Gene mutation eliminating antimycin A-tolerant electron transport in Ustilago maydis

A class of mutants of Ustilago maydis selected on a fungitoxic oxathiin lack of antimycin A-tolerant respiratory system which is present in wild-type cells. This system provides, directly or indirectly, for considerable resistance to antimycin A because growth of mutant cells lacking the system is much more sensitive to the antibiotic than that of the wild type. Antimycin A-sensitive O(2) uptake and growth is found in half of the progeny from crosses of mutant to wild type. All antimycin A-sensitive segregants are somewhat more resistant to oxathiins than the antimycin A-resistant segregants. The respiration of the mutant is strongly inhibited by cyanide and azide at concentrations which stimulate respiration of the wild type. Respiration of both mutant and wild type is about equally inhibited by rotenone. It appears that the mutation alters some component of the respiratory system located between the rotenone inhibition site and the antimycin A inhibition site that permits shift of electron transport to an alternate terminal oxidase when the normal electron transport pathway is blocked.     

In vitro maturation of rabbit reticulocytes: oxygen consumption reaction]

With a simple experimental system the changes of endogenous, antimycin A-suppressed, oligomycin-suppressed and antimycin A-resistant oxygen consumption are studied during the maturation of intact cells of the 6th day of bleeding. All functional characteristics of oxygen consumption decrease during maturation. The rate of decrease is strongly increased by high inorganic phosphate concentrations (125 mM). This effect is most obvious for the oligomycin-suppressed and the endogenous respiration. The degree of uncoupling of non-incubated cells is 14%. During 24 h incubation it rises to 75%. Inorganic phosphate accelerates the increase of uncoupling during maturation. Reticulocytes of the 4th day of bleeding are characterized by a higher respiratory capacity and also by a higher rate of maturation of antimycin A-suppressed and endogenous respiration. The degree of uncoupling does not increase during maturation. This may be attributed to the low lipoxygenase activity of these cells. 25% of the endogenous oxygen consumption of unmatured cells are antimycin A-resistant. This type of respiration declines by 50% in 4 h incubation irrespective of inorganic phosphate concentrations and day of bleeding. In nitrogen all functional characteristics of respiration during the maturation decline more rapidly than in oxygen. The antimycin A-resistant respiration, however decreased more slowly and reached 50% after 12 h. A pH dependence of maturation (maximum at pH 8.4) was found for the endogenous and the antimycin A-suppressed respiration. The degree of uncoupling rises most quickly at pH 7.4. This is possibly related to the pH maximum of lipoxygenase.     

Monitoring the cellular activity of a cultured single cell by scanning electrochemical microscopy (SECM). A comparison with fluorescence viability monitoring

The respiratory activities of cultured HeLa cells were monitored at a single cell level using scanning electrochemical microscopy (SECM) that produces images of the localized distribution of oxygen around the cell. The change in the cellular activity was traced after exposures to KCN, ethyl alcohol and the antibiotic drug, Antimycin A. The results were compared with those from the conventional fluorescence monitoring using Calcein-AM that is sensitive to deformation of the cell membrane. The SECM-based measurement follows the decrease in the cellular activity upon exposure to KCN and Antimycin A more rapidly than the fluorescence-based measurements, demonstrating that SECM is suitable for studying the cellular influence of respiration inhibitors.     

A 36-kDa mitochondrial protein is responsible for cyanide-resistant respiration in Hansenula anomala

Antimycin A-dependent induction of cyanide-resistant respiration in Hansenula anomala was reversibly blocked by carbonylcyanide-m-chlorophenylhydrazone (CCCP). When the cells were pulse-labeled with [35S]methionine in the presence of both antimycin A and CCCP, the radioactivity was incorporated into a 39 kDa mitochondrial protein. Upon removal of CCCP, this protein was processed into a 36 kDa form. The increase in the 36 kDa protein completely paralleled that in cyanide-resistant respiration activity, suggesting that the 39 kDa protein is the precursor of the 36 kDa protein, which is responsible for cyanide-resistant respiration.     

