Respiratory Chain of a Pathogenic Fungus, Microsporum gypseum: Effect of the Antifungal Agent Pyrrolnitrin

Pyrrolnitrin has been reported to inhibit Bacillus megaterium primarily by forming complexes with phospholipids and to block electron transfer of Saccharomyces cerevisiae between succinate or reduced nicotinamide adenine dinucleotide (NADH) and coenzyme Q. We found that pyrrolnitrin inhibited respiration of conidia of Microsporum gypseum. In mitochondrial preparations, pyrrolnitrin strongly inhibited respiration and the rotenone-sensitive NADH-cytochrome c reductase. The rotenone-insensitive NADH-cytochrome c reductase, the succinate-cytochrome c reductase, and the reduction of dichlorophenolindophenol by either NADH or succinate were inhibited to a lesser extent. However, the activity of cytochrome oxidase was not affected by pyrrolnitrin. The extent of reduction of flavoproteins by NADH and succinate, measured at 465 - 510 nm, was unaltered; however, the reduction of cytochrome b, measured at 560 - 575 nm, was partially inhibited by pyrrolnitrin. The level of totally reduced cytochrome b was restored with antimycin A. We, therefore, concluded that the primary site of action of this antifungal antibiotic is to block electron transfer between the flavoprotein of the NADH-dehydrogenase and cytochrome b segment of the respiratory chain of M. gypseum.     

Some effects of decenylsuccinic Acid on isolated corn mitochondria

The effects of decenylsuccinic acid on the swelling and respiratory capacities of mitochondria isolated from etiolated corn (Zea mays L., Wf9 × M14) shoots were studied. Decenylsuccinic acid (0.1 mM to 1.0 mM) inhibited the oxidation of succinate and malate-pyruvate, stimulated the oxidation of reduced nicotinamide adenine dinucleotide, and uncoupled phosphorylation. The swelling of isolated corn mitochondria, as determined by percentage of transmittance changes, was stimulated by decenylsuccinic acid in potassium chloride reaction media and in sucrose reaction media without bovine serum albumin. In a diaphorase (2, 6-dichlorophenolindophenol as acceptor) reaction with intact mitochondria, only the dehydrogenation rate of malate was reduced by the addition of decenylsuccinic acid. The dehydrogenation of reduced nicotinamide adenine dinucleotide or of succinate was either not affected or was stimulated depending on the diaphorase reaction medium. The oxygen uptake of mitochondria oxidizing N, N, N′, N′-tetramethyl-p-phenylenediamine diHCl and ascorbate was inhibited at decenylsuccinic acid concentrations greater than 0.5 mM.     

Changes in the Respiratory, Enzymatic, and Swelling and Contraction Properties of Mitochondria from Colytedons of Phaseolus vulgaris L. during Germination

Mitochondria isolated from cotyledons of germinating wax beans (Phaseolus vulgaris L.) showed fairly good respiratory control on days 1 and 2 after planting. The respiratory control was completely lost from days 3 to 5. During this period mitochondria were shown to be very leaky, losing about 88% of their total nicotinamide adenine dinucleotide to the suspending medium in a short time. The respiratory control was partially recovered by day 7, after which it completely disappeared again. By the use of differential centrifugation, the mitochondria were divided into subfractions by sequential centrifugation: 10,000g for 5 minutes, 25,000g for 5 minutes, and 40,000g for 5 minutes. The 10,000g subfraction was responsible for the recovery of mitochondrial activity (respiratory control value, adenosine diphosphate to oxygen ratio, and rate of oxygen utilization), on day 7. Activities of succinate dehydrogenase, cytochrome oxidase, pyruvate dehydrogenase, and isocitrate dehydrogenase from different mitochondrial subfractions of aging cotyledons were determined. In general, the enzyme activities, adenosine diphosphate to oxygen ratios, and the ability of mitochondria to swell and contract followed the same pattern as for respiratory control.     

Does the respiratory rate in sea urchin embryos increase during early development without proliferation of mitochondria?

