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PubMed1983

Inhibition of an oligomycin-sensitive ATPase by cationic dyes, some of which are atypical uncouplers of intact mitochondria.

The inhibition of an oligomycin sensitive ATPase prepared from bovine heart submitochondrial particles (J.A. Berden and M.M. Voorn-Brouwer, 1978, Biochim. Biophys. Acta 501, 424-439) by a number of cationic dyes has been compared in order to develop a structure-function relationship. Two generalizations emerge from this comparison. First, the most effective dyes have net positive charge at neutral pH; and second, those dyes containing alkyl substituted secondary and tertiary amino groups are more effective than analogs with primary aromatic amino groups. Some of the cationic dyes exhibit uncoupling activity when added to intact rat liver mitochondria, stimulating both State 4 respiration and the latent ATPase activity. The order of effectiveness and concentrations for maximal stimulation of respiration are: coriphosphine (0.3 microM), Nile blue A (0.5 microM), pyronin Y (0.8 microM), and acridine orange (10 microM). Atypically, oligomycin inhibits the stimulation of respiration by these cationic acid uncouplers. The order of effectiveness and concentrations for maximal stimulation of the latent ATPase are: Nile blue A (2 microM), pyronin Y (8 microM), acridine orange (25 microM), and coriphosphine (75 microM). At concentrations greater than those shown for maximal stimulation, the uncoupling dyes inhibited respiration and the latent ATPase. The cationic dyes tested that were not uncouplers are inhibitors of respiration and the latent ATPase of intact mitochondria at all concentrations tested.

Adenosine Triphosphatases↗
PubMed1983

Purification and characterization of the soluble form of mitochondrial adenosine triphosphatase from sweet potato.

The soluble form of mitochondrial adenosine triphosphatase was purified in an electrophoretically and immunologically pure form from sweet potato root tissue. The enzyme consisted of six kinds of subunits with different molecular weights (52,500, 51,500, 35,500, 26,000, 23,000, and 12,000), and its molecular weight was about 370,000. Adenosine triphosphatase associated with the submitochondrial particles was oligomycin-sensitive and heat-labile, whereas the soluble form of the enzyme was oligomycin-insensitive and cold-labile. The enzyme in either the membrane-bound or the soluble form showed negative cooperativity. Both experiments with polyacrylamide gel electrophoresis and immunological methods suggest that some of the subunits, probably those with molecular weights of 52,500 and 51,500, are dissociated from the enzyme protein during storage of the enzyme preparations.

Adenosine Triphosphatases↗
PubMed1988

Studies on the structure of NADH:ubiquinone oxidoreductase complex: topography of the subunits of the iron-sulfur flavoprotein component.

A catalytic component of the bovine mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a soluble NADH dehydrogenase iron-sulfur flavoprotein (FP). FP is composed of three subunits of Mr 51,000, 24,000, and 9,000, and contains FMN and two iron-sulfur clusters. Previous studies by others with the use of various chemical probes had suggested that, except for an access for NADH to the 51-kDa subunit, the FP polypeptides are buried within Complex I and shielded from the medium. In the present study, monospecific antibodies were raised to each of the three FP subunits, and used in conjunction with Complex I, submitochondrial particles (SMP), mitoplasts, and intact mitochondria as sources of antigens. Results of enzyme-linked immunosorbent assays and 125I-protein A labeling experiments indicated that epitopes from the 51-, 24-, and 9-kDa subunits of FP are exposed to the medium in Complex I and SMP, but not in mitoplasts and mitochondria. Appropriate enzymatic assays showed that none of the antibodies inhibited the NADH dehydrogenase activity of isolated FP or the NADH oxidase activity of SMP. These results have been discussed in relation to the structure of Neurospora Complex I deduced from membrane crystals of the isolated enzyme complex by Leonard et al. [K. Leonard, H. Haiker, and H. Weiss (1987) J. Mol. Biol. 194, 277-286].

Animals↗
PubMed1990

Influence of pH on sulfhydryl groups and fluidity of the mitochondrial membrane.

