Characterization of cyanide-insensitive respiration in mitochondria and submitochondrial particles of Moniliella tomentosa

Mitochondria and submitochondrial particles of the osmophilic yeast-like fungus Moniliella tomentosa may respire by means of two pathways: a normal cytochrome pathway, sensitive to cyanide and antimycin A, and an alternative pathway, which is insensitive to these inhibitors but is specifically inhibited by salicylhydroxamic acid. The affinities of both oxidases for succinate and NADH as substrates, for O(2) as terminal electron acceptor, and for AMP as stimulator of the alternative oxidase were determined. 1. Submitochondrial particles of M. tomentosa may also respire by means of a cyanide-sensitive and/or cyanide-insensitive system. 2. The activities of both oxidases as compared with the total activity are roughly the same in submitochondrial particles as in the original mitochondria. 3. The terminal oxidase of the cyanide-insensitive pathway requires a 10-fold higher O(2) concentration for saturation than does cytochrome c oxidase. 4. The apparent K(m) for succinate is about 3 times higher for the alternative than for the normal oxidase when measured in mitochondria, and 4-10 times higher when measured in submitochondrial particles. The apparent K(m) for NADH is roughly the same for both oxidases. 5. The apparent K(m) values of both oxidases for succinate are always lower in submitochondrial particles than in mitochondria. 6. The apparent K(m) for AMP, acting as a stimulator of the alternative oxidase, is the same (25mum) in mitochondria as in sub-mitochondrial particles. These results are discussed in the light of the structure and localization of the components of the alternative oxidase.     

Effects of equisetin on rat liver mitochondria: Evidence for inhibition of substrate anion carriers of the inner membrane

The effect of equisetin, an antibiotic produced byFusarium equiseti, has been studied on mitochondrial functions (respiration, ATPase, ion transport). Equisetin inhibits the DNP-stimulated ATPase activity of rat liver mitochondria and mitoplasts in a concentration-dependent manner; 50% inhibition is caused by about 8 nmol equisetin/mg protein. The antibiotic is without effect either on the ATPase activity of submitochondrial particles or on the purified F₁-ATPase. It inhibits both the ADP- or DNP-activated oxygen uptake by mitochondria in the presence of glutamate + malate or succinate as substrates, but only the ADP-stimulated respiration is inhibited if the electron donors are TMPD + ascorbate. It does not affect the NADH or succinate oxidation of submitochondrial particles. Equisetin inhibits in a concentration-dependent manner the active Ca²⁺-uptake of mitochondria energized both by ATP or succinate without affecting the Ca²⁺-uniporter itself. The antibiotic inhibits the ATP-uptake by mitochondria (50% inhibition at about 8 nmol equisetin/mg protein) and the P_i and dicarboxylate carrier. It does not lower the membrane potential at least up to 200 nmol/mg protein concentration. The data presented in this paper indicate that equisetin specifically inhibits the substrate anion carriers of the mitochondrial inner membrane.Abbreviations EGTA ethyleneglycol bis/-aminoethylether/-N, N-tetraacetic acid - DNP 2, 4-dinitrophenol - TMPD N,N,N,N,tetramethyl-p-phenylenediamine - CCP carbonylcyanide-m-chlorophenyl hydrazone - TPP tetraphenyl-phosphonium - Hepes /4,(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid/     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles.

1. Beef heart mitochondria have a cytochrome c1:c:aa3 ratio of 0.65:1.0:1.0 as isolated; Keilin-Hartree submitochondrial particles ahve a ratio of 0.65:0.4:1.0. More than 50% of the submitochondrial particle membrane is in the 'inverted' configuration, shielding the catalytically active cytochrome c. The 'endogenous' cytochrome c of particles turns over at a maximal rate between 450 and 550 s-1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300-400 s-1, at 28 degrees-30 degrees C, pH 7.4. 2. Ascorbate plus N,N,N',N'-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5-547 nm and 550-556.5 nm, respectively. 3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate + N,N,N',N'-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c1/c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

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].     