The composition of free fatty acids and mitochondrial activity in seedlings of winter cereals under cold shock

Cold shock (-4° C, 1 h) induces qualitative and quantitative changes in the composition of free fatty acids in mitochondria of winter-hardy cereals (Secale cereale L.,Triticum aestivum L.). The amount of these compounds and the degree of their unsaturation increases. Simultaneously, a marked change occurs in the oxidative and phophorylative activities of the mitochondria: respiratory control decreases, respiration in state 4 and antimycin A-resistant respiration increase. The changes in mitochondrial activity are presumed to be caused by endogenous free fatty acids, the amount of which is increased because of phospholipase activity.     

Essential role of ferrous iron in cyanide-resistant respiration in Hansenula anomala

Antimycin A-dependent induction of cyanide-resistant respiration in Hansenula anomala was completely blocked by o-phenanthroline, alpha,alpha'-dipyridyl, or 8-hydroxyquinoline. Pulse-labeling of the cells with [35S]methionine in the presence of both antimycin A and o-phenanthroline indicated that the 36-kDa protein previously reported to be involved in cyanide-resistant respiration [(1989) J. Biochem. 105, 864-866] was formed in mitochondria even under these conditions. The addition of Fe2+, but not Fe3+, ions to these cells in the presence of cycloheximide resulted in the rapid expression of cyanide-resistant respiration activity. These results suggest that in the presence of both antimycin A and o-phenanthroline an inactive form of the 36-kDa protein was formed and Fe2+ ions converted it to the active form. It is also likely that Fe2+ ions are involved in the reaction mechanism of cyanide-resistant respiration.     

Calcium Inhibits Phase Shifting of the Circadian Conidiation Rhythm of Neurospora crassa by the Calcium Ionophore A23187

Effects of the calcium ionophore, A23187, and antimycin A on the circadian conidiation rhythm of Neurospora crassa were examined. A23187 at a concentration of 1 mum in medium not containing divalent cations delayed the phase by 10 hours at CT 10 and advanced it by 5 hours at CT 14 (CT 12 corresponds to the time that discs are transferred from light to dark). This phase shifting was completely inhibited by addition of 0.1 millimolar CaCl(2) but not by MgCl(2) at any concentrations examined.Antimycin A inhibited respiration by 90% at a concentration of 0.2 micrograms per milliliter and lowered the ATP content by 85%. Antimycin A alone caused small phase advances but in combination with A23187 resulted in a large phase delay at CT 10. This phase shifting was not reversed by addition of CaCl(2) lower than 10 millimolar.     

Endogenous and endobiotic induced reactive oxygen species formation by isolated hepatocytes.

The rat hepatocyte catalyzed oxidation of 2',7'-dichlorofluorescin to form the fluorescent 2,7'-dichlorofluorescein was used to measure endogenous and xenobiotic-induced reactive oxygen species (ROS) formation by intact isolated rat hepatocytes. Various oxidase substrates and inhibitors were then used to identify the intracellular oxidases responsible. Endogenous ROS formation was markedly increased in catalase-inhibited or GSH-depleted hepatocytes, and was inhibited by ROS scavengers or desferoxamine. Endogenous ROS formation was also inhibited by cytochrome P450 inhibitors, but was not affected by oxypurinol, a xanthine oxidase inhibitor, or phenelzine, a monoamine oxidase inhibitor. Mitochondrial respiratory chain inhibitors or hypoxia, on the other hand, markedly increased ROS formation before cytotoxicity ensued. Furthermore, uncouplers of oxidative phosphorylation inhibited endogenous ROS formation. This suggests endogenous ROS formation can largely be attributed to oxygen reduction by reduced mitochondrial electron transport components and reduced cytochrome P450 isozymes. Addition of monoamine oxidase substrates increased antimycin A-resistant respiration and ROS formation before cytotoxicity ensued. Addition of peroxisomal substrates also increased antimycin A-resistant respiration but they were less effective at inducing ROS formation and were not cytotoxic. However, peroxisomal substrates readily induced ROS formation and were cytotoxic towards catalase-inhibited hepatocytes, which suggests that peroxisomal catalase removes endogenous H(2)O(2) formed in the peroxisomes. Hepatocyte catalyzed dichlorofluorescin oxidation induced by oxidase substrates, e.g., benzylamine, was correlated with the cytotoxicity induced in catalase-inhibited hepatocytes.     