Fluidity of the red blood cell membrane decreases as pH changes from 8 to 7.5. In rat liver mitochondrial (RLM) membrane fluidity precipitously declines as pH drops from 7.35 toward 7.0. With dithionitrobenzoate (Nbs2), reaction rates of mitochondrial -SH groups from rat liver and heart (RHM) and in beef heart submitochondrial particles are reduced at pH 7.0 as compared to 7.35. Similar results are obtained with the lipophilic fluorescence dye monobromobimane (MB). Bromobimane Q (MQ), which predominantly labels superficially located -SH groups, does not detect differences in -SH reaction rate between pH 7.35 and 7.0. Oligomycin diminishes the amount of reactive -SH groups in RLM titrated with Nbs2 only at pH 7.35, whereas with MB a decrease caused by oligomycin is found at pH 7.35 and pH 7.0. With MQ, an increase in reaction rate is observed for both pH values after pretreatment with oligomycin. Using 4-maleimido-TEMPO mobilization of -SH groups is found with oligomycin at pH 7.0, whereas at pH 7.35 they are immobilized. Phosphate significantly stimulates reaction rates of -SH groups at pH 7.0 in RHM and RLM. In RHM inhibition of succinate oxidation by oxaloacetate as well as the efflux of NAD(P)H is enhanced at pH 7.0, indicating increased permeability in both directions. Decreases in pH, fluidity, and thiol reactivity are important factors in hypoxic/ischemic membrane damage.

Animals↗
PubMed1990

Characterization of tonoplast-enriched vesicles isolated by gradient density fractionation of suspension-cultured cells.

A tonoplast-enriched microsomal fraction was isolated from Catharanthus roseus cells. It was characterized by structural and functional criteria. This fraction presented a homogeneous size distribution as shown by quasi-elastic light scattering and a homogeneous density on self-generated gradients of Percoll. The mean diameter of the vesicles was estimated to be 0.30 micron and the buoyant density around 1.04 g/ml. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of its polypeptide pattern was in good agreement with the one obtained for tonoplast purified from isolated vacuoles. According to enzymatic assays and inhibition tests, this fraction possessed pyrophosphate and ATP-dependent proton pumps and very low contamination by submitochondrial particles, endoplasmic reticula and Golgi membranes. In light of our previous published results on tonoplast purified from isolated vacuoles, the very low extent of AMP hydrolysis by the microsomal fraction is interpreted as supplementary proof in favor of a tonoplast-enriched fraction.

Adenosine Triphosphatases↗
PubMed1992

Effects of adenosine administration on the function and membrane composition of liver mitochondria in carbon tetrachloride-induced cirrhosis.

The effect of chronic carbon tetrachloride (CCl4) administration on liver mitochondria function and the protective action of adenosine on CCl4-induced damage were assessed in rats made cirrhotic by long-term exposure to the hepatotoxin (8 weeks). The CCl4 treatment decreased the ADP-stimulated oxygen consumption, respiratory control, and ADP/O values, mainly for substrates oxidation of site I, in isolated mitochondria. This impaired mitochondrial capacity for substrate oxidation and ATP synthesis was accompanied by an important diminution (approximately 30 mV) of membrane electrical potential. Disturbances of the mitochondrial membrane, induced by CCl4 treatment, were also evidenced as increased mitochondria swelling and altered oscillatory states of mitochondrial volume, both energy-linked processes. The deleterious effects of CCl4 on mitochondrial function were also reflected as a deficient activity of the malate-aspartate shuttle that correlated with abnormal distribution of cholesterol and phospholipids in membranes obtained from submitochondrial particles. Adenosine treatment of CCl4-poisoned rats partially prevented the alterations in mitochondria membrane composition and prevented, almost completely, the impairment of mitochondria function induced by CCl4. Although the nature of the protective action of adenosine on CCl4-induced mitochondria injury remains to be elucidated, such action at this level might play an important role in the partial prevention of liver damage induced by the CCl4.

