Carbon-13 nuclear magnetic resonance spectroscopy of high-spin iron(III) porphyrin compounds

1. There was no apparent correlation between the rate of respiration and rate of accumulation of proline in Candida albicans cells. 2. In contrast to normal cells, the respiration in the starved cells became completely cyanide insensitive. The starvation of cells in the presence of cycloheximide prevented the cells from becoming cyanide insensitive. The addition of Fe(III), however, accelerated the process. 3. Oxidizable substrates e.g. NADH, acetate and glucose, when added to cyanide-insensitive starved cells, exhibited 40--280% stimulation in respiration rate. However, this enhancement in oxidation by various substrates was not coupled to a simultaneous increase in the proline uptake or in intracellular ATP levels. 4. There was 6-fold stimulation in proline uptake when cyanide-insensitive cells were preincubated with 50 mM glucose. The preincubation of starved cells resulted in a partial restoration of cyanide sensitivity and increased intracellular ATP levels. The preincubation of starved cells with other oxidizable substrates resulted in a partial restoration of cyanide sensitivity but had no stimulatory effect on intracellular ATP levels and proline accumulation. 5. Both the enhanced uptake and ATP levels in glucose preincubated cells were found to be completely abolished by iodoacetate. 6. It is proposed that the increased proline uptake in cells preincubated with glucose was mainly due to the production of glycolytic energy.     

Some Specific Features of the Respiration of Hydrocarbon-utilizable Candida Yeasts

The respiration of both glucose-grown and hydrocarbon-grown cells of Candida tropicalis pK 233 harvested in the stationary phases was not inhibited by cyanide when glucose was used as oxidation substrate, but the former was rather stimulated in the presence of cyanide. When n-alkanes were used as oxidation substrate, cyanide lowered the respiratory activities of both cells to about 50%. With respect to the susceptibility to cyanide, the younger cells growing on n-alkanes were less sensitive in hydrocarbon oxidizing ability than the older cells, whereas the older cells growing on glucose or n-alkanes were more resistant in glucose oxidizing ability than the younger cells. Acetate was oxidized by both glucose-grown and hydrocarbon-grown cells of the yeast. Laurate was oxidized by hydrocarbon-grown cells, but not by glucose-grown cells. The respiration on laurate was inhibited completely by 3.3 mM of cyanide. In general, hydrocarbon-grown cells of Candida tropicalis pK 233 were more sensitive to various respiratory inhibitors than glucose-grown cells, although the oxidation substrates had a significant effect.

The respiration of both glucose-grown and hydrocarbon-grown cells of C. albicans, C. guilliermondii and C. lipolytica harvested in the stationary phases was also resistant to cyanide when glucose was used as oxidation substrate. But the respiration on n-alkanes of these cells was inhibited significantly by 3.3 mM of cyanide except for C. albicans.     

The Disappearance of Intermediates involved in Glucose Oxidation during the Starvation of the Fungus Zygorhyncus moelleri

The respiration of carbohydrate-starved cells of Zygorhyncusmoelleri showed a lag period of 2 to 3 hours when glucose wasadded before the rate of oxygen consumption became constant.Analysis of the rates of oxygen uptake from the time of additionof the sugar until they became constant showed that the lagperiod could not be ascribed entirely to a low concentrationof free glucose in the cells in the period immediately followingthe addition of glucose to the medium. The analysis was, however,consistent with the supposition that the synthesis of an intermediarymetabolite on the glucose oxidation pathway was necessary beforethe oxidation could proceed with a maximum speed. The lengthof the lag period could be reduced by adding extracts of cellstogether with glucose; extracts of unstarved cells were moreeffective than those of starved cells in shortening the lagperiod. Various known substances were also effective in thisrespect, acetate and ethanol being the most active. These resultsare discussed together with earlier work on the lag period.     

