The respiratory chain of Paramecium tetraurelia in wild type and the mutant Cl1. II. Cyanide-insensitive respiration. Function and regulation.

1. The cyanide-insensitive respiration in Paramecium tetraurelia was found to be located in mitochondria. 2. Sensitivity of the mitochondrial respiration to cyanide depended on growth conditions. Under standard conditions of growth, 15--20% of respiration was insensitive to 1 mM cyanide. Full resistance to 1 mM cyanide was observed by growing cells in the presence of erythromycin (100--400 microgram/ml) 0.2 mM cyanide. The mitochondrial respiration of the mutant Cl1 harvested during the exponential phase of growth was largely insensitive to cyanide (more than 80%). 3. Pyruvate was oxidized at the same rate by wild type mitochondria and mitochondria of the mutant Cl1. In contrast, succinate oxidation was 2--3 times faster in mitochondria of the mutant Cl1 than in wild type mitochondria. 4. The cyanide-insensitive respiration was inhibited by 1 mM salicylhydroxamic acid to nearly 100%. Other efficient respiratory inhibitors included amytal and heptylhydroxyquinoline. Antimycin was not inhibitory even at concentrations as high as 5 microgram/mg protein, a finding consistent with the lack of antimycin binding sites.     

The alternate respiratory pathway of Candida albicans

Candida albicans contains a cryptic cyanide and antimycin A insensitive respiratory system. This alternate oxidase was found (i) at all growth rates from =0.05 to 0.26 in a chemostat culture and (ii) in both mycelial and yeast forms of the organism. Neither chloramphenicol nor cycloheximide prevented the expression of the alternate oxidase. Salicyl-hydroxamic acid was a potent inhibitor of the cyanide insensitive respiration. The respiration of mitochondria grown in the presence of antimycin A was not inhibited by cyanide or antimycin A but was inhibited by salicylhydroxamic acid.Abbreviations KCN potassium cyanide - SHAM salicyl hydroxamic acid     

Cytochemical localization of peroxidase activity in the miracidium of Schistosoma mansoni.

Distribution of peroxidase activity in the mitochondria of the miracidium of the blood fluke, Schistosoma mansoni, was investigated cytochemically using the diaminobenzidine (DAB) technique. Re-action product was localized in the mitochondria of this larvae stage at pH 7.4 and 9.7. The reaction was peroxide-dependent and insensitive to either potassium cyanide, sodium azide, or 3-amino-1,2,4-triazole at the concentrations used. The reaction was inactivated by heat and by pretreatment with methanol-nitro-ferricyanide, and inhibitor of peroxidase. A perioxide-independent reaction was also observed in the mitochondria. This latter reaction was sensitive to potassium cyanide and sodium azide. It is hypothesized that the peroxidase either may act where peroxide is an electron acceptor in a flavoprotein-linked system or may be a vestige of a more primitive pathway. No peroxidase activity was observed in the mitochondria of other stages of the life cycle of the worm.     

Adenosine 5''-monophosphate-stimulated cyanide-insensitive respiration in mitochondria of Moniliella tomentosa.

Mitochondria of the yeastlike fungus Moniliella tomentosa oxidize reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, succinate, isocitrate, and lactate. These oxidations are completely inhibited by cyanide or antimycin A in mitochondria isolated from cells grown in the standard medium. On the other hand, the oxidation of all substrates, except lactate, is almost completely insensitive to cyanide or antimycin A in mitochondria from cells grown in the presence of ethidium bromide. In this instance, the oxidation is mainly mediated by an alternate oxidase which can be blocked by salicyl hydroxamic acid. The alternate oxidase can be specifically stimulated by adenosine 5'-monophosphate and this provides a new method for the characterization of the alternate oxidase in mitochondria of M. tomentosa.     