During early development of the sea urchin, the respiratory rate, enhanced upon fertilization, is maintained up to hatching (pre-hatching period) and then gradually increases to a maximum at the gastrula stage (post-gastrula period). Except for a short duration after fertilization, respiration in embryos is strongly inhibited by CN⁻ and antimycin A. During the whole span of early development, the amounts of proteins, cytochromes and the specific activities of cytochrome c oxidase and reduced nicotinamide adenine dinucleotide (NADH) cytochrome c reductase in mitochondria are practically the same as in unfertilized eggs. A marked augmentation of mitochondrial respiration after hatching probably occurs without net increase in whole mitochondrial intrinsic capacities. Carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP) or tetramethyl p-phenylenediamine (TMPD) enhances the respiratory rate in the pre-hatching period but hardly augments the respiration in the post-gastrula period. In the presence of both FCCP and TMPD, the respiratory rate in the pre-hatching period was as high as in the post-gastrula period. Probably, electron transport in the mitochondrial respiratory chain is regulated by acceptor control and limitation of cytochrome c reduction in the pre-hatching period and released from those regulations in the post-gastrula period. Acceptor control of respiration is experimentally reproduced in isolated mitochondria by making adenine nucleotide levels as those levels in the pre-hatching period.     

The effects of drought stress on respiration of isolated corn mitochondria

Mitochondria were isolated from etiolated corn shoots (Zea mays L.) that were stressed to a measured water potential. The rates of mitochondrial respiration in state III, state IV, and without phosphate or ADP on a milligram protein basis decreased as water stress increased with succinate, malatepyruvate, or reduced nicotinamide adenine dinucleotide as substrates. Coupling (as determined by respiratory control and ADP/O ratios) did not decrease with increasing water stress. At water potentials greater than −35 bars all respiration had ceased.     

Oxidative phosphorylation in mitochondria isolated from small intestinal epithelium of thiamphenicol-treated rats and control rats

Treatment of rats with thiamphenicol in a dose of 125 mg/kg per day for 60–64 h causes specific inhibition of mitochondrial protein synthesis, leading to a drastic decrease of the cytochrome c oxidase activity in intestinal epithelium. At the same time the mitochondrial ATPase activity becomes resistant to inhibition by oligomycin. Experiments with isolated intestinal mitochondria revealed that respiration in state 3 is diminished by 55% with succinate (5 mM) and by 40% with pyruvate (10 mM) plus L-malate (2 mM) as the substrates, both as compared to intestinal mitochondria isolated from control rats. P : O ratios as well as respiratory control indices are comparable in the two groups of animals. Uncoupled respiration is inhibited by 35% with succinate as the substrate, while the succinate cytochrome c reductase activity remains unaltered. No inhibition of uncoupled respiration with pyruvate plus L-malate as the substrates was observed. The ATP-P_i exchange activity in the mitochondria from the treated animals is diminished by about 75%. It is concluded that in the mitochondria of the treated animals the inhibition of the coupled respiration (state 3) is caused by the limitation of the ATP-generating capacity and that electron transport is rate limiting only with the rapidly oxidized substrates such as succinate, if respiration is uncoupled.     

Preparation and some properties of submitochondrial particles from tightly coupled mung bean mitochondria

Osmotic shock was found to be better than freezing and thawing, a French press, or sonic oscillation for the preparation of submitochondrial particles from mung bean (Phaseolus aureus) hypocotyl mitochondria. Particles prepared by osmotic shock rapidly oxidize reduced nicotinamide adenine dinucleotide and succinate, but they oxidize malate slowly. NADH oxidation was slightly stimulated by cytochrome c, ATP, and ADP; succinate oxidation was markedly increased by ATP, slightly by ADP and cytochrome c; and malate oxidation required the addition of NAD⁺ NADH oxidation is inhibited weakly by amytal, completely by antimycin A and KCN, but not by rotenone. Chlorsuccinate, malonate, antimycin A, and KCN inhibit succinate oxidation. The action of antimycin A and KCN is incomplete, while chlorsuccinate and malonate were competitive inhibitors. Antimycin A combined stoichiometrically with particle protein in the ratio of 0.23 millimicromole per milligram of protein.     

Effects of guanidine inhibitors on mung bean mitochondria

The effects of phenylethylbiguanidide, decamethylenediguanidide, and octylguanidine have been studied with mung bean hypocotyl mitochondria (Phaseolus aureus var. Jumbo) supplied with malate, reduced nicotinamide adenine dinucleotide, succinate, or ascorbate-tetramethyl-p-phenylenediamine as substrates. The guanidines act as energy transfer inhibitors, all three inhibiting all three phosphorylation sites. Phenylethylbiguanidide causes only partial inhibition even at relatively high concentrations. Decamethylenediguanidide inhibits about 70% of the malate respiration, 55% of the succinate respiration, and 35% of the ascorbate-tetramethyl-p-phenylenediamine respiration.     