Cyanide-resistant respiration in Torulopsis candida

The effect of cyanide and the inhibitors of cyanide-resistant oxidase--hydroxamic acids on endogenous respiration and oxidation of a number of substrates by Torulopsis candida resting cells taken at different stages of growth on glucose and hexadecane was studied and made it possible to arrive at the following conclusions. 1. The effect of cyanide on endogenous respiration of T. candida differs during its growth on glucose and hexadecane. On hexadecane, irrespective of the growth phage, cyanide inhibits endogenous respiration by 70--75%. On glucose, cyanide inhibits endogenous respiration not more than by 35% and only at the exponential growth phase whereas it stimulates endogenous respiration in the course of other growth stages. 2. The effect of cyanide on respiration of the resting cells of T. candida which oxidize glucose, hexadecane, primary alcohols and tetradecanoic acid hardly depends on the growth stage. It is determined mainly by the nature of a substrate to be oxidized. 3. Hydroxamic acid have no effect on the cell respiration in the absence of cyanide. However, in its presence, they entirely inhibit both endogenous respiration and oxidation of the aforementioned substrates. 4. Under almost all above experimental conditions, the sensitivity of cell respiration to cyanide changes only slightly at different stages of growth on either glucose or hexadecane. This feature markedly distinguishes T. candida among other cyanide-resistant yeasts.     

Lysosomal acid proteinase of rabbit liver

1. The interference mechanism of carbonyl cyanide m-chlorophenylhydrazone with the respiratory process and with phosphorylation coupled to respiration has been investigated in resting cells of Escherichia coli. 2. Preincubation of the cells with carbonyl cyanide m-chlorophenylhydrazone in the absence of substrate caused strong inhibition of succinate oxidation. The inactivation of the respiratory system proved to be time-dependent and temperature-dependent and could be arrested by adding the substrate. Inhibition of incorporation of ³²P into acid-soluble organic phosphate esters exceeded the inhibition of oxygen uptake. 3. In contrast with succinate, the rate of oxidation of glucose was increased by carbonyl cyanide m-chlorophenylhydrazone. The sensitivity of other substrates to the inhibitor was less than that of succinate. 4. Various observations are described in support of the view that respiratory inhibition induced by carbonyl cyanide m-chlorophenylhydrazone is a result of its interference with ATP synthesis. The capacity of a given substrate to increase intracellular ATP concentration appeared to be directly related to its resistance to inhibition. In cell-free extracts carbonyl cyanide m-chlorophenylhydrazone still suppressed ³²P incorporation but had no effect on respiration. 5. Carbonyl cyanide m-chlorophenylhydrazone-induced stimulation of glucose oxidation and the acceleration of succinate oxidation by ADP or AMP in cells rendered permeable to nucleotides are tentatively interpreted as an indication that a certain part of respiration in E. coli is under phosphate-acceptor-mediated control.     

The Assimilation of Ammonia by Nitrogen-starved Cells of Chlorella vulgaris: Part I. The Correlation of Assimilation with Respiration

The addition of ammonium sulphate to a suspension of nitrogen-starvedChlorella cells is followed immediately by the rapid assimilationof ammonia and a large increase of the respiration rate. Theassimilation of ammonia and the high rate of respiration continueuntil either all the ammonia has been assimilated or some carbonreserve within the cells has been exhausted. Which happens firstdepends on the amount of ammonia added and the quantity of cellspresent. The respiration which accompanies, ammonia assimilationis sensitive to cyanide and it has a respiratory quotient of0•75 compared with 1•2–1•3 for normal reapiration.The addition of glucose to nitrogen-starved cells when ammoniais being assimilated does not increase either the rate of respirationor the rate of assimilation. The rates of reapiration and ammoniaassimilation by normal cells are markedly increased by the additionof glucose. Light has little effect on the rate of ammonia assimilationby nitrogen-starved cells, but doubles the assimilation rateof normal cells.     

