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PubMedDate not supplied

[Inhibition of mitochondrial ATP-synthetase by redox-agents].

Effects of phenol and phenothiazine on ATP synthesis and electron transport in submitochondrial particles were studied. Nitrophenols and phenothiazines inhibited ATP synthesis without notable effect on electron transport. On the contrary chlorphenols equally decreased the velocities of electron transport and ATP synthesis. The inhibitors studied showed the properties of electron acceptors in relation to the radicals, their acceptor properties corresponding to their ability to inhibit ATP synthesis.

Adenosine Triphosphatases↗
PubMed1991

Tryptophan fluorescence in electron-transfer flavoprotein:ubiquinone oxidoreductase: fluorescence quenching by a brominated pseudosubstrate.

We have studied the intrinsic fluorescence of the 12 tryptophan residues of electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF:QO). The fluorescence emission spectrum (lambda ex 295 nm) showed that the fluorescence is due to the tryptophan residues and that the contribution of the 22 tyrosine residues is minor. The emission maximum (lambda m 334 nm) and the bandwidth (delta lambda 1/2 56 nm) suggest that the tryptophans lie in hydrophobic environments in the oxidized protein. Further, these tryptophans are inaccessible to a range of ionic and nonionic collisional quenching agents, indicating that they are buried in the protein. Enzymatic or chemical reduction of ETF:QO results in a 5% increase in fluorescence with no change of lambda m or delta lambda 1/2. This change is reversible upon reoxidation and is likely to reflect a conformational change in the protein. The ubiquinone analogue Q0(CH2)10Br, a pseudosubstrate of ETF:QO (Km = 2.6 microM; kcat = 210 s-1), specifically quenches the fluorescence of one tryptophan residue (Kd = 1.6-3.2 microM) in equilibrium fluorescence titrations. The ubiquinone homologue UQ-2 (Km = 2 microM; kcat = 162 s-1) and the analogue Q0(CH2)10OH (Km = 2 microM; kcat = 132 s-1) do not quench tryptophan fluorescence; thus the brominated analogue acts as a static heavy atom quencher. We also describe a rapid purification for ETF:QO based on extraction of liver submitochondrial particles with Triton X-100 and three chromatographic steps, which results in yields 3 times higher than previously published methods.

Acrylamide↗
PubMed1989

Mitochondrial electron transport-linked tocopheroxyl radical reduction.

alpha-Tocopherol (vitamin E) is a lipophilic chain-breaking antioxidant which inhibits lipid peroxidation in isolated mitochondrial membranes and protects membranes from oxidative damage. The primary oxidation product of vitamin E is the tocopheroxyl radical. Reduction of the tocopheroxyl radical can occur by reactions with water-soluble anti-oxidants such as ascorbate or glutathione, resulting in the recycling of vitamin E. Physiological concentrations of vitamin E are too low to allow detection of tocopheroxyl radical by ESR. After dietary supplementation with vitamin E, a 10-20-fold increase in the rat liver mitochondrial membrane content of vitamin E was achieved and this allowed for direct detection of the tocopheroxyl radical by ESR, after treatment with an oxidizing system composed of lipoxygenase and arachidonic acid. By using submitochondrial particle membranes, it was shown that NADH, succinate, and reduced cytochrome c-linked oxidation reduce the tocopheroxyl radical, preventing both accumulation of the radical and vitamin E consumption. As the electron transport chain can reduce tocopheroxyl radical it may have an important physiological role in recycling vitamin E.

Animals↗
PubMedDate not supplied

Mitochondrial antibodies in primary biliary cirrhosis: species and nonspecies specific determinants of M2 antigen.

Sera from patients with primary biliary cirrhosis reacted with four major bands in beef heart mitochondria and ATPase extract when analyzed by immunoblot after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These four immunologically reactive bands corresponded to protein bands with molecular weights of about (a) 80,000; (b) 63,000; (c) 56,000; and (d) 43,000 to 46,000. An additional immunoreactive band was found with some high-titered primary biliary cirrhosis sera at 36,000. No association with any ATPase subunits was found, except for band c which migrated between the alpha- and beta-subunit of ATPase. Most ATPase fractions did not contain this band c, indicating that M2 determinants, as defined by immunoblot, are not identical with any ATPase subunit. Species and nonspecies-specific determinants of M2 were identified using mitochondria from rat liver and human heart and liver. Antigenic bands a, c and d were nonspecies-specific. Band b and e occurred only in beef heart. An additional determinant at about 38,000 was detected using human heart and liver mitochondria. Primary biliary cirrhosis sera showed a typical reaction with two protein bands of Escherichia coli, one at about 85,000 to 90,000 and the other at 60,000. Antibodies against both determinants could be absorbed with submitochondrial particles of beef heart showing that E. coli shares cross-reacting determinants with mitochondria. Sera from 56 primary biliary cirrhosis patients were tested using beef heart mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphatases↗
PubMed2005

Possible involvement of glutamic and/or aspartic acid residue(s) and requirement of mitochondrial integrity for the protective effect of creatine against inhibition of cardiac mitochondrial respiration by methylglyoxal.