Adenosine↗
PubMed1975

Inhibition of electron transfer from ferrocytochrome b to ubiquinone, cytochrome c1 and duroquinone by antimycin.

The effect of antimycin on (i) the respiratory activity of the KCN-insensitive pathway of mitochondria of Neurospora grown on chloramphenicol (chloramphenicol-grown) with durohydroquinone and succinate or NADH as substrate, (ii) the electron transfer from the b-type cytochromes to ubiquinone with durohydroquinone as electron donor as well as (iii) the electron transfer from the b-type cytochromes to duroquinone with succinate as electron donor in chloramphenicol-grown Neurospora and beef heart submitochondrial particles was studied. All experiments were performed in the uncoupled state. 1. The respiratory chain of chloramphenicol-grown Neurospora mitochondria branches at ubiquinone into two pathways. Besides the cytochrome oxidase-dependent pathway, a KCN-insensitive branch equiped with a salicylhydroxamate-sensitive oxidase exists. Durohydroquinone, succinate or NADH are oxidized via both pathways. The durohydroquinone oxidation via the KCN-insensitive pathway is inhibited by antimycin, wheras the succinate or NADH oxidation is not. The titer for ful inhibition is one mol antimycin per mol cytochrome b-563 or cytochrome b-557. 2. The electron transfer from durohydroquinone to ubiquinone, which takes place in the KCN-inhibited state, does not occur in the antimycin-inhibited state. 3. The reduction of duroquinone by succinate in the presence of KCN is inhibited by antimycin. The titer for full inhibition is one mol antimycin per mol cytochrome b-566 or cytochrome b-562 for beef heart (or cytochrome b-563 or cytochrome b-557 for Neurospora). 4. When electron transfer from the b-type cytochromes to cytochrome C1, ubiquinone and duroquinone is inhibited by antimycin, the hemes of cytochrome b-566 and cytochrome b-562 (or cytochrome b-563 and cytochrome b-557) are in the reduced state. 5. The experimental results suggest that the two b-type cytochromes form a binary complex the electron transferring activity of which is inhibited by antimycin, the titer for full inhibition being one mol of antimycin per mol of complex. The electron transfer from the b-type cytochromes to ubiquinone is inhibited in a non-linear fashion.

Animals↗
PubMed1975

EPR studies on the respiratory chain of wild-type Saccharomyces cerevisiae and mutants with a deficiency in succinate dehydrogenase.

1. Three nuclear mutants of Saccharomyces cerevisiae deficient in succinate dehydrogenase have been isolated. Two of these mutants are allelic. 2. The amount of covalently bound flavin of submitochondrial particles of the two allelic mutants is about 14% and that of the third mutant about 50% of the amount in wild-type particles. The turnover number of succinate dehydrogenase of particles is decreased in all mutants. The turnover number of fumarate reductase is increased in the two allelic mutants, but decreased in the third mutant. 3. EPR spectra, measured at 82 degrees K, show that the amplitude of the g equals 1.93 signal in particles of the two allelic mutants is less than 10% of that in wild-type particles. It is concluded that iron-sulphur centres other than those of succinate dehydrogenase make only a negligible contribution to the line at g equals 1.93 in wild-type particles. 4. EPR measurements below 20 degrees K show that the amplitude of the signal at g equals 2.01 detected in oxidized particles is decreased in particles of the two allelic mutants. 5. A signal with lines at g equals 2.027 and g equals 1.933 is detected at low temperatures in all particle preparations, even in those from a cytoplasmic petite mutant. It is suggested that this signal is derived from a contaminant and not from the inner membrane.

Cytochromes↗
PubMed1975

A protective function of superoxide dismutase during respiratory chain activity.

