Fumarate reductase and other mitochondrial activities in Trypanosoma cruzi

Subcellular fractions obtained from Trypanosoma cruzi epimastigotes broken by freezing and thawing were assayed for fumarate reductase activity with reduced methyl viologen as electron donor and fumarate as electron acceptor under anaerobic conditions. Two distinct activities were detected: one in the mitochondrial membranes, 115 mU(mg protein)-1, accounting for 96% of the total and the other in the cytosol, 3 mU(mg protein)-1, accounting for 3% of the total. The activity of membrane-bound fumarate reductase correlated statistically with either the activity or the amount of mitochondrial markers such as succinate and NADH dehydrogenases, cytochromes b + c558, cytochrome a611 and 5,7-diene sterols in the obtained subcellular fractions (580 X g, 12 000 X g, and 105 000 X g sediments and supernatant). Mitochondrial fumarate reductase was inhibited by succinate, malonate, cyanide, and 2-thenoyltrifluoroacetone (TTFA); whereas the soluble enzyme was inhibited by succinate and not by TTFA. The 12 000 X g sediment (mitochondrial membranes) showed after dithionite addition, absorption maxima at 611, 560 and 530 nm accounting for the presence of cytochrome b560, c558 and a611. A CO-binding cytochrome o was also detected. A scheme of the T. cruzi mitochondrial respiratory chain is presented.     

Mechanisms of respiration and phosphorylation in Ascaris muscle mitochondria

In Ascaris muscle mitochondria the major respiratory chain-linked phosphorylation activity is accomplished by a NADH-linked reduction of fumarate to succinate. Oxygen can also be employed as a terminal electron acceptor via a cyanide- and salicyl-hydroxamate-resistant terminal oxidase. As in fumarate-dependent electron transport this process appears to be coupled to energy conservation at phosphorylation site I. The branchpoint from which electrons are taken from the main respiratory chain to either the alternative oxidase or fumarate reductase is likely to be on the oxygen side of the NADH dehydrogenase segment.Malate and succinate are the only substrates which appreciably support respiration in the mitochondrion of the nematode. Regardless of the presence or absence of oxygen malate is utilized by an oxidation-reduction reaction resulting in the formation of pyruvate, acetate, succinate, propionate and CO₂. In addition, aerobically, hydrogen peroxide is formed as the product of oxygen reduction. Succinate accumulation was found to be significantly higher in the anaerobic as compared to the aerobic incubation mixtures. This effect was accompanied by an increase in anaerobic malate consumption. ATP generation and the formation of pyruvate, acetate and propionate were found to be similar in the presence and absence of oxygen.In malate-supported respiration of intact Ascaris mitochondria reducing equivalents (NADH) are produced exclusively through pyruvate and acetate formation. These enzymatic reactions are functionally coupled to the electron transport-linked reductions of fumarate to succinate and oxygen to hydrogen peroxide, respectively. In accordance with the position of the redox potentials of the fumarate/succinate and O₂/H₂O₂ couples, anaerobic and aerobic respiration was found to be associated with relatively low energy conservation efficiencies. Thus one molecule of ATP was conserved per 2e^? transferred to fumarate or oxygen, respectively. No evidence could be obtained for a significant activity of energy conservation sites II and III and electron transfer through the alternative oxidase pathway was shown not to be coupled to phosphorylation.     

Rotenone-insensitive NADH dehydrogenase is a potential source of superoxide in procyclic Trypanosoma brucei mitochondria

The rotenone-insensitive NADH dehydrogenase isolated from mitochondria of the procyclic form of Trypanosoma brucei has the ability to produce superoxide anions (Biochemistry 41 (2002) 3065). Superoxide production by the purified enzyme was 60% inhibited by diphenyl iodonium (DPI), stimulated significantly by ubiquinone analogues, and unaffected by metal ions. Production of reactive oxygen species (ROS) in intact cells was not affected by addition of rotenone with proline and malate as substrates; however, addition of rotenone inhibited 41% ROS production with succinate as substrate. These results suggest that complex I is not involved in production of ROS and that succinate-linked reversed electron transport occurs in trypanosome mitochondria. Superoxide formation in mitochondria with NADH as substrate was stimulated by antimycin A but was unaffected by myxothiazol plus stigmatellin, indicating that bc(1) complex is not a source of superoxide. DPI and fumarate inhibited by 68 and 36%, respectively, the rate of superoxide production with NADH as substrate. Addition of both fumarate and DPI blocked 70% superoxide production in mitochondria, a total inhibition similar to that observed with DPI addition alone. These results suggest that the rotenone-insensitive NADH dehydrogenase in addition to NADH fumarate reductase is a potential source of superoxide production in procyclic trypanosome mitochondria.     