Effects of quercetin and F1 inhibitor on mitochondrial ATPase and energy-linked reactions in submitochondrial particles

Quercetin (3,3′,4′,5,7-pentahydroxyflavone) shares certain properties with the mitochondrial ATPase inhibitor protein. At low concentrations it inhibits both soluble and particulate mitochondrial ATPase and has no effect on oxidative phosphorylation in submitochondrial particles. Unlike the mitochondrial inhibitor protein quercetin inhibits the ATP-dependent reduction of NAD⁺ by succinate in fully reconstituted submitochondrial particles. A comparison of various flavones indicates that the hydroxyl groups at the 3′ and perhaps 3 position are important for the inhibition of ATPase activity.     

Specificity of the Organic Acid Activation of Alternative Oxidase in Plant Mitochondria

The claim that succinate and malate can directly stimulate the activity of the alternative oxidase in plant mitochondria (A.M. Wagner, C.W.M. van den Bergen, H. Wincencjusz [1995] Plant Physiol 108: 1035-1042) was reinvestigated using sweet potato (Ipomoea batatas L.) mitochondria. In whole mitochondria, succinate (in the presence of malonate) and both L- and D-malate stimulated respiration via alternative oxidase in a pH- (and NAD+)-dependent manner. Solubilized malic enzyme catalyzed the oxidation of both L- and D-malate, although the latter at only a low rate and only at acid pH. In submitochondrial particle preparations with negligible malic enzyme activity, neither L- nor D-malate stimulated alternative oxidase activity. However, even in the presence of high malonate concentrations, some succinate oxidation was observed via the alternative oxidase, giving the impression of stimulation of the oxidase. Neither L-malate nor succinate (in the presence of malonate) changed the dependence of alternative oxidase activity on ubiquinone reduction state in submitochondrial particles. In contrast, a large change in this dependence was observed upon addition of pyruvate. Half-maximal stimulation of alternative oxidase by pyruvate occurred at less than 5 [mu]M in submitochondrial particles, one-twentieth of that reported for whole mitochondria, suggesting that pyruvate acts on the inside of the mitochondrion. We suggest that malate and succinate do not directly stimulate alternative oxidase, and that reports to the contrary reflect intra-mitochondrial generation of pyruvate via malic enzyme.     

Mode of inhibition of mitochondrial energy transduction by chlorophenoxyisobutyrate

1. alpha-p-Chlorophenoxyisobutyric acid, the ethyl ester of which is widely used as an antihypercholesterolaemic drug, is an inhibitor of energy-transfer reactions in isolated rat liver mitochondria. 2. The compound at lower concentrations (<4.0mumol/mg of mitochondrial protein) inhibits state 3 oxidation, stimulates state 4 oxidation, abolishes respiratory control and stimulates the latent adenosine triphosphatase activity of mitochondria. The inhibition imposed on state 3 oxidation is relieved by dinitrophenol. 3. At higher concentrations it inhibits coupled phosphorylation as well as dinitrophenol-stimulated adenosine triphosphatase activity. The inhibition of state 3 oxidation under these conditions is not reversed by uncouplers. 4. The three coupling sites of phosphorylation exhibit differential susceptibility to inactivation by this compound. Coupled phosphorylation at the first site is abolished at a drug concentration of 3.0mumol/mg of protein. The third site is inactivated when the concentration of the drug reaches 5.0mumol/mg of protein. The second site is the most refractory and drug concentrations of the order of 10.0mumol/mg of protein are required effectively to inhibit phosphorylation at this site. 5. The compound also inhibits ATP-dependent reversal of electron transport as well as the adenosine triphosphatase activity in submitochondrial particles. 6. The oxidation of NADH and succinate in these particles is not inhibited. 7. These properties indicate that the compound acts as an ;inhibitory uncoupler' of energy-transfer reactions in isolated mitochondria.     