(1) Aerobic incubation of heart muscle submitochondrial particles in phosphate buffer after treatment with NADH causes a progressive and substantial inhibition of the NADH oxidation system. Succinate oxidation remains almost unaffected by NADH treatment. (2) The loss of NADH oxidase activity is due to an inhibition of the respiratory chain-linked NADH dehydrogenase. This inhibition of the enzyme is very similar to that caused by combination of the organic mercurial mersalyl with NADH dehydrogenase. (3) The inhibition of NADH oxidation is largely prevented by compounds that are known to react with superoxide ions (02-.), including superoxide dismutase, cytochrome c, tiron and Mn2+. EDTA also has a protective effect, but a number of other metal chelating agents, and several proteins, including catalase, are without effect. (4) It is concluded that the inhibition of NADH oxidation of NADH oxidation by superoxide ions or by mersalyl is reversible and is therefore not due to the loss of oxidoreduction components from the respiratory chain or to an irreversible change in protein conformation. (6) The function of mitochondrial superxide dismutase is discussed in relation to the key role of NADH dehydrogenase in energy-conserving reactions and the formation of hydrogen peroxide during mitochondrial oxidations.

Animals↗
PubMed1975

Membrane potentials in mitochondrial preparations as measured by means of a cyanine dye.

Changes in the fluorescent intensity of the dye 3,3'-dipropylthiodicarbocyanine iodide were measured in suspensions of hamster liver mitochondria upon the development of a K+ diffusion potential by the addition of valinomycin and upon the development of the energized state by the addition of succinate or ATP. The changes (large decreases) seen with the addition of succinate or ATP (inhibitable by NaCN and oligomycin respectively) were comparable to those recorded upon the addition of valinomycin to mitochondria suspended in media containing low concentrations of K+. The change observed with succinate was partially reversed by the addition of either 2,4-dinitrophenol or ADP. Oligomycin prevented the reversal seen with ADP. Decreases in fluorescent intensity were also recorded when succinate was added to suspensions of inner membranes (prepared from rat liver mitochondria) containing the dye. With submitochondrial particles (also from rat liver mitochondria), however, increases in fluorescent intensity were seen upon the addition of succinate or ATP. These observations are consistent with the idea that a large negative (internal) potential develops across the inner membrane of the mitochondrion during energization and with other aspects of the chemiosmotic hypothesis.

Adenosine Diphosphate↗
PubMed1975

Antimycin-insensitive mutants of Candida utilis II. The effects of antimycin on Cytochrome b.

1. Cytochrome b-562 is more reduced in submitochondrial particles of mutant 28 during the aerobic steady-state respiration with succinate than in particles of the wild type. When anaerobiosis is reached, the reduction of cytochrome b is preceded by a rapid reoxidation in the mutnat. A similar reoxidation is observed in the wild type in the present of low concentrations of antimycin. 2. In contrast to the wild type, inhibition of electron transport in the mutant has a much higher antimycin titre than effects on cytochromes b (viz., aerobic steady-state reduction; reduction in the presence of substrate, cyanide and oxygen; the 'red shift' and lowering of E'-o of cytochrome b-562). Moreover, the titration curve of electron transport is hyperbolic whereas the curves for the reduction are sigmoidal. The conclusion is, that in both mutant and wild type, the actions of antimycin on electron transport and cytochromes b are separable. 3. The red shift in the mutant is more extensive than in the wild type. 4. Cytochrome b-558 and cytochrome b-566 (that absorbs in mutant and wild type at 564.5 nm) do not respond simultaneously to addition of antimycin, indicating that they are two separate cytochromes. 5. The difference between the effect of antimycin on electron transport and cytochromes b reduction is also found in intact cells of the mutant. 6. A model is suggested for the wild-type respiratory chain in which (i) the cytochromes b lie, in an uncoupled system, out of the main electron-transfer chain, (ii) antimycin induces a conformation change in QH-2-cytochrome c reductase resulting in effects on cytochrome b and inhibition of electron transport, (iii) a second antimycin-binding site with low affinity to the antibiotic is present, capable of inhibiting electron transport.

Antimycin A↗
PubMed1976

Purification and properties of a low molecular weight protein factor of mitochondrial energy-linked functions.