Effects of inhibitors on the oxygen kinetics of Nippostrongylus brasiliensis

Endogenous respiration of the parasitic nematode Nippostrongylus brasiliensis and the succinate oxidase activity of isolated mitochondria were partially inhibited by antimycin A; the remaining respiratory activity was sensitive to salicylhydroxamic acid (SHAM). Sub-millimolar concentrations of SHAM markedly stimulated respiration by 60% in whole N. brasiliensis and isolated mitochondria; stimulation by SHAM was not observed in the presence of antimycin A. Little change in the relative fluxes of electrons through the classical, antimycin A-sensitive pathway and the alternative SHAM sensitive pathway was observed between low and high O2 concentrations; this may suggest that the O2 affinities of both pathways are similar. O2 dependence of respiration showed O2 thresholds above which respiration decreases; in the absence of inhibitors whole N. brasiliensis and isolated mitochondria had threshold values around 60 microM O2. Increased O2 threshold values were observed in the presence of SHAM and antimycin A. The apparent Km values for O2 of whole N. brasiliensis and isolated mitochondria were 31 +/- 2 microM O2 and 3.5 +/- 0.2 microM O2 respectively; this difference in apparent Km values may reflect the presence of O2 gradients in the whole worm. The Km and O2 inhibition threshold values observed for whole N. brasiliensis are in good agreement with the proposed range of O2 concentrations thought to exist within the worm's natural environment. H2O2 production was detected in respiring uncoupled mitochondria, but H2O2 could not be detected in the medium surrounding whole N. brasiliensis. SHAM-stimulated respiration was accompanied by increased H2O2 production which was prevented by the addition of antimycin A.     

Electron transfer complexes of Ascaris suum muscle mitochondria: I. Characterization of NADH-cytochrome c reductase (complex I-III), with special reference to cytochrome localization

An NADH-cytochrome c reductase (complex I-III) was isolated from Ascaris suum muscle mitochondria. The enzyme preparation catalyzed the reduction of 1.68 mumol cytochrome c min-1 mg-1 protein at 25 degrees C with NADH but not with NADPH, and retained its sensitivity to rotenone, piericidin A and 2-heptyl-4-hydroxyquinoline-N-oxide as with the submitochondrial particles. The isolated complex I-III, essentially free of succinate-cytochrome c reductase and cytochrome c oxidase, consisted of fourteen polypeptides with apparent molecular weights ranging from 76 000 to 12 000. The complex I-III contained three cytochromes, b-559.5, b-563 and c1-550.5 and Pigment-558 at concentrations of 1.28, 0.211, 1.23 and 0.321 nmol mg-1 protein, respectively. Cytochrome b-558, a major constituent cytochrome of Ascaris mitochondria and previously suggested to participate in the fumarate reductase system, was not fractionated in the complex I-III. Localization of the cytochromes in Ascaris electron transfer complexes is discussed.     

Characterization of the dihydroorotate dehydrogenase as a soluble fumarate reductase in Trypanosoma cruzi

Trypanosoma cruzi, a protozoan causing Chagas' disease, excretes a considerable amount of succinate even though it uses the TCA cycle and the aerobic respiratory chain. For this reason, it was believed that unknown metabolic pathways participate in succinate production in this parasite. In the present study, we examined the molecular properties of dihydroorotate dehydrogenase (DHOD), the fourth enzyme of de novo pyrimidine biosynthetic pathway, as a soluble fumarate reductase (FRD) because our sequence analysis of pyr genes cluster showed that the amino acid sequence of T. cruzi DHOD is quite similar to that of type 1A DHOD of Saccharomyces cerevisiae, an enzyme that uses fumarate as an electron acceptor and produces succinate. Biochemical analyses of the cytosolic enzyme purified from the parasite and of the recombinant enzyme revealed that T. cruzi DHOD has methylviologen-fumarate reductase (MV-FRD) activity. In addition, T. cruzi DHOD was found to catalyze electron transfer from dihydroorotate to fumarate by a ping-pong Bi-Bi mechanism. The recombinant enzyme contained FMN as a prosthetic group. Dynamic light scattering analysis indicated that T. cruzi DHOD is a homodimer. These results clearly indicated that the cytosolic MV-FRD is attributable to T. cruzi DHOD. The DHOD may play an important role in succinate/fumarate metabolism as well as de novo pyrimidine biosynthesis in T. cruzi.     

Anaerobic respiration of Escherichia coli in the mouse intestine

The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coli is the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coli uses to maximize its competitiveness and achieve the highest possible population in the intestine.     