Respiration-department uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles.

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitchondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATP-ase activity was stimulated at higher concentrations of uncouplers. 2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response. 3. The addition of succinate, NADH or ascorbate/N,N,N'-N'-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin. 4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin. 5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity. 6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.     

Effect of luliberin on the activities of mitochondrial respiratory enzymes]

Luliberin, a luteinizing hormone-releasing hormone, was shown to inhibit the respiratory enzymes of rat liver mitochondria and submitochondrial particles prepared from beef heart mitochondria. At the hormone concentration of 8.10(-6) M the NADH-oxidase activity of the submitochondrial particles was inhibited by 50%. The fragments of the hormone and its analogs and pyroglutamic acid, oxytocin and bradikinin possessed practically no inhibiting effects. In the case of submitochondrial particles the inhibition was only observed in the presence of Ca2+ and was significantly decreased after addition of bovine serum albumin and phospholipase inhibitors -- butacaine and dicaine. It is assumed that the effect of luliberin on the respiratory chain is mediated through mitochondrial phospholipase.     

The antioxidant functions of cytochrome c

Low (C(1/2) = 1.5 x 10(-7) M) concentrations of horse cytochrome c strongly inhibit H(2)O(2) production by rat heart mitochondria under conditions of reverse electron transfer from succinate to NAD(+). The effect is abolished by binding of cytochrome c with liposomes and is not prevented by SOD. Yeast cytochrome c is much less effective than the horse protein whereas acetylated horse cytochrome c is without effect. H(2)O(2) formation stimulated by antimycin A is resistant to added cytochrome c. In inside-out submitochondrial vesicles, H(2)O(2) production is suppressed by all three cytochrome c samples tested, but at higher concentrations (C(1/2) is about 5 x 10(-7) M). In vesicles, SOD abolishes the cytochrome c inhibition. We conclude that extramitochondrial cytochrome c is competent in down-regulation of the Complex I H(2)O(2) production linked to the reverse electron transfer. Such an effect is absent in the inside-out submitochondrial vesicles where another antioxidant cytochrome c function can be observed, i.e. the oxidation of O(2-*) to O(2). A possible role of cytochrome c in the antioxidant defence is discussed.     

Intramitochondrial positions of ubiquinone and iron-sulphur centres determined by dipolar interactions with paramagnetic ions.

E.p.r. (electron-paramagnetic-resonance) spectra of ubisemiquinone (QH) organic radicals and all of the known iron-sulphur centres were studied in normal and 'nickle-plated' pigeon heart mitochondria, submitochondrial particles and submitochondrial particles from which succinate dehydrogenase had been removed. Incubation of pigeon heart mitochondria, submitochondrial particles or succinate dehydrogenase-depleted submitochondrial particles with substrate in the presence of pure O2 results in the accumulation of Q-H. In mitochondria, the e.p.r. spectrum of Q-H is characterized by in-homogeneous line broadening. A heterogeneous population of semiquinones appears to be partly responsible for these effects in mitochondria. Additon of Ni(II) to mitochondria renders saturation of the Q-H resonance more difficult. On the other hand, the resonance in either submitochondrial particles or succinate dehydrogenase-depleted particles is narrower than the same spectrum in mitochondria, and saturates like a homogeneous line. The presence of Ni(II) in either of these preparations, further, has no effect on either the A-H spectrum or the saturation curve. Therefore QH appears to be situated on the exterior surface of the mitochondrion. Likewise, the e.p.r. spectra and saturation curves of iron-sulphur centre N-2 exhibit characteristics of inhomogeneous line broadening, not only in intact mitochondria but also in both submitochondrial particles and succinate dehydrogenase-depleted particles. Because of the small pool size of centre N-2, this effect is likely to arise from a spin interaction with some other component in the membrane. Ni(II) has no effect on the saturation in centre N-2 in mitochondria or submitochondrial particles, and only a marginal effect in the succinate dehydrogenase-depleted preparation. These results are indeterminate with respect to the position of centre N-2 in the membrane; but suggest that its distance from the succinate dehydrogenase binding site is on the order of 1 nm. All of the other ferredoxin-type iron-sulphur centres in both preparations were not affected by paramagnetic ions. Homogeneous e.p.r. spectra and saturation curves are observed for both of the HiPIP-type (high-potential iron-sulphur protein-type) iron-sulphur centres in mitochondrial centres S-3 and bc-3. Addition of No(II) to intact mitochondria results in a dipolar interaction with centre bc-3. No effect was observed on centre S-3 in either preparation. A comprehensive model is presented for the structure of the respiratory electron-transport system in mitochondria, based on e.p.r. relaxation studies in the present and the preceding paper. There is no direct evidence for transmembrane electron flow through any of the known energy-coupling sites in mitochondria, so that direct hydrogen atom transfer across the membrane (as a combination of H+ translocation coupled to electron flow) does not occur...     