1. A soluble protein with a molecular weight of 11-12-10(3) has been isolated from bovine-heart mitochondria, which stimulates the following ATP-dependent reactions of submitochondrial particles treated with 0.6 mM EDTA and 1 M NH4OH: reverse electron transfer from succinate to NAD, transhydrogenation from NADH to NADP, and ATP-Pi exchange. The factor has no effect on the NADH oxidase, succinate oxidase and ATPase activities of the particles. 2. The stimulatory effect of the factor in the ATP-dependent reduction of NAD by succinate is 12 mumol-min-1-mg-1 of the factor protein. However, the NH4OH-EDTA treated particles are saturated for maximal activation of the above reaction by very small amounts of the factor (about 20-40 mug factor per mg particle). 3. Electrophoresis of the factor preparation on polyacrylamide gels showed a single protein band plus a nonprotein material which moved at the dye front and was weakly stained with Coomassie Blue. The protein was shown to be required for activation of the particles; whether the fast-moving, nonprotein material is also required is not known. 4. The factor is inhibited by mercurials and N-ethylmaleimide. The former, but not the latter, inhibition is completely reversed by 1,4-dithiothreitol. 5. The NH4OH-EDTA treated particles are also stimulated by rutamycin up to about 0.1 nmol of rutamycin per mg particle; higher rutamycin concentrations inhibit. Depending on the particle preparation, the factor stimulates up to about 3 nmol per mg particle, but does not inhibit at higher concentrations. In addition, under certain conditions in which appropriate concentrations of rutamycin fail to stimulate the particles, the factor still does.

4-Chloromercuribenzenesulfonate↗
PubMed1977

bc1-Complex from beef heart. One-step purification by hydroxyapatite chromatography in Triton X-100, polypeptide pattern and respiratory chain characteristics.

A new simple method for the purification of the bc1-complex has been developed. The polypeptide composition of the complex was analysed by dodecyl sulfate-polyacrylamide gel electrophoresis. The content of chain components and phospholipids was determined. The b-type cytochromes were further characterized by their absorbance spectra and midpoint potentials. (1) Starting from a Triton X-100 extract of submitochondrial particles supplemented with antimycin, the bc1-complex is purified by adsorption chromatography on hydroxyapatite with citrate as specific eluant. (2) The complex splits in dodecyl sulfate into five main polypeptides with apparent molecular weight of 47, 44, 31, 11 and less than 10 kdalton. (3) The purified complex has a heme-b content of 8.0 mumol/g protein and a cytochrome c1 content of 3.8 mumol/g protein. (4) The cytochromes show the typical absorbance spectra of cytochromes b-562 and b-565 and are present in approximately equal amounts with midpoint potentials of Em7 = + 100 mV and Em7 = + mV respectively. Carbon monoxide does not bind to the cytochromes. (5) The nonheme iron protein content of the complex is diminished to 0.6 mumol/g protein. (6) The use of the nonionic surfactant Triton X-100 leads to a complete loss of lipids and ubiquinone of the bc1-complex. (7) The complex contains no succinate dehydrogenase as indicated by the absence of the 69 kdalton subunit in the dodecyl sulfate gel electrophoresis. In addition, it lacks an ubiquinone cytochrome c reductase activity and other electron transferring activities. This may be inferred from an inhibition by antimycin and depletion of ubiquinone and phospholipids. The highly purified and relative stable complex can be prepared giving 50% yield and may be suitable for protein chemistry studies.

Animals↗
PubMed1977

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. II. The beef heart mitochondrial system.

1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP. 2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled. 3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors. 4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules. 5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.

Adenosine Diphosphate↗
PubMed1977

The number of Fe atoms in the iron-sulphur centers of the respiratory chain.