Dinitrocresol, Cyanide, and the Pasteur Effect in Yeast

At pH 5·0 the respiration of yeast is stimulated by lowconcentrations of 3:5-dinitro-o-cresol, reaching a peak levelof 170 per cent, at 10⁵ M. Concentrations above this inhibitoxygen uptake and cause aerobic fermentation to appear, whichin turn reaches a peak value and is then inhibited. The rateof carbohydrate breakdown, or glycolysis, calculated from therates of respiration and aerobic fermentation, increases steadilyup to 3 x 10^–5 M., at which concentration it is 5 timesfaster than the control: higher concentrations depress the rateof glycolysis. The rate of fermentation under nitrogen is abouttwice that of respiration, and it is inhibited over the sameconcentration range as aerobic fermentation. It was found earlier that oxidative assimilation of glucoseby yeast is progressively inhibited by increasing concentrationsof dinitrocresol, and it is now shown that this parallels theincrease in the rate of aerobic glycolysis. It is argued thatdinitrocresol is here acting as an uncoupling agent and thatboth oxidative assimilation and the rate of glycolysis are controlledby the level of energy-rich phosphate. With cyanide there is no stimulation of oxygen uptake, aerobicfermentation only appears when respiration becomes inhibited,and after an initial slight decrease the rate of glycolysisrises to 575 per cent. of the control value at 5 x 10^–4M. It is suggested that the rate of glycolysis only increaseswhen respiration has been inhibited sufficiently to reduce therate of formation of energy-rich phosphate.     

Response to Phosphate Deficiency in Bean (Phaseolus vulgarisL.) Roots. Respiratory Metabolism, Sugar Localization and Changes in Ultrastructure of Bean Root Cells

Bean plants (Phaseolus vulgarisL. ‘Zlota Saxa’)were cultured on complete (+P) or phosphate-deficient (-P) nutrientmedium. A large increase in glucose concentration was foundin the meristematic zone of -P roots compared to control roots.The increased glucose concentration in the meristematic zonedid not influence total respiration rate. Glucose or uncoupler(carbonyl cyanide m-chlorophenylhydrazone) failed to increasethe respiration rate in -P root segments, but stimulated respirationin +P roots. The ultrastructure of cortical cells from the meristematicroot zone showed marked differences between +P and -P plants.Large vacuoles, invaginations of the plasmalemma and condensedforms of mitochondria were dominating features in cortical cellsof -P roots. Analysis of extracts after treating roots withdimethylsulfoxide (DMSO) indicated different localization ofsugars in the cell compartments. In roots of -P plants, mostof the reducing sugars were detected in the cytoplasm fractionwhile most sucrose was in the vacuole. Observations of the effectof 10% DMSO on cell ultrastructure indicated partial destructionof the plasmalemma but not the tonoplast. The localization ofreducing sugars in secondary vacuoles or plasmalemma invaginationsin the cells from the meristematic region of -P roots is discussed.Copyright1998 Annals of Botany Company. Bean (Phaseolus vulgarisL.), roots, Pi deficiency, respiration, meristematic zone, ultrastructure, sugar efflux, reducing sugars and sucrose localization.     

Similarities between the Actions of Ethylene and Cyanide in Initiating the Climacteric and Ripening of Avocados

A continuous exposure of intact avocados (Persea americana) to 400 μl/l of cyanide results in a rapid increase in the rate of respiration, followed by a rise in ethylene production, and eventual ripening. The pattern of changes in the glycolytic intermediates glucose 6-phosphate, fructose diphosphate, 3-phosphoglyceric acid, and phosphoenolpyruvate during the rapid rise in respiration in both ethylene and cyanide-treated fruits is similar to that found in fruits made anaerobic where a 2.3- to 3-fold increase in the rate of glycolysis is observed. It is suggested that both during the climacteric and in response to cyanide, glycolysis is enhanced. It is proposed that cyanide implements the diversion of electrons to the cyanide-resistant electron path through structural alterations which are independent of the simultaneous inhibition of cytochrome oxidase.     