Cysteine biosynthesis, in concert with a novel mechanism, contributes to sulfide detoxification in mitochondria of Arabidopsis thaliana

In higher plants, biosynthesis of cysteine is catalysed by OAS-TL [O-acetylserine(thiol)lyase], which replaces the activated acetyl group of O-acetylserine with sulfide. The enzyme is present in cytosol, plastids and mitochondria of plant cells. The sole knockout of mitochondrial OAS-TL activity (oastlC) leads to significant reduction of growth in Arabidopsis thaliana. The reason for this phenotype is still enigmatic, since mitochondrial OAS-TL accounts only for approximately 5% of total OAS-TL activity. In the present study we demonstrate that sulfide specifically intoxicates Complex IV activity, but not electron transport through Complexes II and III in isolated mitochondria of oastlC plants. Loss of mitochondrial OAS-TL activity resulted in significant inhibition of dark respiration under certain developmental conditions. The abundance of mitochondrially encoded proteins and Fe-S cluster-containing proteins was not affected in oastlC. Furthermore, oastlC seedlings were insensitive to cyanide, which is detoxified by β-cyano-alanine synthase in mitochondria at the expense of cysteine. These results indicate that in situ biosynthesis of cysteine in mitochondria is not mandatory for translation, Fe-S cluster assembly and cyanide detoxification. Finally, we uncover an OAS-TL-independent detoxification system for sulfide in mitochondria of Arabidopsis that allows oastlC plants to cope with high sulfide levels caused by abiotic stresses.     

Evidence for a structural interacti on betwen ATP synthetase and cytochrome c oxidase in mitochondria

The reaction of cyanide with the oxidized form of cytochrome c oxidase in mitochondria is strongly inhibited by adenosine triphosphate (ATP). This inhibition is strictly dependent on the ATP concentration and is insensitive to changes in the concentrations of adenosine diphosphate (ADP) and orthophosphate. It is completely prevented by oligomycin or uncouplers of oxidative phosphorylation. The ATP is kinetically competitive with respect to cyanide and has a measured inhibitor constant of less than 2 μm The stoichiometry is one ATP/cyanide. This ATP effect is proposed to result from a structural interaction of ATP synthetase with cytochrome c oxidase, such that the formation of an ATP complex of the synthetase results in a decrease in the affinity of the oxidized form of cytochrome c oxidase for cyanide in the formation of an intermediate in the overall measured cyanide reaction.     

Mitochondria and other calcium buffers of squid axon studied in situ

Continuous nondestructive monitoring of intracellular ionized calcium in isolated squid axons by differential absorption spectroscopy (using arsenazo III and antipyrylazo III) was used to study uptake of calcium by carbonyl cyanide, p-trifluoromethoxy-phenylhydrazone (FCCP)- and (or) cyanide (CN)-sensitive and insensitive constituents of axoplasm. Known calcium loads imposed on the axon by stimulation produced proportional increments of free axoplasmic calcium. Measurement of increments in ionized calcium as a function of load confirmed earlier reports of buffering in normal and FCCP- and (or) CN-poisoned axons. Measurement of rates of calcium uptake by presumed mitochondria showed little uptake at ambient Ca below 200--400 nM, with sigmoidal rise to about 20--30 mumol/kg axoplasm per min (calculated to be about 200 mmol/kg mitochondrial protein per min) at 50 micrometer, indicating a functional threshold for presumed mitochondrial uptake well above physiological ionized calcium concentration. Treatment of stimulated axons with cyanide, to release calcium from presumed mitochondria, showed that the sensitivity to cyanide decreased progressively with time after stimulation (t 1/2 = 3--10 min) implying transfer of sequestered calcium into a less metabolically labile form.     

Partial Purification and Characterization of the Quinol Oxidase Activity of Arum maculatum Mitochondria

The menadiol oxidase activity of Arum maculatum mitochondria has been solubilized and fractionated. A preparation has been obtained which has an increased specific activity and a greatly decreased polypeptide composition when compared to the mitochondria. This preparation retains normal inhibitor sensitivities in that the oxidation of menadiol remains insensitive to cyanide and is inhibited by aromatic hydroxamates. Metal analyses of the preparation showed that only iron was closely correlated with the oxidase activity. No unusual lipid components were detected in the preparation. The results are discussed in relation to chemical quinol oxidation mechanisms and to several recent hypotheses concerning the nature of the higher plant alternative oxidase.     