An alternate respiratory pathway in Candida albicans

Usual concentrations of antimycin A, rotenone and EDTA, individally or in combination, reduced aerobic growth rate and cell yield of Candida albicans to about half its normal level and to about the levels of previously-described acetate-negative, cytochrome-complete and aa₃-deficient variants which were little affected by the inhibitors. Anaerobic conditions (not affected by antimycin A) reduced growth rate and cell yield of all cultures-including that of a nonrespiring aa₃, b-deficient mutant-to low, equal levels. Antimycin A but not rotenone prevented growth of the normal strain on ethanol medium. Cyanide and antimycin A blocked most of the respiration of the normal strain and cytochrome-complete variant, but did not affect that of the cytochrome aa₃-deficient mutant. Rotenone and EDTA did not affect respiration of any of the cultures. SHAM blocked cyanide- and antimycin A-insensitive respiration and prolonged the lag phases of the three respiring cultures, especially in the presence of antimycin A, but alone increased oxygen-uptake rate of the cytochromecomplete cultures while curtailing that of the cytochrome aa₃-deficient mutant. Resting cells, especially wild-type, grown in medium containing antimycin A exhibited lowered oxygen-uptake rate, which was increased upon the addition of cyanide or antimycin A. Antimycin A stimulated, but cyanide inhibited, respiration of cytochrome-complete cultures grown in the presence of rotenone but did not affect that of the cytochrome aa₃-deficient mutant. SHAM inhibited respiration of all antimycin A- or rotenone-grown cultures. The high rate of respiration of C. albicans in the presence of inhibitors for three sites of electron transport in the conventional oxidative pathway, the inhibition of this respiration by SHAM and its loss by the absence of cytochrome b, indicate an alternate oxidative pathway in this organism which crosses the conventional one at cytochrome b.This work was supported by Public Health Service Graduate Dental Training Grant DE 00144 and the Graduate School and the Department of Microbiology, Southern Illinois University.     

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.     

Fasciola hepatica: cytochrome c oxidoreductases and effects of oxygen tension and inhibitors

Studies were made on the mechanism of respiration in Fasciola hepatica (Trematoda). Respiration was found to be dependent on the oxygen tension. The respiratory enzyme systems, NADH-cytochrome c oxidoreductase (EC 1.6.2.1), succinate-cytochrome c oxidoreductase (EC 1.3.99.1) NADH oxidase and cytochrome c-oxygen oxidoreductase (EC 1.9.3.1) were detected in a mitochondrial preparation, the NADH oxidase activity being markedly stimulated by addition of mammalian cytochrome c. Amytal and rotenone inhibited NADH oxidase activity. Antimycin A inhibited succinoxidase activity only at relatively high concentrations. Azide was inhibitory at high concentrations. However, cyanide was found to stimulate respiration. Hydrogen peroxide was found to be an end product of respiration in F. hepatica.     