We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661-669,2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3'-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine.

Alamethicin↗
PubMed1990

Evidence that the blockade of mitochondrial respiration by the neurotoxin 1-methyl-4-phenylpyridinium (MPP+) involves binding at the same site as the respiratory inhibitor, rotenone.

It has been postulated that 1-methyl-4-phenylpyridinium (MPP+) blocks mitochondrial respiration by combining at the same site as rotenone, a potent inhibitor of NADH oxidation in mitochondria, known to act at the junction of NADH dehydrogenase and coenzyme Q (CoQ). The present experiments show that MPP+ and two of its analogs indeed act in a concentration dependent manner to prevent the binding of [14C]-rotenone to submitochondrial particles (ETP) and significantly decrease the inhibition of electron transport caused by rotenone. It therefore appears that MPP+ binds at the same site as rotenone or an adjacent site, supporting the hypothesis that its neurotoxic action is due to the inhibition of mitochondrial respiration.

1-Methyl-4-phenylpyridinium↗
PubMed1988

Analysis of NADH dehydrogenases from plant [mung bean (Phaseolus aureus)] mitochondrial membranes on non-denaturing polyacrylamide gels and purification of complex I by band excision.

The present paper describes the analysis of plant mitochondrial NADH dehydrogenases using a recently developed non-dissociating gradient polyacrylamide-gel-electrophoresis technique [Kuonen, Roberts & Cottingham (1986) Anal. Biochem. 153, 221-226]. Solubilized mung-bean (Phaseolus aureus) submitochondrial particles were analysed on 3-22% (w/v) gradient polyacrylamide gels containing 0.1% Triton X-100 and stained for multiple NADH dehydrogenase activities. A rotenone-sensitive NADH dehydrogenase (Complex I) was identified on the basis of co-migration with the purified mammalian enzyme. The polypeptide composition of the plant enzyme was further analysed by band excision and SDS/polyacrylamide-gel electrophoresis.

Cytochrome Reductases↗
PubMed1984

Photodamage to intact erythrocyte membranes at high laser intensities: methods of assay and suppression.

A simple hemolytic assay of the photodamage suffered by individual intact erythrocytes upon localized exposure to high laser intensities such as those encountered in fluorescence photobleaching recovery (FPR) experiments has been characterized. At incident beam powers over 100,000 W/cm2 at 514 or 568 nm, hemoglobin absorption induces thermal-shock lysis. Below the thermal-shock threshold, other intracellular chromophores appear to catalyze membrane contraction and lysis with linear dose kinetics in anaerobic preparations. A variety of chemical agents that inhibit photodamage to red cell ghost membranes at low intensities (less than 450 W/cm2) do not benefit intact cells at realistic FPR bleaching doses of 10,000 to 1,000,000 W/cm2. However, we find that sample deoxygenation dramatically photostabilizes erythrocytes, thus we have developed a physiologically compatible in situ deoxygenation method utilizing submitochondrial particles. The extent to which this and other deoxygenation procedures modify fluorophore photolability has been determined. FPR measurements become practical on intact erythrocytes when a combination of low bleach power, thorough deoxygenation, and long wavelength excitable (approximately 650 nm) fluorescence probes is employed; the equivalent of 60,000 photobleaches per cell can then be tolerated without detectable damage.

Animals↗
PubMed1994

Tissue specificity of mitochondrial F0F1-ATPase activity of Lilium longiflorum plant.

A large difference was found in the activities of oligomycin-sensitive mitochondrial F0F1-ATPase isolated from different tissues of Lilium longiflorum plants. The enzyme activity of F0F1-ATPase in pollen was the highest, while that in bulbs was the lowest. When ATPases were cross-reconstituted from F1-ATPases and F1-depleted submitochondrial particles (SMP), ATPases reconstituted from F1-depleted pollen SMP showed the higher activity regardless of the source of F1-ATPase. Fatty acid compositions of phospholipids in SMP were also different between bulbs and pollen. These suggest that the F0 portion and/or its environment are important for regulation of F0F1-ATPase activity in L. longiflorum plant.

Enzyme-Linked Immunosorbent Assay↗
PubMed1996

Ubiquinol-cytochrome c oxidoreductase. The redox reactions of the bis-heme cytochrome b in ubiquinone-sufficient and ubiquinone-deficient systems.