Properties of Higher Plant Mitochondria. I. Isolation and Some Characteristics of Tightly-coupled Mitochondria from Dark-grown Mung Bean Hypocotyls

The mitochondria isolated from dark-grown mung bean hypocotyls oxidize succinate, l-malate, and externally added reduced nicotine adenine dinucleotide (NADH) with good respiratory control. While the pattern of respiration resembles that of animal mitochondria, there are 4 basic differences between the respiratory properties of mung bean and animal mitochondria: A) the ability to oxidize NADH, B) the pattern of succinate and malate oxidation, C) the rate of oxygen uptake, and D) the adenosine-5′-diphosphate to oxygen ratios.     

Cytochrome c Effect on Respiration of Heart Mitochondria: Influence of Various Factors

The effect of exogenous cytochrome c on respiration rate of the rat and human heart mitochondria was assessed in situ, using permeabilized fibers. It was (i) much more pronounced in State 2 and 4 than in State 3 with all the respiratory substrates (pyruvate+malate, succinate, palmitoyl-CoA+carnitine and octanoyl-L-carnitine), (ii) different with different substrates, (iii) much higher after ischemia in both metabolic states, particularly in the case of succinate oxidation compared to pyruvate+malate, (iv) the highest in State 4 with succinate as a substrate. Similar results were obtained with the isolated rat and rabbit heart mitochondria. The differences in the degree of stimulation of mitochondrial respiration by cytochrome c and, thus, sensitivity of cytochrome c test in evaluation of the intactness/injury of outer mitochondrial membrane are probably determined by the differences in the cytochrome c role in the control of mitochondrial respiration in the above-described conditions.     

Effect of trifluoperazine on skeletal muscle mitochondrial respiration

The effect of trifluoperazine on the respiration of porcine liver and skeletal muscle mitochondria was investigated by polarographic and spectroscopic techniques. Low concentrations of trifluoperazine (88 nmol/mg protein) inhibited both the ADP- and Ca²⁺-stimulated oxidation of succinate, and reduced the values of the respiratory control index and the $\text{ADP}\text{O}$ and $\text{Ca}{}^{\mathrm{2+}}\text{O}$ ratio. High concentrations inhibited both succinate and ascorbate plus tetramethyl-p-phenylenediame (TMPD) oxidations, and uncoupler (carbonyl cyanide p-trifluromethoxyphenylhydrazone) and Ca²⁺-stimulated respiration. Porcine liver mitochondria were more sensitive to trifluoperazine than skeletal muscle mitochondria. Trifluoperazine inhibited the electron transport of succinate oxidation of skeletal muscle mitochondria within the cytochrome b-c₁ and cytochrome c₁-aa₃ segments of the respiratory chain system. 233 nmol trifluoperazine/mg protein inhibited the aerobic steady-state reduction of cytochrome c₁ by 92% with succinate as substrate, and of cytochrome c and cytochrome aa₃ by 50–60% with ascorbate plus TMPD as electron donors. Trifluoperazine can thus inhibit calmodulin-independent reactions particularly when used at high concentrations.     

Paragonimus westermani possesses aerobic and anaerobic mitochondria in different tissues,adapting to fluctuating oxygen tension in microaerobic habitats

We previously showed that adult Paragonimus westermani, the causative agent of paragonimiasis and whose habitat is the host lung, possesses both aerobic and anaerobic respiratory chains, i.e., cyanide-sensitive succinate oxidase and NADH-fumarate reductase systems, in isolated mitochondria (Takamiya et al., 1994). This finding raises the intriguing question as to whether adult Paragonimus worms possess two different populations of mitochondria, one having an aerobic succinate oxidase system and the other an anaerobic fumarate reductase system, or whether the worms possess a single population of mitochondria possessing both respiratory chains (i.e., mixed-functional mitochondria). Staining of trematode tissues for cytochrome c oxidase activity showed three types of mitochondrial populations: small, strongly stained mitochondria with many cristae, localised in the tegument and tegumental cells; and two larger parenchymal cell mitochondria, one with developed cristae and the other with few cristae. The tegumental and parenchymal mitochondria could be separated by isopycnic density-gradient centrifugation and showed different morphological characteristics and respiratory activities, with low-density tegumental mitochondria having cytochrome c oxidase activity and high-density parenchymal mitochondria having fumarate reductase activity. These results indicate that Paragonimus worms possess three different populations of mitochondria, which are distributed throughout trematode tissues and function facultatively, rather than having mixed-functional mitochondria.     