Citreoviridin, a specific inhibitor of the mitochondiral adenosine triphosphatase.

1. Citreoviridin was a potent inhibitor of the soluble mitochondrial ATPase (adenosine triphosphatase) similar to the closely related aurovertins B and D. 2. Citreoviridin inhibited the following mitochondrial energy-linked reactions also: ADP-stimulated respiration in whole mitochondria from ox heart and rat liver; ATP-driven reduction of NAD+ by succinate; ATP-driven NAD transhydrogenase and ATPase from ox heart submitochondrial particles. 3. The dissociation constant (KD) calculated by a simple law-of-mass-action treatment for the citreoviridin--ATPase complex was 0.5--4.2micron for ox-heart mitochondrial preparations and 0.15micron for rat liver mitochondria. 4. Monoacetylation of citreoviridin decreased its inhibitory potency (KD=2--25micron, ox heart; KD=0.7micron, rat liver). Diacetylation greatly decreased the inhibitory potency (KD=60--215micron, ox heart). 5. Hydrogenation of citreoviridin monoacetate diminished its inhibitory potency considerably. 6. No significant enhancement of fluorescence was observed when citreoviridin interacted with the mitochondrial ATPase.     

2,3-butanedione monoxime unmasks Ca(2+)-induced NADH formation and inhibits electron transport in rat hearts

We used 2,3-butanedione monoxime (BDM) to suppress work by the perfused rat heart and to investigate the effects of calcium on NADH production and tissue energetics. Hearts were perfused with buffer containing BDM and elevated perfusate calcium to maintain the rates of cardiac work and oxygen consumption at levels similar to those of control perfused hearts. BDM plus calcium hearts displayed higher levels of NADH surface fluorescence, indicating calcium activation of mitochondrial dehydrogenases. These hearts, however, displayed 20% lower phosphocreatine levels. BDM suppressed the rates of state 3 respiration of isolated mitochondria. Uncoupled respiration was suppressed to a lesser degree, and the state 4 respiration rates were not affected. Double-inhibitor experiments with liver mitochondria using BDM and carboxyatractyloside (CAT) were used to identify the site of inhibition. BDM at low levels (0-5 mM) suppressed respiration. In the presence of CAT at levels that inhibit respiration by 60%, low levels of BDM were without effect. Because these effects were not additive, BDM does not inhibit adenine nucleotide transport. This was supported by an assay of adenine nucleotide transport in liver mitochondria. BDM did not inhibit ATP hydrolysis by submitochondrial particles but strongly suppressed reversed electron transport from succinate to NAD(+). Oxidation of NADH by submitochondrial particles was inhibited by BDM but oxidation of succinate was not. We conclude that BDM inhibits electron transport at site 1.     

On the mechanism of action of alkylguanidines on oxidative phosphorylation in mitochondria.