1. From the 57Fe hyperfine interaction in EPR spectra of reduced submitochondrial particles from the yeast Candida utilis, grown with 57Fe, it is concluded that all Fe-S centers in these particles detectable in spectra at 35-80 K are [2Fe-2S]2-(2-; 3-) centers. These are the centers 1 of NADH and succinate dehydrogenase, the Rieske Fe-S center and possibly center 2 of succinate dehydrogenase. 2. The signals of the reduced particles detectable only at temperatures below 20 K are [4Fe-4S]2-(2-; 3-) clusters. These are the centers 2,3 and 4 of NADH dehydrogenase. 3. EPR spectra of the [2Fe-2S]3- centers of Complex I and II, but not that of Complex III, display a great inequality of the Fe nuclei in the effective hyperfine interaction in the x-y direction.

Candida↗
PubMed1978

EPR studies of higher plant mitochondria. II. Center S-3 of succinate dehydrogenase and its relation to alternative respiratory oxidations.

1. An electron paramagnetic resonance study of the high potential iron sulfur (HiPIP-type) Center S-3 of higher plant mitochondria is described. This center is the major HiPIP-type center associated with plant mitochondria and it displays physical properties which are similar to its mammalian counterpart. It has a pH-independent midpoint potential of +65 +/- 10 mV between pH 6.0 and 8.5. 2. The behavior of Center S-3 in a variety of steady-state conditions suggests that it is of physiological significance in electron transport. Furthermore, it can be shown that the alternative oxidase, which is present in many higher plant mitochondria, tends to keep this center oxidized in the presence of succinate and cyanide. This indicates that the alternative oxidation site is on the electron-donating side of the Center S-3. 3. Salicylhydroxamic acid, an inhibitor of the alternative pathway, does not affect the midpoint potential, signal size or shape, or temperature and power saturation profiles of Center S-3, suggesting that direct autoxidation of this center cannot account for alternative oxidase activity. This is further confirmed by the finding that the presence of succinate dehydrogenase is not necessary for alternative oxidase activity with NADH as respiratory substrate in submitochondrial particles.

Binding Sites↗
PubMed1978

Studies on the ATPase complex from beef-heart mitochondria. I. Isolation and characterization of an oligomycin-sensitive and an olgiomycin-insensitive ATPase complex from beef-heart mitochondria.

1. A new method for the isolation of the oliogomycin-sensitive ATPase from beef-heart mitochondria is described. 2. A Triton-soluble ATPase complex was isolated as a by-product of the standard procedure, or as the main product when the submitochondrial particles were pretreated with 1% Triton. The ATPase activity of this complex is sensitive neither to oligomycin nor to dicyclohexylcarbodiimide. 3. The ATPase activity of the oligomycin-sensitive ATPase complex is nearly completely dependent on added phospholipids. The highest activation was found with asolectin. 4. The oligomycin-sensitive complex can be integrated into phospholipid vesicles resulting in an ATP- and Mg2+-dependent energization of the vesicles as monitored with the fluorescent dye 9-amino-6-chloro-2-methoxyacridine. 5. Aurovertin-binding studies based on fluorescence measurement reveal the presence of 1.5 mumol aurovertin-binding sites per g protein for the oligomycin-sensitive complex and about 2.2 mumol for the oligomycin-insensitive complex. 6. The preparation of the oligomycin-sensitive complex contains at least 6--7 polypeptides in addition to those derived from F1. One of these polypeptides, with an apparent molecular weight of 31 000, is virtually absent from the oligomycin-insensitive complex. 7. Some of these polypeptides have been identified and isolated.

Adenosine Triphosphatases↗
PubMed1979

The orientation of iron-sulfur clusters and a spin-coupled ubiquinone pair in the mitochondrial membrane.

Oriented multilayers made from beef heart and yeast mitochondria and submitochondrial particles were studied using electron paramagnetic resonance. EPR signals from membrane-bound iron-sulfur clusters and from a spin-coupled ubiquinone pair are highly orientation dependent, implying that these redox centers are fixed in the membrane at definite angles relative to the membrane plane. Typically the iron-iron axis (gz) of the binuclear iron-sulfur clusters is in the membrane plane. This finding is discussed in terms of the protein structure. The tetranuclear iron-sulfur clusters can have their gz axis either perpendicular or parallel to the membrane plane, but intermediate orientation was not observed.

Animals↗