Biogenesis of Mitochondria in Imbibed Peanut Cotyledons: Influence of the Axis

Development of mitochondrial activities (state 3 respiration,respiratory control ratio, ADP/O ratio) in peanut cotyledonsoccurs over the first 5 d from the start of imbibition. Mitochondriain cotyledons with the axis attached develop better than inthose from which the axis has been removed. Initially, mitochondriaare deficient in cytochrome c, but after 2 d from the startof imbibition this deficiency is overcome. Mitochondrial developmentin attached cotyledons, as measured by state 3 respiration,respiratory control ratio, ADP/O ratio, and succinate dehydrogenaseand cytochrome oxidase activities, is severely impaired by cycloheximide.This indicates that de novo synthesis of proteins is necessaryfor mitochondria and their enzymes to develop, a situation whichis in sharp contrast to the situation in pea cotyledons. Electronmicroscope studies also show that there is an increase in thenumbers of mitochondria in peanut cotyledons with time afterthe start of imbibition. Two patterns of mitochondrial developmentexist in legumes: in imbibed peanut cotyledons respiratory activitiesincrease due to biogenesis of mitochondria, whereas in pea cotyledonsthe increases are due to improvement of pre-existing organelles     

Effects of Gramicidin on Corn Mitochondria

The effects of gramicidin D, S, and J on corn mitochondria respiration and swelling were studied. Only gramicidin D was found to have any pronounced effect on mitochondrial swelling. In buffered KCl gramicidin D produced a rapid, respiration-independent swelling which was not reversed with respiratory inhibitors or substrate exhaustion. The respiration rate of exogenous reduced nicotinamide adenine dinucleotide was stimulated by all three gramicidins, but the effects on malate-pyruvate and succinate respiration depended on the type of gramicidin and the reaction media. The respiration effects of gramicidin D may be due to action at specific sites for each substrate.     

Effects of fusaric acid on respiration in maize root mitochondria

The effects of fusaric acid, a phytotoxin produced byFusarium pathogens, on the metabolism of isolated maize root mitochondria and on maize seed germination and seedling growth were investigated. The phytotoxin inhibited basal and coupled respiration when succinate and α-ketoglutarate were the substrates. Coupled respiration dependent on NADH was inhibited, but basal respiration was not. Consistently, succinate cytochromec oxidoreductase activity was decreased whereas NADH cytochromec oxidoreductase was not affected. The ATPase activities of carbonyl cyanide p-trifluoro-methoxyphenyl hydrazone stimulated mitochondria and of freeze-thawing disrupted mitochondria were inhibited. These results indicate that the phytotoxin impairs the respiratory activity of maize mitochondria by at least three mechanisms: (1) it inhibits the flow of electrons between succinate dehydrogenase and coenzyme Q, (2) it inhibits ATPase/ATP-synthase activity and (3) it possibly inhibits α-ketoglutarate dehydrogenase. Seed germination and seedling growth were also affected by fusaric acid with the most pronounced effect on root development. These effects can possibly contribute to the diseases ofFusarium- infected plants     

The Respiratory Chain of Plant Mitochondria: VIII. Reduction Kinetics of the Respiratory Chain Carriers of Mung Bean Mitochondria with Reduced Nicotinamide Adenine Dinucleotide

Addition of 90 micromolar reduced nicotinamide adenine dinucleotide (NADH) in the presence of cyanide to a suspension of aerobic mung bean (Phaseolus aureus) mitochondria depleted with ADP and uncoupler gives a cycle of reduction of electron transport carriers followed by reoxidation, as NADH is oxidized to NAD⁺ through the cyanide-insensitive, alternate oxidase by excess oxygen in the reaction medium. Under these conditions, cytochrome b₅₅₃ and the nonfluorescent, high potential flavoprotein Fp_ha of the plant respiratory chain become completely reduced with half-times of 2.5 to 2.8 seconds for both components. Reoxidation of flavoprotein Fp_ha on exhaustion of NADH is more rapid than that of cytochrome b₅₅₃. There is a lag of 1.5 seconds after NADH addition before any reduction of ubiquinone can be observed, whereas there is no lag perceptible in the reduction of flavoprotein Fp_ha and cytochrome b₅₅₃. The half-time for ubiquinone reduction is 4.5 seconds, and the extent of reduction is 90% or greater. About 30% of cytochrome b₅₅₇ is reduced under these conditions with a half-time of 10 seconds; both cytochrome b₅₆₂ and the fluorescent, high potential flavoprotein Fp_hf show little, if any, reduction. The two cytochromes c in these mitochondria, c₅₄₇ and c₅₄₉, are reduced in synchrony with a half-time of 0.8 second. These two components are already 60% reduced in the presence of cyanide but absence of substrate, and they become completely reduced on addition of NADH. These results indicated that reducing equivalents enter the respiratory chain from exogenous NADH at flavoprotein Fp_ha and are rapidly transported through cytochrome b₅₅₃ to the cytochromes c; once the latter are completely reduced, reduction of ubiquinone begins. Ubiquinone appears to act as a storage pool for reducing equivalents entering the respiratory chain on the substrate side of coupling site 2. It is suggested that flavoprotein Fp_ha and cytochrome b₅₅₃ together may act as the branching point in the plant respiratory chain from which forward electron transport can take place to oxygen through the cytochrome chain via cytochrome oxidase, or to oxygen through the alternate, cyanide-insensitive oxidase via the fluorescent, high potential flavoprotein Fp_hf.     