The nature of the oxidation states of sweet potato mitochondria

Sweet potato mitochondria exhibited respiratory control duringthe oxidation of malate and succinate with ADP/O ratios approachingthe theoretical P/O values. Prior to the addition of ADP themitochondria showed a considerable rate of substrate oxidation,defined as the basic respiration, which was of the same magnitudeas state 4 respiration. Electrons from state 4 and the basicrespiration were at least partially mediated by the cytochromechain, as shown by effects of cyanide, azide and amytal, andby spectrophotometric evidence. The nature of ATPase was studied and the influence of inhibitorsof ATPase activity on oxidation helped to establish the relationshipbetween the several states of oxidation and ATPase activity.The ADP/O ratio and ADP-stimulated respiration were slightlydecreased by fluoride, while state 4, the basic respirationand ATPase activity were effectively inhibited. Chlorpromazineinhibited DNP-stimulated ATPase activity, respiration uncoupledby DNP and all the states of malate oxidation. However, state4 and basic respiration were less sensitive than was state 3of malate oxidation to 0.3 mM chlorpromazine. It was concluded that mitochondrial ATPase played a role inthe basic respiration and in state 4 oxidation. ¹Present address: Department of Biochemistry Tel-Aviv University,Tel-Aviv, Israel (Received August 1, 1969; )     

Some Effects of Temperature and Substrate Content upon Respiration and the Carbon Balance of Field Beans (Vicia faba L.)

The effects of substrate content and temperature upon the productionof carbon dioxide in the dark were investigated in Vicia fabaand Sorghum vulgare, using the time courses for carbon dioxiderelease. No endogenous rhythms were found. With V. faba at highsubstrate contents, a steady rate of respiration was measuredat low temperatures, suggesting that the rate of respirationis limited by a third factor such as enzyme activity. This steadyrate eventually decreased rapidly. There was a first order rateof decrease with time at higher temperatures. Low substratecontents gave a complex decay. The rate of decrease of the rateof respiration was affected by the initial state of the plantsover the same range of rates. Reasons for this are discussed.The temperature sensitivity of respiration was investigated.The respiration of plants with high substrate contents had alower temperature sensitivity than those with low substratecontents. This was further investigated by measuring the stoichiometryof carbon dioxide production in the dark from total solublecarbohydrate (as hexose equivalent). It is likely that incompletehexose respiration, which occurs at high substrate contents,is less temperature sensitive than complete hexose respirationor the respiration of some other substrate. Vicia faba, Sorghum oulgare, carbon dioxide, respiration, temperature, substrate content     

Action of Respiratory Inhibitors on Seed Germination and Oxygen Uptake in Avena fatua L.

The effects of sodium azide, potassium cyanide (cytochrome oxidaseinhibitors), and salicylhydroxamic acid (SHAM; an alternativerespiration inhibitor) on germination and respiration of Avenafatua L. seeds were studied. Azide and cyanide released seeddormancy at similar concentrations and treatment durations.Cyanide, however, stimulated germination of seeds with littleafter-ripening, whereas azide had no effect under similar conditionsunless the seeds were after-ripened for several months; theduration of after-ripening required for seeds to respond toazide varied with seed batch. There was also a greater lag priorto germination in the case of azide, compared to cyanide treatedseeds. SHAM inhibited the stimulation of germination and respirationby azide, but not by cyanide. Furthermore, respiration induced by azide or cyanide could notbe inhibited by the subsequent application of SHAM. These findingssuggest that the respiration stimulated by azide and cyanideis not alternative (SHAM-sensitive) and, therefore, this respiratorypathway cannot be involved in the stimulation of germinationby cytochrome oxidase inhibitors. While embryos excised fromcontrol, azide or cyanide pretreated seeds had the capacityto perform alternative respiration, the actual contributionof this pathway was negligible. A large proportion of respirationof embryos excised from azide or cyanide pretreated seeds wasresidual, i.e. insensitive to both SHAM and cyanide. Alternative respiration, azide, cyanide, dormancy, salicylhydroxamic acid, wild oats     

Effect of sulfur, copper, and iron deficiency on the development of cyanide resistant respiration and the cytochrome composition of Pseudomonas aeruginosa

