfer from succinate to cytochromes b, one of which is blocked by antimycin and the other one by BAL. These two pathways form a cyclic loop of electron transport, in accordance with the Mitchell's "protonmotive CoQ-cycle" hypothesis. The traditional scheme of linear arrangement of the respiratory carriers in the succinate--cytochrom c reductase span of the electron transfer chain, as well as the Wikstrom--Berden's branched model, do not provide satisfactory explanation of the mode of BAL inhibitory action.

Animals↗
PubMed1984

[Localization of the redox-center of cytochrome b562 on the internal (M-) side of the mitochondrial membrane].

In the inside-out submitochondrial particles, cytochrome b-562 is readily reduced by a hydrophilic redox mediator Ru(NH3)6(2)+; this reaction is not inhibited by antimycin and myxothiazol. In mitochondria, cytochromes b do not virtually interact with Ru(NH3)6(2)+. The accessibility of cytochrome b-562 to Ru(NH3)6(2)+ in submitochondrial particles and its inaccessibility in mitochondria suggest the localization of the hemoprotein redox center on the inner surface of the mitochondrial membrane.

Animals↗
PubMedDate not supplied

[The effect of adrenaline on the activity and functional mobility of transhydrogenase in the inner mitochondrial membrane].

Adrenaline injection to rats increased activity of liver mitochondrial transhydrogenase. Transhydrogenase in the adrenaline activated submitochondrial particles showed increased sensitivity to thermal and proteolytic inactivations as well as to inhibition by dicyclohexyl carbodiimide. Substrates of the reaction NAD+ and NADPH, which induce different conformational changes of the enzyme molecule in membranes, alternated to a greater extent the sensitivity to these treatments of transhydrogenase in the adrenaline activated submitochondrial particles as compared with controls. Activation of transhydrogenase by catecholamines appears to occur due to increase of the enzyme molecule mobility in the inner mitochondrial membrane.

Animals↗
PubMed1997

Further studies on the recoupling effect of 6-ketocholestanol upon oxidative phosphorylation in uncoupled liver mitochondria.

The effect of 6-ketocholestanol was studied on CCCP-induced uncoupling in liver mitochondria, submitochondrial particles and cytochrome oxidase proteoliposomes. It was found that 6-ketocholestanol prevents and reverses uncoupling induced by nM concentrations of CCCP on the three systems assayed. As it was reported on kidney mitochondrial membranes [Chavez et al. (1996) FEBS Lett. 379, 305-308], the recoupling effect caused by 6-ketocholestanol on submitochondrial particles and proteoliposomes could be due to a diminution of membrane fluidity.

Animals↗