Oxidative interactions between fatty acid peroxy radicals and quinones: Possible involvement in cyanide-resistant electron transport in plant mitochondria

The interactions between duroquinol, linoleic acid, and lipoxygenase have been followed spectrophotometrically in the uv (210–340 nm) in a simple reaction medium (5 mm [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] buffer, pH 6.0). Duroquinol is oxidized by reacting with the peroxy radicals of linoleic acid generated in the presence of lipoxygenase. This oxidation is insensitive to cyanide but is sensitive to salicylhydroxamic acid, propylgallate, and disulfiram, the known inhibitors of lipoxygenase and of the cyanide-resistant electron transport pathway of plant mitochondria. This reaction is proposed as a model for the functioning of this pathway in plant mitochondria.     

Malic enzyme activity and cyanide-insensitive electron transport in plant mitochondria.

In the presence of exogenous NAD⁺, malate oxidation by cauliflower mitochondria takes place essentially via an electron transport pathway that is insensitive to rotenone, antimycin and cyanide but is strongly sensitive to salicyl hydroxamic acid. It bypasses all phosphorylation sites. NAD⁺ is reduced by an enzyme identified as malic enzyme (L-malate:NAD oxidoreductase (decarboxylating), EC 1.1.1.39). The NADH produced is reoxidized by an internal rotenone-insensitive NADH dehydrogenase that yields electrons directly to the cyanide-insensitive pathway.     

The oxidation of external NADH by an intermembrane electron transfer in mitochondria from the ubiquinone-deficient mutant E3-24 of Saccharomyces cerevisiae

Cells of the E3-24 mutant of the strain D273-10B of Saccharomyces cerevisiae, grown in a fermentable substrate not showing catabolite repression of respiration (2% galactose), are able to respire, in spite of their ubiquinone deficiency in mitochondrial membranes. Mitochondria isolated from these mutant cells oxidize exogenous NADH through a pathway insensitive to antimycin A but inhibited by cyanide. Addition of methanolic solutions of ubiquinone homologs stimulates the oxidation rate and restores antimycin A sensitivity in both isolated mitochondria and whole cells. Mersalyl preincubation of isolated mitochondria inhibits both NADH oxidation and NADH-cytochrome c oxido-reductase activity (assayed in the presence of cyanide) with the same pattern. Electrons resulting from the oxidation of exogenous NADH reduce both cytochrome b5 and endogenous cytochrome c. The increase in ionic strength stimulates NADH oxidation, which is also coupled to the ATP synthesis with an ATP/O ratio similar to that obtained with ascorbate plus N,N,N',N'-tetramethyl-p-phenylendiamine (TMPD) as substrate. The effect of cyanide on these activities and on NADH-induced endogenous cytochrome c reduction is also comparable. These results support the existence in vivo and in isolated mitochondria of a energy-conserving pathway for the oxidation of cytoplasmatic NADH not related to the dehydrogenases of the inner membrane, the ubiquinone, and the b-c1 complex, but involving a cytochrome c shuttle between the NADH-cytochrome c reductase of the outer membrane and cytochrome oxidase in the inner membrane.     

Microspectrofluorometry of the protonation state of ellipticine, an antitumor alkaloid, in single cells.

The protonation state and intracellular distribution of ellipticine were investigated in single human mammary T47D cells by confocal laser microspectrofluorimetry. In the cell nucleus, only the protonated form of ellipticine was detected as a direct consequence of its apparent pK increase upon DNA binding. Both protonated and neutral forms were present in the aqueous cytoplasm, where the pH is close to the drug pK. When cells were incubated in high concentrations of K+, a condition that depolarizes the plasma membrane potential, ellipticine cellular accumulation was reduced. In the cytoplasm, ellipticine was mainly bound to mitochondria, and its protonation equilibrium was shifted toward the neutral form. The fluorescence spectrum of ellipticine bound to mitochondria was insensitive to valinomycin, whereas it was markedly shifted toward the protonated form after carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or nigericin addition. Similar studies with ellipticine bound to isolated mitochondria suggest that it behaves as a fluorescent probe of mitochondrial pH in both isolated mitochondria and single living cells.     