The effect of deficiency in sulfur, copper and iron in the growth medium on cyanide resistant respiration and cytochrome composition was studied in Pseudomonas aeruginosa and Candida lipolytica. It has been shown that: cyanide resistant respiration was observed at the stationary growth phase when the two microorganisms were cultivated in a complete medium; this respiration was detected already at the phase of decelerated growth in the case of copper deficiency; iron deficiency inhibited cyanide resistant respiration in the bacterium but stimulated its appearance in the yeast; sulfur deficiency inhibited the manifestation of cyanide resistant respiration in the both microorganisms; limitation of the bacterial growth with iron resulted in the accumulation of an iron complex (identical to pyoverdin in its spectral characteristics) in the cultural broth; the deficiency of sulfur, copper and iron inhibited the synthesis of all cytochromes in the bacterium; copper deficiency inhibited only the synthesis of a + a3 in the yeast; iron deficiency inhibited the synthesis of all cytochromes in the yeast; sulfur deficiency had virtually no effect on the content of cytochromes in the yeast. A possible nature of cyanide resistant oxidases in these microorganisms is discussed.     

Characteristics of Chlorophyll Formation in the Green Alga Golenkinia

Cells of the alga Golenkinia are bleached by growth in darknessin media containing sodium acetate. Re-greening of these cellsis light dependent; neither glucose nor intermediates of chlorophyllsynthesis can substitute. The amount of chlorophyll synthesizedis proportional to the light intensity between darkness and1,000 lux and to the duration of the exposure. Initially, onlychlorophyll a is synthesized. After 9–12 hr illumination,formation of chlorophyll b and carotenoids begins. Chlorophyllproduction apparently occurs in two stages: (1) the first 12–16hr of greening. This stage is sensitive to cyanide, azide oranaerobiosis and relatively resistant to DCMU. (2) the second16–24 hr of greening. This stage is sensitive to DCMUand relatively resistant to inhibitors of respiration. Glucosestimulates greening in both stages. The metabolic requirementsof chlorophyll synthesis are discussed. (Received December 17, 1980; Accepted June 25, 1981)     

Rates of Respiration and of Increase in Structural Dry Matter in Young Wheat, Ryegrass and Maize Plants in Relation to Temperature, to Water Stress and to Their Sugar Content

A method to determine the rate of conversion of reserve materialsinto structural dry matter in living, whole plants is presented.It is based on a brief measurement of plant respiration. Therate of increase in structural dry matter and of decrease ofreserve materials is calculated by subtracting the maintenancerespiration component from the total respiration. The remainder,the growth respiration rate, is multiplied by a factor thatis derived from the biochemical composition of the structuraldry matter formed. This method is applied to determine the relations of the rateof conversion of reserve material into structural dry matterto temperature, water stress and the level of reserve carbohydratesin plants of three species. The weakest part of the method is in the determination of therate of maintenance respiration. Consequences of different assumptionsconcerning the rate of adjustment of this respiration componentto modified environmental conditions are discussed. Lolium perenne L, Triticum aestivum L, Zea mays L, ryegrass, wheat, maize, respiration, maintenance respiration, water stress, sugar content, structured dry matter     

Metabolism of cyanide by Phanerochaete chrysosporium

The oxidation of veratryl alcohol (3,4-dimethoxybenzyl alcohol) by lignin peroxidase H2 (LiP H2) from the white rot fungus Phanerochaete chrysosporium was strongly inhibited by sodium cyanide. The I50 was estimated to be about 2-3 microM. In contrast, sodium cyanide binds to the native enzyme with an apparent sodium cyanide dissociation constant Kd of about 10 microM. Inhibition of the veratryl alcohol oxidase activity of LiP H2 by cyanide was reversible. Ligninolytic cultures of P. chrysosporium mineralized cyanide at a rate that was proportional to the concentration of cyanide to 2 mM. The N-tert-butyl-alpha-phenylnitrone-cyanyl radical adduct was observed by ESR spin trapping upon incubation of LiP H2 with H2O2 and sodium cyanide. The identity of the spin adduct was confirmed using 13C-labeled cyanide. Six-day-old cultures of the fungus were more tolerant to sodium cyanide toxicity than spores. Toxicity measurements were based on the effect of sodium cyanide on respiration of the fungus as determined by the metabolism of [14C]glucose to [14C]CO2. We propose that this tolerance of the mature fungus was due to its ability to mineralize cyanide and that this fungus might be effective in treating environmental pollution sites contaminated with cyanide.     