The effect of octylguanidine and oligomycin on the oxygen uptake of rat liver mitochondria and on the ATPase activity of "sonic" submitochondrial particles has been studied. 1. Octylguanidine inhibits state 3 respiration with glutamate-malate and succinate as substrates, but much lower concentrations are required to inhibit oxygen uptake with the former substrates. State 4 respiration is unaffected by octylguanidine. 2. The titration-curve for the octylguanidine inhibition of glutamate-malate oxidation is hyperbolic and apparently biphasic, half-maximal inhibition is obtained at 30 muM octylguanidine. The octylguanidine-curve for inhibition of succinate oxidation is sigmoid with half-maximal inhibition at about 250 muM. 3. Octylguanidine and oligomycin show additive inhibitory action on state 3 respiration with both glutamate plus malage and succinate as respiratory substrates. 4. Concentrations of oligomycin or octylguanidine, which added separately are ineffective on state 3 respiration, become inhibitory when the two inhibitors are added together. 5. Octylguanidine inhibits the ATPase activity of sonic submitochondrial particles with a hyperbolic titration-curve analogous to that obtained for oligomycin inhibition. The inhibitory actions of octylguanidine and oligomycin on the ATPase activity are additive. 6. It is concluded that octylguanidine acts directly on the ATPase complex and that its binding at the action site is mutually exclusive with the binding of oligomycin. A kinetic explanation is given for the reported higher sensitivity of site I phosphorylation to octylguanidine.     

Respiration-dependent uncoupler-stimulated ATPase activity in castor bean endosperm mitochondria and submitochondrial particles

1. The uncoupler-stimulated ATPase activity of castor bean endosperm mitochondria and submitochondrial particles has been studied. The rate of ATP hydrolysis catalyzed by intact mitochondria was slow and little enhanced by addition of uncouplers at the concentration required for uncoupling the oxidative phosphorylation. ATPase activity was stimulated at higher concentrations of uncouplers.

2. 1-Anilinonaphthalene 8-sulfonate fluorescence was decreased when the mitochondria were oxidizing succinate. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone and antimycin reversed the succinate-induced fluorescence diminution. ATP did not induce the fluorescence response.

3. The addition of succinate, NADH or ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as electron donor induced high ATPase activity in the presence of low concentrations of uncouplers. Stimulating effect of uncouplers was completely abolished by further addition of antimycin.

4. Submitochondrial particles were prepared by sonication. The particles catalyzed a rapid hydrolysis of ATP and carbonylcyanide-p-trifluoromethoxyphenylhydrazone at 10^-8 M did not stimulate the ATPase activity. Addition of succinate induced uncoupler-stimulated ATPase activity. The effect of succinate was completely abolished by further addition of antimycin.

5. The treatment of submitochondrial particles by trypsin or high pH also induced uncoupler-stimulated ATPase activity.

6. The above results were interpreted to indicate that ATPase inhibitor regulated the back-flow reaction of mitochondrial oxidative phosphorylation.     

The respiratory chain in a ubiquinone-deficient mutant of Saccharomyces cerevisiae.

1. Two allelic mutants of Saccharomyces cerevisiae with a deficiency in the biosynthesis of ubiquinone have been isolated. The properties of one particular mutant strain were investigated. Submitochondrial particles of this strain contain maximally 3% of the amount of ubiquinone in wild-type particles; the amounts of other components of the respiratory chain are essentially normal. 2. The respiratory rates of mutant cells, mitochondria and submitochondrial particles are low with ubiquinone-dependent substrates, but are restored to normal levels by addition of Q-1; the restored respiration is antimycin sensitive. Intact cells and mitochondria show respiratory control both in the absence and presence of Q-1. 3. The NADH:Q-1 oxidoreductase of submitochondrial particles of the mutant followspseudo first-order kinetics in [Q-1]. QH2-1 inhibits competitively with respect to Q-1, the Ki for QH2-1 being equal to the Km for Q-1. 4. Succinate dehydrogenase in both wild-type and mutant submitochondrial particles can be activated by NADH. 5. The turnover number of succinate dehydrogenase in the mutant, measured with phenazine methosulphate as primary electron acceptor, is about one-half that of wild-type particles. The turnover numbers measured with Q-1 as electron acceptor are about the same in the two types of particles. 6. The kinetics of redox changes in cytochrome b, in the presence of antimycin and oxygen, are distinctly different in the mutant and wild-type particles. They indicate that ubiquinone plays an important role in the phenomenon of the increased reducibility of cytochrome b induced by antimycin plus oxygen.     