Antimycin and myxothiazol are stoichiometric inhibitors of complex III (ubiquinol-cytochrome c oxidoreductase), exerting their highest degree of inhibition at I mol each/mol of complex III monomer. Phenomenologically, however, they each inhibit three steps in the redox reaction of the bis-heme cytochrome b in submitochondrial particles (SMP), and all three inhibitions are incomplete to various extents. (i) In SMP, reduction of hemes bH and bL by NADH or succinate is inhibited when the particles are treated with both antimycin and myxothiazol. Each inhibitor alone allows reduced bH and bL to accumulate, indicating that each inhibits the reoxidation of these hemes. (E)-Methyl-3-methoxy-2-(4')-trans-stilbenyl)acrylatc in combination with antimycin or 2-n-heptyl-4-hydroxyquinoline-N-oxide in combination with myxothiazol causes less inhibition of b reduction than the combination of antimycin and myxothiazol. (ii) Reoxidation of reduced b, is inhibited by either antimycin or myxothiazol (or 2-n-heptyl-4-hydroxyquinoline-N-oxide, (E)-methyl-3-methoxy-2-(4'-trans-stilbenyl)acrylate, or stigmatellin). (iii) Reoxidation of reduced bH is also inhibited by any one of these reagents. These inhibitions are also incomplete, and reduced bL is oxidized through the leaks allowed by these inhibitors at least 10 times faster than reduced bH. Heme bH can be reduced in SMP via cytochrome c, and the Rieske iron-sulfur protein by ascorbate and faster by ascorbate + TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). Energization of SMP by the addition of ATP affords reduction of bL as well. Reverse electron transfer to bH and bL is inhibited partially by myxothiazol, much more by antimycin. Ascorbate + TMPD also reduce bH in ubiquinone-extracted SMP in which the molar ratio of ubiquinone to cytochrome b has been reduced 200-fold from 12.5 to aproximately 0.06. Reconstitution of the extracted particles with ubiquinone-10 restores substrate oxidation but does not improve the rate or the extent of b, reduction by ascorbate + TMPD. These reagents also partially reduce cytochrome b in SMP from a ubiquinone-deficient yeast mutant. The above results are discussed in relation to the Q-cycle hypothesis.

Animals↗
PubMed1980

Substrate kinetics of the alternative oxidase of Neurospora crassa.

The kinetics of the succinate oxidation by cyanide-sensitive and cyanide-insensitive submitochondrial particles of Neurospora crassa cells suggest that both respiratory pathways use the same complex II. This is confirmed by comparing the kinetics of the reductase activities of the isolated succinate-ubiquinone oxidoreductase (complex II) of cyanide-sensitive and cyanide-insensitive cells respectively. No alternative-oxidase activity was found to be associated with the isolated complex II of cyanide-insensitive cells.

Chloramphenicol↗
PubMed1987

The hydrophobic cationic cyanine dye inhibits oxidative phosphorylation by inhibiting ADP transport, not by electrophoretic transfer, into mitochondria.

The effect of the divalent cationic cyanine dye tri-S-C4(5) on oxidative phosphorylation in rat liver mitochondria was examined. The dye at about 100 n mols per mg mitochondrial protein inhibited state 3 respiration and ATP synthesis almost completely. However, it had no effect on submitochondrial particles, like other hydrophobic cations. The dye inhibited the transport of ADP into mitochondria mediated by the adenine nucleotide translocator. Thus, the inhibition of oxidative phosphorylation by the cationic dye was concluded to be due to its action on the adenine nucleotide translocator, not to its electrophoretic transfer into the inner space of mitochondria according to the inside-negative electrochemical potential.

Adenosine Diphosphate↗
PubMed1991

Acetaminophen toxicity results in site-specific mitochondrial damage in isolated mouse hepatocytes.

Exposure of isolated mouse hepatocytes to a toxic concentration of acetaminophen (5 mM) resulted in damage to the mitochondrial respiratory apparatus. The nature of this damage was investigated by measuring respiration stimulated by site-specific substrates in digitonin-permeabilized hepatocytes after acetaminophen exposure. Respiration stimulated by succinate at energy-coupling site 2 was most sensitive to inhibition and was decreased by 47% after 1 h. Respiration supported by NADH-linked substrates (site 1) was also decreased but to a lesser extent, while there was no decrease in the rate of ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD)-supported respiration (site 3). The loss of mitochondrial respiratory function was accompanied by a decrease in ATP levels and ATP/ADP ratios in the cytosolic compartment and was preceded by a loss of reduced glutathione in both the cytosol and mitochondria. All these effects occurred well before the loss of cell membrane integrity. The putative toxic metabolite of acetaminophen, N-acetyl-p-benzoquinonimine (NAPQI), produced a similar pattern of respiratory dysfunction in isolated hepatic mitochondria. Respiration stimulated by succinate- and NADH-linked substrates was very sensitive to 50 microM NAPQI, while ascorbate + TMPD-supported respiration was unaffected. The interaction between NAPQI and the respiratory chain was further investigated using submitochondrial particles. Succinate dehydrogenase (associated with respiratory complex II) was found to be very sensitive to NAPQI, while NADH dehydrogenase (respiratory complex I) was inhibited to a lesser extent. Our results indicate that a loss of the ability to utilize succinate- and NADH-linked substrates due to attack of the respiratory chain by NAPQI causes a disruption of energy homeostasis in acetaminophen hepatotoxicity.