δ-N-[methyl-14C]arginine: A simplified preparation

A method is described for quantitative assay of cyanide which is based on measuring the rate of rise in absorbancy at 325 nm when cyanide forms an addition compound with NAD⁺ at pH 10. It was found that the rate of rise in absorbancy is proportional with the concentration of cyanide within the range of 0.05–4.00 mm. By applying this method it could be shown that in biological systems in which the interface between the aqueous phase and the atmosphere is large the effective concentration of cyanide, added to inhibit the respiration, decreases rather rapidly during incubation.     

The effect of anthracyclines on heart mitochondria respiration during the action of creatine phosphokinase

It was shown that during glutamate+malate oxidation in the presence of creatine, antitumour anthracycline antibiotics strongly inhibit the rate of oxygen uptake by rat heart mitochondria; ADP excess activated the respiration up to the initial level, i.e., that observed after the first addition of ADP. Carboxyatractyloside addition to a system containing creatine (or hexokinase+glucose) results in the stimulation of rubomycin-induced mitochondrial respiration. Substitution of carboxyatractyloside by oligomycin gives very similar results. It is supposed that anthracycline antibiotics exert a manyfold effect on heart mitochondrial membranes which results in impaired compartmentation of enzymatic systems providing for oxidative phosphorylation.     

The enzyme activity of energy metabolism in antibiotic-resistant staphylococci

The rates of lactate accumulation and glucose oxidation by the clinic strains of Staphylococcus aureus were determined. The inhibition grade of the glucose oxidation by cyanide indicates that respiratory chain of staphylococci participates in the oxidizing processes. During glucose oxidation the respiratory chain generates the difference of electric potentials -156 divided by -179 mV. The comparative assessment have shown that the antibiotic-resistant staphylococci are characterized by a higher rate of glucose oxidation as well as by value of the greater transmembrane difference of electric potentials.     

Mechanism of Superoxide Anion Generation in Intact Mitochondria in the Presence of Lucigenin and Cyanide

In the presence of cyanide and various respiratory substrates (succinate or pyruvate + malate) addition of high concentrations of lucigenin (400 microM; Luc2+) to rat liver mitochondria can induce a short-term flash of high amplitude lucigenin-dependent chemiluminescence (LDCL). Under conditions of cytochrome oxidase inhibition by cyanide the lucigenin-induced cyanide-resistant respiration (with succinate as substrate) was not inhibited by uncouplers (FCCP) and oligomycin. Increase in transmembrane potential (Deltaphi) value by stimulating F0F1-ATPase functioning (induced by addition of MgATP to the incubation medium) caused potent stimulation of the rate of cyanide-resistant respiration. At high Deltaphi values (in the presence of MgATP) cyanide resistant respiration of mitochondria in the presence of succinate or malate with pyruvate was insensitive to tenoyltrifluoroacetone (TTFA) or rotenone, respectively. However, in both cases respiration was effectively inhibited by myxothiazol or antimycin A. Mechanisms responsible for induction of LDCL and cyanide resistant mitochondrial respiration differ. In contrast to cyanide-resistant respiration, generation of LDCL signal, that was suppressed only by combined addition of Complex III inhibitors, antimycin A and myxothiazol, is a strictly potential-dependent process. It is observed only under conditions of high Deltaphi value generated by F0F1-ATPase functioning. The data suggest lucigenin-induced intensive generation of superoxide anion in mitochondria. Based on results of inhibitor analysis of cyanide-resistant respiration and LDCL, a two-stage mechanism of autooxidizable lucigenin cation-radical (Luc*+) formation in the respiratory chain is proposed. The first stage involves two-electron Luc2+ reduction by Complexes I and II. The second stage includes one-electron oxidation of reduced lucigenin (Luc(2e)). Reactions of Luc(2e) oxidation involve coenzyme Q-binding sites of Complex III. This results in formation of autooxidizable Luc*+ and superoxide anion generation. A new scheme for lucigenin-dependent electron pathways is proposed. It includes formation of fully reduced form of lucigenin and two-electron-transferring shunts of the respiratory chain. Lucigenin-induced activation of superoxide anion formation in mitochondria is accompanied by increase in ion permeability of the inner mitochondrial membrane.