Activation of electron transport at the terminal site of the respiratory chain by the submitochondrial fluid from the rat and guinea pig liver

Supermitochondrial liquid (SL) of rat and guinea-pig liver increases the activity of 2, 3, 5 triphenyltetrasolium chloride (TPC) and tetrasolium violet (TV) reduction at succinate, NADH and NADPH oxidation by mitochondria (MC). SL contains an activating factor A, being evidently of a protein nature and factor B, increasing the activating activity of factor A. NAD, NADP, NADH and NADP at 5 x 10(-5)-1 x 10(-4) M concentration activate the TPC and TV reduction at succinate oxidation by mitochondria. TPC and TV reduction at succinate and NADP oxidation by mitochondria makes antimicin and cyanide sensitive. SL does not influence succinate dehydrogenase activity, when used as electron acceptors of ferricyanide, blue Vurster, cytochrome C, blue and violet nitrotetrasolium. Activation of electron transfer chair between cytochrome C and oxygen is supposed to be responsible for such an effect.     

The metabolic effects and uptake of ethidium bromide by rat liver mitochondria.

The uptake of ethidium bromide by rat liver mitochondria and its effect on mitochondria, submitochondrial particles, and F₁ were studied. Ethidium bromide inhibited the State 4-State 3 transition with glutamate or succinate as substrates. With glutamate, ethidium bromide did not affect State 4 respiration, but with succinate it induced maximal release of respiration. These effects appear to depend on the uptake and concentration of the dye within the mitochondrion. In submitochondrial particles, the aerobic oxidation of NADH is much more sensitive to ethidium bromide than that of succinate. Ethidium bromide partially inhibited the ATPase activity of submitochondrial particles and of a soluble F₁ preparation. Ethidium bromide behaves as a lipophilic cation which is concentrated through an energy-dependent process within the mitochondria, producing its effects at different levels of mitochondrial function. The ability of mitochondria to concentrate ethidium bromide may be involved in the selectivity of the dye as a mitochondrial mutagen.     

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.     

Binding and inhibitory effect of 2-heptyl-4-hydroxyquinoline-N-oxide in the presence of ubiquinone-3 in Saccharomyces cerevisiae

1. 2-Heptyl-4-hydroxyquinoline-N-oxide (HpHOQnO) binds to oxidized mitochondria of Saccharomyces cerevisiae with a dissociation constant of 5 x 10(-8) M and with a ratio of 1 mol per mol cytochrome b. 2. After addition of 171 micro M ubiquinone-3 to oxidized mitochondria, 0.3 mol HpHOQnO bind to 1 mol cytochrome b. The binding strength of the inhibitor is not essentially affected. 3. Reduction of mitochondria with succinate results in a strong decrease of the HpHOQnO binding. The dissociation constant could not be calculated on the basis of the applied method. 4. On the basis of the inhibitory effect of HpHOQnO on reduced mitochondria, a dissociation constant of 5 x 10(-6) M is calculated. 5. The respiratory electron flow to oxygen is about 100 times as sensitive to HpHOQnO as the electron flow to cytochrome c. 6. Ubiquinone-3 reverses the inhibition by HpHOQnO and stimulates the electron flow. 7. The different influence of ubiquinone-3 in binding and in inhibition experiments is discussed as a result of redox conditions.