Induction by Oxygen of Respiration and Phosphorylation of Anaerobically Grown Escherichia coli

The changes occurring in the respiratory enzymes of anaerobically grown Escherichia coli strain B and E. coli 15 T⁻A⁻U⁻bar during exposure to oxygen were studied. Reduced nicotinamide adenine dinucleotide (NADH) oxidase activity reached its peak soon after O₂ exposure; cytochrome content and succinate oxidase activity increased more slowly, and these increases paralleled each other. The activities of isocitrate and malate dehydrogenases also increased, but the increase was less than that of the succinate and NADH oxidases; exposure to O₂ had no effect on the succinate and NADH dehydrogenase activities. On the other hand, the glycolytic activity decreased slowly after O₂ exposure. The incorporation of ³²P into acid-soluble organic phosphate esters paralleled the respiratory rate during the first 60 min after O₂ exposure, but continued to increase after the respiration reached a plateau. The sensitivity of ³²P incorporation to the uncoupler carbonyl cyanide m-chlorophenylhydrazone also increased with time. The observed relationship between the development of the respiratory chain and the energy-conserving mechanism during O₂ exposure is discussed. Synthesis of the respiratory enzymes upon exposure to oxygen was dependent on concomitant protein and ribonucleic acid synthesis but not on deoxyribonucleic acid synthesis.     

Hydrogenase Activity and the H2-Fumarate Electron Transport System in Bacteroides fragilis

Hydrogenase activity and the H₂-fumarate electron transport system in a carbohydrate-fermenting obligate anaerobe, Bacteroides fragilis, were investigated. In both whole cells and cell extracts, hydrogenase activity was demonstrated with methylene blue, benzyl viologen, flavin mononucleotide, or flavin adenine dinucleotide as the electron acceptor. A catalytic quantity of benzyl viologen or ferredoxin from Clostridium pasteurianum was required to reduce nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate with H₂. Much of the hydrogenase activity appeared to be associated with the soluble fraction of the cell. Fumarate reduction to succinate by H₂ was demonstrable in cell extracts only in the presence of a catalytic quantity of benzyl viologen, flavin mononucleotide, flavin adenine dinucleotide, or ferredoxin from C. pasteurianum. Sulfhydryl compounds were not required for fumarate reduction by H₂, but mercaptoethanol and dithiothreitol appeared to stimulate this activity by 59 and 61%, respectively. Inhibition of fumarate reduction by acriflavin, rotenone, 2-heptyl-4-hydroxyquinoline-N-oxide, and antimycin A suggest the involvement of a flavoprotein, a quinone, and cytochrome b in the reduction of fumarate to succinate. The involvement of a quinone in fumarate reduction is also apparent from the inhibition of fumarate reduction by H₂ when cell extracts were irradiated with ultraviolet light. Based on the evidence obtained, a possible scheme for the flow of electrons from H₂ to fumarate in B. fragilis is proposed.     

The effect of diabetes on protein synthesis and the respiratory chain of rat skeletal muscle and kidney mitochondria

The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa₃ and a 27% decrease in cytochrome b, both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro, nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b, c + c₁, and aa₃, the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : V. On the Reduction of Nonheme Iron and the Cytochromes by Nicotinamide Adenine Dinucleotide and Succinate

The sensitivity of nicotinamide adenine dinucleotide (NADH) oxidase and succinoxidase to metal chelators, the generation of an electron paramagnetic resonance (EPR) signal upon addition of these substrates, and the rate of formation of the EPR signal relative to the rate of the cytochrome reduction suggest the participation of nonheme iron proteins in the respiratory process of Escherichia coli. The most inhibitory metal chelator, thenoyltrifluoro acetone, inhibited the reduction of nonheme iron and cytochromes but did not prevent the reoxidation of the reduced forms. The EPR signal, dehydrogenase, and oxidase activities evoked by NADH are considerably greater than the corresponding activities evoked by succinate. Because both substrates can reduce almost all of the cytochromes, a model in which fewer succinate dehydrogenase-nonheme iron protein complexes are linked to a common cytochrome chain than NADH dehydrogenase-nonheme iron protein complexes is considered likely.