The action of the sesquiterpenic benzoquinone, perezone, on electron transport in biological membranes

Perezone (2-(1,5-dimethyl-4-hexenyl)-3-hydroxymethyl-p-benzoquinone) is a sesquiterpenic benzoquinone isolated from roots of plants of the genus Perezia. It exhibits oxido-reduction characteristics which suggest that the compound can be used for studies of the electron transfer chain of rat liver mitochondria. Perezone at 50 microM inhibits mitochondrial electron transport through a process which differs from that of rotenone, amytal, and Antimycin A. The inhibition is temperature dependent; at 35 degrees C it fails to inhibit valinomycin-induced mitochondrial respiration, but at 20 degrees C it inhibits respiration by 80-90%. Perezone is an electron-donor and electron-acceptor compound that behaves similarly to naphtoquinone. It mediates electron transport from a reaction center preparation isolated from Rhodopseudomonas sphaeroides and added cytochrome c. The low respiration of rat liver mitochondria depleted of coenzyme Q10 (CoQ) is increased by perezone. The electron transport activity of perezone was also demonstrated with CoQ-deficient yeast mutant E3-24.     

A cytoplasmic gene for partial suppression of a nuclear pleiotropic respiratory deficient mutant in the petite negative yeast Schizosaccharomyces pombe

Summary The nuclear pleiotropic respiratory-deficient mutant pet1 (previously M126) exhibits cytochromes aa₃ and b deficiencies accompanied by loss of the oligomycin-sensitivity of the mitochondrial ATPase. The mutant pet1, unable to grow on glycerol, exhibits in addition sensitivity of Antimycin A of the growth on glucose. The latter phenotypic trait symbolized by ANA^S-D, exhibits a high frequency (2 to 4×10^-5) of spontaneous suppression into Antimycin A-resistant strains. Mutagenesis with MnCl₂ increases by a factor of 10² the frequency of ANA^R-D derivatives. This suppression is partial since none of the suppressed strains is able to grow on glycerol even when respiratory functions and cytochromes activities are restored as in the pet1 [SUP2] strain. In the latter strain it is concluded that the extralocus suppressor gene [SUP2] is responsible for the ANA^R-D trait. Tetrad analysis in a cross homozygous for pet1 demonstrates a non-Mendelian segregation pattern for the SUP2 suppressor gene. In stable diploids, homozygous for pet1, the [SUP2] suppressor exhibits a mitotic segregation pattern. Furthermore the transmission of the [SUP2] gene is decreased by ethidium bromide treatment. Therefore, the [SUP2] suppressor gene responsible for partial suppression of the nuclear pleiotropic phenotype in mutant pet1 is of cytoplasmic heredity.     

Hydrogen peroxide production in uncoupled mitochondria of the parasitic nematode worm Nippostrongylus brasiliensis.

1. Mitochondria from the parasitic nematode worm Nippostrongylus brasiliensis produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 2. Antimycin A inhibits respiration and H2O2 production by 70 and 65% respectively; the residual activities can be attributed to alternative electron-transport pathway(s). 3. o-Hydroxydiphenyl and 1,3,5-trihydroxybenzene, inhibitors of alternative electron transport, inhibit respiration by 37% and H2O2 production by 26%. 4. Another inhibitor of alternative electron transport, salicylhydroxamic acid, shows a complex mode of action; low concentrations (less than 0.5 mM) stimulate respiration and H2O2 production, whereas 2 mM-salicylhydroxamic acid inhibited respiration by 35% and stopped H2O2 production completely. 5. O2 thresholds were observed for the inhibition of respiration at O2 concentrations greater than 57.7 microM and inhibition of H2O2 production (greater than 20.5 microM-O2); apparent Km values for oxygen were 5.5 microM and 3.0 microM respectively. 6. In the presence of antimycin A the O2-inhibition thresholds and apparent Km values for O2 of respiration and H2O2 production matched closely, suggesting that the alternative oxidase is a likely site of H2O2 production. 7. These results are discussed in relation to O2 toxicity to N. brasiliensis.     