Acetaminophen↗
PubMed2005

Semisynthesis and biological activity of aminoacyl triesters of squamocin, an annonaceous acetogenin.

A number of aminoacyl triesters of squamocin 1, a cytotoxic acetogenin isolated from the seeds of Annona reticulata, have been synthesized in two to three steps from protected (l)-aminoacids and squamocin 1 using standard coupling/deprotection procedures. These semisynthetic analogs were tested on submitochondrial particles (SMP) for their complex I inhibitory activities, and against KB 3-1 cells in vitro. All triesters derivatives exhibited a complete extinction of activity at the enzymatic level, correlated to a reduced though modulated cytotoxicity in comparison with squamocin 1. This activity can apparently be considered as a function of the amphipathy of the analogs, the more amphiphilic ones being the more cytotoxic.

Annona↗
PubMed1996

Purification and characterization of a 43-kDa rotenone-insensitive NADH dehydrogenase from plant mitochondria.

A 43-kDa NAD(P)H dehydrogenase was purified from red beetroot mitochondria. An antibody against this dehydrogenase was used in conjunction with the membrane-impermeable protein cross-linker 3,3'-dithiobis(sulfosuccinimidylpropionate) to localize the dehydrogenase on the matrix side of the inner membrane. Immunoblotting showed that the dehydrogenase was found in mitochondria isolated from several plant species but not from rat livers. Antibodies against the purified dehydrogenase partially inhibited rotenoneinsensitive internal NADH oxidation by inside-out submitochondrial particles. The level of rotenone-insensitive respiration with NAD-linked substrates correlated with the amount of 43-kDa NAD(P)H dehydrogenase present in mitochondria isolated from different soybean tissues. Based on these results, we conclude that the 43-kDa NAD(P)H dehydrogenase is responsible for rotenone-insensitive internal NADH oxidation in plant mitochondria.

Animals↗
PubMedDate not supplied

Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase.

We show that urea inhibits the ATPase activity of MgATP submitochondrial particles (MgATP-SMP) with Ki = 0.7 M, probably as a result of direct interaction with the structure of F0F1-ATPase. Counteracting compounds (sorbitol, mannitol or inositol), despite slightly (10-20%) inhibiting the ATPase activity, also protect the F0F1-ATPase against denaturation by urea. However, this protection was only observed at low urea concentrations (less than 1.5 M), and in the presence of three polyols, the Ki for urea shift from 0.7 M to 1.2 M. Urea also increases the initial activation rate of latent MgATP-SMP in a dose-dependent-manner. However, when the particles (0.5 mg/ml) were preincubated in the presence of 1 M, 2 M or 3 M urea, a decrease in the activation level occurred after 1 h, 30 and 10 min, respectively. At high MgATP-SMP concentration (3 mg/ml) a decrease in activation was observed after 2 h, 1 h and 20 min, respectively. These data indicate that the effect of urea on the activation of MgATP-SMP depends on time, urea and protein concentrations. It was also observed that polyols suppress the activation of latent MgATP-SMP in a dose-dependent manner, and protect the particles against urea denaturation during activation. We suppose that a decrease in membrane mobility promoted by interactions of polyols with phospholipids around the F0F1-ATPase may also increase the compactation of protein structure, explaining the inhibition of natural inhibitor protein of ATPase (IF1) release and the activation of the enzyme.

Adenosine Triphosphate↗
PubMed1983

Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments.

The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer.

Animals↗
PubMed1982

[Inhibition of H2O2 and O2-. generation in the respiratory chain, treated with 2,3-dimercaptopropanol].

Inhibition of electron transport in bovine heart submitochondrial particles resulting from treatment with British antilewisite is accompanied by suppression of superoxide radical and H2O2 generation at the b-c1 site of the respiratory chain. In conjunction with the previously obtained data, the results suggest that the effect of British antilewisite is directed towards the step of ubihydroqinone oxidation to ubisemiquinone by the Rieske FeS-protein.

Animals↗