Transport and metabolism of D-lactate in Jerusalem artichoke mitochondria

We report here initial studies on D-lactate metabolism in Jerusalem artichoke. It was found that: 1) D-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-D-lactoyl-glutathione. 2) Externally added D-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) D-lactate was metabolized inside mitochondria by a flavoprotein, a putative D-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added D-lactate by means of a D-lactate/H(+) symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial D-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

The effect of phosphate deficiency on mitochondrial activity and adenylate levels in bean roots

Bean plants ( Phaseolus vulgaris ) were grown for 16–20 days with or without phosphate in Knop nutrient medium. It was found in previous experiments that for roots grown on a P_i-deficient medium respiration is mainly carried out by the cyanide-insensitive pathway. Mitochondria isolated from—P_i, roots had poor respiratory control and their respiration exhibited 62% inhibition by cyanide and was inhibited (30%) by salicylhydroxamic acid (SHAM). In contrast, mitochondria obtained with control (+P_i) roots had respiratory control and ADP/O ratios typical for succinate as the substrate; their respiration was inhibited to 95% by cyanide and insensitive to SHAM. The integrity of mitochondrial membranes was similar in both types of mitochondria. Cytochrome oxidase activity, however, was about 20% lower in -P_i mitochondria, but the cytochrome composition was the same in both types of mitochondria. The cytochrorae pathway was not operating at full capacity in mitochondria isolated from—P_i roots but the alternative oxidation pathway participated in a great part in mitochondrial respiration, similar to in vivo whole roots. The participation of the non-phosphorylating., alternative pathway decreased the respiratory control ratio in mitochondria and had an effect on the total adenine nucleotide pool and energy charge values which were lower (16 and 13% respectively) in -P_i roots. About 50% lower ADP and 20% lower ATP levels were observed whereas AMP levels were several times higher.     

The Effect of Respiratory Inhibitors on NADH, Succinate and Malate Oxidation in Corn Mitochondria

The effect of a series of respiratory inhibitors on the oxidation of NADH in state 4 and state 3 conditions was studied with corn shoot mitochondria. Comparisons were made using malate and succinate as substrates. The inhibitors, rotenone, amytal, antimycin A and cyanide, inhibited oxidation of NADH in state 3 but rotenone and amytal did not inhibit oxidation in state 4. The inhibition by antimycin A was partially overcome by the presence of cytochrome c. The results indicate the presence of alternative pathways available for NADH oxidation depending on the metabolic condition of the mitochondria. Under state 4 conditions, NADH oxidation bypasses the amytal and rotenone sensitive sites but under state 3 conditions a component of the NADH respiration appears to be oxidized by an internal pathway which is sensitive to these inhibitors. Still a third pathway for NADH oxidation is dependent on the addition of cytochrome c and is insensitive to antimycin A. Succinate oxidation was sensitive to cyanide and antimycin A under both state 4 and state 3 conditions as well as amytal and rotenone under state 3 conditions but was not inhibited by amytal and rotenone under state 4 conditions. Malate oxidation was inhibited by cyanide, rotenone and amytal under both state 4 and state 3 conditions. Antimycin A inhibited state 3 but did not appreciably alter state 4 rates of malate oxidation. With all substrates tested inhibition by antimycin A was greatly facilitated by preswelling the mitochondria for 10 min. This was interpreted to indicate that swelling increases the accessibility of antimycin A to the site of inhibition.     