Wirkungen sichtbaren Lichtes auf Saccharomyces cerevisiae

Zusammenfassung Ein mögliches Mitwirken des Pasteur-Effektes bei der lichtinduzierten Veränderung der Gärungsintensität wird untersucht. Mit steigenden Konzentrationen von Antimycin A im Nährmedium wird die Atmung der Hefezellen zunehmend ausgeschaltet. Während sich dabei in den Dunkelkontrollen die Gärungsintensität gemäß der Rückläufigkeit des Pasteur-Effektes bis zu einem Endwert erhöht, wird bei den belichteten Zellen die ohne Antimycin nur geringfügig reduzierte oder wenig geförderte Gärung immer stärker — wiederum bis zum Erreichen eines Endwertes — gehemmt.Bei niedriger Lichtintensität wird ohne Antimycinzugabe — wie schon früher gefunden — eine mit der Dauer der Belichtung ansteigende Gärungsförderung bei gleichzeitig fortschreitender Hemmung der Atmung beobachtet. Wird dagegen die Atmung durch Antimycin ausgeschaltet, so erfolgt statt einer Förderung eine zunehmende Hemmung der Gärung.Die Ergebnisse werden als Beweis dafür angesehen, daß die Gärungsintensität unter Lichteinwirkung eine Resultante aus zwei gegenläufig wirkenden Prozessen darstellt. Diese beruhen einerseits auf einer hemmenden Wirkung des Lichtes, andererseits auf der teilweisen Rückläufigkeit des Pasteur-Effektes infolge der lichtinduzierten Atmungshemmung, wodurch eine teilweise Enthemmung der Gärung erfolgt.

---

Summary A possible cooperation of the Pasteur effect in light-induced changes of the rate of fermentation in yeast cells was investigated. Increasing concentrations of antimycin A in the culture medium progressively decrease respiration. In the dark this results in an increase of fermentation — up to a saturation value — due to decreased regulation by the Pasteur effect. In the light, on the other hand, where fermentation in the absence of antimycin A is either slightly stimulated or slightly decreased, increasing concentrations of antimycin A lead to a progressive inhibition of fermentation — again down to a saturation value.At low light intensities and without antimycin the degree of fermentation is enhanced with the time of exposure to light while respiration is decreased simultaneously. However, when respiration has been fully inhibited by antimycin A, light does not enhance, but decreases fermentation progressively with time.These findings provide evidence that in the light the rate of fermentation by yeast is determined by two counteracting processes: these are on the one hand the inhibitory action of light, on the other hand a stimulation mediated by the partial reduction of the Pasteur effect on account of light-inhibited respiration.

     

Effects of thyroid hormones on the bypasses of the antimycin A block in the bc1 complex of rat liver mitochondria.

The effect of thyroid hormones on the electron flow through the bc1 complex of rat liver mitochondria was studied using two dye bypasses of the Antimycin A block of the bc1 complex by the method of Alexandre and Lehninger (Biochim. Biophys. Acta 767:120; 1984). Bypass respiration rates with both DCIP (2,6-dichlorophenolindophenol) and TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride) were elevated in the hyperthyroid rats and depressed in the hypothyroid groups compared to the euthyroid controls. T3 treatment of hypothyroid rats returned the bypass rates to control levels in 24 hours with the TMPD dye but not for the DCIP. This further demonstrates that different portions of the bc1 complex respond individually to the thyroid state.     

The O2-dependence of respiration and H2O2 production in the parasitic nematode Ascaridia galli.

1. Respiration in the parasitic nematode worm Ascaridia galli was inhibited at O2 concentrations in excess of 255 microM, and an apparent Km,O2 of 174 microM was determined. 2. Mitochondria-enriched fractions isolated from the tissues of A. galli have much lower apparent Km,O2 values (approx. 5 microM). They produce H2O2 in the energized state; higher rates of H2O2 production were observed in the presence of the uncoupler carbonyl cyanide m-chlorophenylhydrazone. 3. Antimycin A inhibited respiration in muscle tissue mitochondria by 10%, but had no effect on respiration in gut + reproductive tissue mitochondria; the major portion of respiration in both types of mitochondria could be attributed to an alternative electron-transport pathway. 4. o-Hydroxydiphenyl, an inhibitor of alternative electron-transport pathways, inhibits respiration by 98% and completely inhibits the production of H2O2 in gut-plus-reproductive-tissue mitochondria; respiration and H2O2 production in muscle tissue mitochondria were inhibited by 90 and 86% respectively. 5. Another inhibitor of alternative electron transport, salicylhydroxamic acid, had the same effect as o-hydroxydiphenyl on H2O2 production and respiration in gut-plus-reproductive-tissue mitochondria. However, its effect on muscle tissue mitochondria was complex; a low concentration (0.35 mM) stimulated H2O2 production, whereas 3 mM inhibited respiration by 87% and prevented H2O2 production completely. 6. The similarities between the apparent Km,O2 values for H2O2 production and respiration in muscle mitochondria and in gut-plus-reproductive-tissue mitochondria suggests that the site of H2O2 production on the alternative electron-transport chain is cytochrome 'o'. 7. These results are discussed in relation to potential O2 toxicity in A. galli.     

Relationship between superoxide anion radical generation and aging in the housefly, Musca domestica

The objective of this study was to elucidate the possible cause of increased oxidative stress observed in the adult housefly during aging. The hypothesis that increased production of oxygen radicals may be a cause of the increased oxidative stress was tested by comparison of 8-day and 15-day old flies, which represent the stage of full maturation and the beginning of the dying phase, respectively. Rates of both antimycin A-resistant respiration of isolated mitochondria and O2 generation at ubiquinone-cytochrome b site by submitochondrial particles increased during aging and were associated with life expectancy of flies. Flies destined to die earlier than their cohorts of the same age exhibited a relatively higher rate of O2- production. Age-related increase in O2- generation was not associated with corresponding changes in ubiquinone content of mitochondria.     

Studies on the respiratory system of Aspergillus oryzae I. Development of respiratory activity during germination in the presence and absence of antimycin A

Active growth of Aspergillus oryzae was observed when conidiawere inoculated into a medium containing antimycin A. Immediatelyafter adding antimycin A, to young mycelia germinated in itsabsence, growth stopped, but began again after several hours.This restored growth was antimycin A-insensitive. Percentagegermination was the same in the presence and absence of thisdrug. It seems that drug-resistant germination and growth donot result from selection of resistant cells but result frominduction of antimycin A-insensitive mitochondria in the wholepopulation. Endogenous respiration of cells germinated in theabsence of antimycin A was inhibited by this drug, whereas thatof cells grown in the presence of antimycin A was completelyinsensitive. Antimycin A-sensitivity of cellular respirationseems to determine the effect of this drug on mycelial growth.Mitochondria were isolated from mycelia grown in the presenceand absence of this drug. The difference in antimycin A-sensitivityin endogenous respiration was attributed to a difference inproperties of the mitochondrial respiratory systems. ¹Present address: Department of Chemistry, Institute of MedicalScience, University of Tokyo, Tokyo, Japan (Received December 21, 1969; )     

Light-Mediated Nitrite Accumulation during Denitrification by Pseudomonas sp. Strain JR12

The effect of light on the denitrifying characteristics of a nonphotosynthetic denitrifier, Pseudomonas sp. strain JR12, was examined. Already at low light intensities, nitrite accumulated as a result of light inhibition of nitrite but not of nitrate reduction rates. Exposure of this bacterium to light caused a photooxidation of cytochrome c, an intermediate electron carrier in its respiratory pathway. Photoinhibition of nitrite reduction was reversible, as nitrite reduction rates returned to preillumination levels when light-exposed cells were returned to dark conditions. Antimycin A reversed the inhibitory effect of light on nitrite reduction by preventing a reversed electron flow. Aerobic respiration by this bacterium was not affected by light.