Measurements of membrane potentials in plant mitochondria with the safranine method

The positively charged dye, safranine, has been used as an indicator of membrane potentials in mung bean (Phaseolus aureus) and Voodoo lily (Sauromatum guttatum) mitochondria under a variety of metabolic conditions. The spectral response of safranine has been calibrated with respect to a K⁺ diffusion potential and was found to be linearly related to the developed potential within the range of 50 to 160 millivolts. Both respiration and ATP hydrolysis gave rise to a membrane potential of approximately 135 millivolts. Respiratory inhibitors such as cyanide and antimycin depolarized the potential, whereas rotenone has little effect. No potentials were developed during NADH supported cyanide insensitive respiration. It is concluded that safranine may be a useful spectrophotometric probe of the mitochondrial membrane potential.     

Substrates respired by mitochondrial fractions of two isolates of the nematode Aphelenchus avenae and the effects of electron transport inhibitors

Electron transport has been assayed and compared in two isolates (M and F) of the free-living (model) nematode Aphelenchus avenae. Of the substrates tested only alpha-glycerophosphate and succinate were utilised to any significant extent by both isolates. Comparative data on respiratory rates, respiratory control ratios and ADP:O ratios for various substrates are given. Succinate oxidation by isolate-F mitochondria was ca 80-90% sensitive to antimycin A while that of isolate M was almost completely refractory to antimycin A. The response to other electron transport inhibitors suggests the operation of (a) azide/cyanide sensitive, (b) azide/salicylhydroxamic acid (SHAM) insensitive but carbon monoxide sensitive and (c) SHAM-sensitive terminal oxidases to varying degrees in the mitochondria of these two isolates of A. avenae.     

Ascaris suum: Oxidative phosphorylation in mitochondria from developing eggs and adult muscle

Respiration and the phosphorylating capability of mitochondria isolated from one-celled fertilized eggs, 10-day vermiform embryos, 21-day infective larvae, and adult body wall muscle from Ascaris suum were compared with that of rat liver mitochondria. Although oligomycin-sensitive ATPase and O₂ consumption/ mitochondrion in the presence of succinate and malate was lower in eggs than in liver, other properties such as respiratory control, ADP:O and P:O ratios at sites I, II, III, and the sensitivity of respiration to cyanide, azide, oligomycin, rotenone, and malonate were similar. In muscle mitochondria, the oligomycin-sensitive ATPase and O₂ consumption/ mitochondrion were sharply reduced, respiratory control was poor, and electron transport at sites II and III in particular was inefficiently coupled with phosphorylation. In addition, about 60% of the respiration was insensitive to cyanide or azide but sensitive to salicylhydroxamic acid. The results support earlier evidence that the free-living eggs of A. suum are aerobes. The adult parasite, while continuing to ferment actively in the presence of oxygen, nevertheless possesses one or more electron transport systems that are inefficiently coupled with aerobic phosphyorylations. The physiological significance of these systems has yet to be elucidated.     

Transport and metabolism of d-lactate in Jerusalem artichoke mitochondria

We report here initial studies on d-lactate metabolism in Jerusalem artichoke. It was found that: 1) d-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-d-lactoyl-glutathione. 2) Externally added d-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) d-lactate was metabolized inside mitochondria by a flavoprotein, a putative d-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added d-lactate by means of a d-lactate/H⁺ symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial d-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

The expression,function and regulation of mitochondrial alternative oxidase under biotic stresses

To survive, plants possess elaborate defence mechanisms to protect themselves against virus or pathogen invasion. Recent studies have suggested that plant mitochondria may play an important role in host defence responses to biotic stresses. In contrast with animal mitochondria, plant mitochondria possess a unique respiratory pathway, the cyanide‐insensitive alternative pathway, which is catalysed by the alternative oxidase (AOX). Much work has revealed that the genes encoding AOX, AOX protein and the alternative respiratory pathway are frequently induced during plant–pathogen (or virus) interaction. This raises the possibility that AOX is involved in host defence responses to biotic stresses. Thus, a key to the understanding of the role of mitochondrial respiration under biotic stresses is to learn the function and regulation of AOX. In this article, we focus on the theoretical and experimental progress made in the current understanding of the function and regulation of AOX under biotic stresses. We also address some speculative aspects to aid further research in this area.