Evidence for the occurrence of respiratory electron transport in adult Brugia pahangi and Dipetalonema viteae

Mixed-sex adult stages of Brugia pahangi and Dipetalonema viteae, in the absence of exogenous substrate, consumed oxygen at rates of 4.18 +/- 0.38 and 2.12 +/- 0.20 ngatoms O2 min-1 mg-1 dry wt. respectively. When calculated on a unit dry weight basis the endogenous O2 consumption rates (E-QO2) of mature adult male macrofilariae of B. pahangi and D. viteae were significantly greater than those of mature females, although the E-QO2 calculated per individual worm was essentially similar irrespective of sex. When assayed as separate unisexual groups, the oxygen uptake of male and female macrofilariae of both species was inhibited by classical inhibitors of respiratory electron transport (RET), and showed classical substrate bypass phenomena in response to succinate and ascorbate, N,N,N',N'-tetramethyl-p-phenylenediamine with respect to the RET inhibitors rotenone (inhibitor of complex I) and antimycin A (inhibitor of complex III). Since male worms elicited similar responses to the classical RET inhibitors as did mixed-sex and/or adult female populations, the possibility that developmental stages contained within the female filariids were contributing in any significant manner to the overall responses observed with the RET inhibitors can be discounted. Such responses as observed with live-intact macrofilariae are normally elicited only by mitochondrial preparations and suggest that the cuticles of both species are permeable to rotenone, succinate, antimycin A, N,N,N',N'-tetramethyl-p-phenylenediamine, azide and cyanide. The uncoupler 2,4-dinitrophenol stimulated the endogenous rate of oxygen consumption (E-QO2) of intact B. pahangi at 33-160 microM, indicating the probable occurrence of RET-coupled oxidative phosphorylation. Higher concentrations of 2,4-dinitrophenol proved inhibitory. Respiratory studies on subcellular fractions substantiated the responses elicited by the intact parasites, suggesting the presence of antimycin A-sensitive and -insensitive RET pathways capable of utilising alpha-glycerophosphate, succinate, and malate as substrates. Both B. pahangi and D. viteae macrofilariae therefore probably possess branched RET-pathways bifurcating on the substrate side of RET-complex III. The rates of substrate oxidation in terms of QO2 mg-1 mitochondrial protein compare well with those observed with other nematode parasites.     

Rhodoquinone requirement of the Hymenolepis diminuta mitochondrial electron transport system

The occurrence of rhodoquinone as a mitochondrial membrane component was demonstrated in adult Hymenolepis diminuta. Chromatographic separation of pentane extracts, from lyophilized mitochondrial membranes, coupled with spectral analyses of separated material demonstrated the presence of rhodoquinone. The presence of ubiquinone was not apparent. Rhodoquinone content of membranes was about 1.2 micrograms (mg protein)-1. The rhodoquinone requirement of the H. diminuta electron transport system was demonstrated both in terms of the less active NADH oxidase and the physiologically required, NADH-dependent fumarate reductase employing lyophilized mitochondrial membranes as the source of activities. Pentane extraction of membranes virtually abolished the oxidase and fumarate reductase systems. Supplementation of pentane-treated membranes with H. diminuta rhodoquinone restored oxidase and fumarate reductase activities to levels simulating those of lyophilized membranes. Ubiquinone did not substitute for rhodoquinone. The rhodoquinone-reconstituted membranes displayed rotenone sensitivity. These findings represent the first direct demonstration of the rhodoquinone requirement of helminth electron transport-coupled oxidase and fumarate reductase.     

Elaboration of mitochondrial function during Trypanosoma brucei differentiation

The development of various mitochondrial functions has been studied using an in vitro system in which Trypanosoma brucei LUMP 1026 bloodstream trypomastigotes differentiate to procyclic trypomastigotes. During differentiation, antimycin A sensitive respiration begins to develop prior to cyanide sensitive respiration, with sensitivity levels equivalent to established procyclic trypomastigotes attained by 20–24 days in culture. In the early stage of differentiation, inhibitor sensitivity is dependent upon the order of inhibitor addition to the cells. Stimulation of oxygen uptake by the artificial electron donor ascorbate (plus tetramethyl-p-phenylenediamine as carrier) closely parallels the development of cyanide sensitivity. Cytochrome aa₃ is not observed spectroscopically until nine days in culture. Specific activities of two mitochondrial enzymes, succinate dehydrogenase and succinate cytochrome c reductase, increase from nil in bloodstream trypomastigotes to significant levels in established procyclic trypomastigotes by 24–30 days in culture.     

Identification of the gene encoding the sole physiological fumarate reductase in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a Gram-negative, nonfermentative rod with a complex electron transport system which facilitates its ability to use a variety of terminal electron acceptors, including fumarate, for anaerobic respiration. CMTn-3, a mutant isolated by transposon (TnphoA) mutagenesis, can no longer use fumarate as an electron acceptor; it lacks fumarate reductase activity as well as a 65-kDa soluble tetraheme flavocytochrome c. The sequence of the TnphoA-flanking genomic DNA of CMTn-3 did not align to those for fumarate reductase or related electron transport genes from other bacteria. Sequence analysis of the MR-1 genomic database demonstrated that an open reading frame encoding a 65-kDa tetraheme cytochrome c with sequence similarity to the fumarate reductase from S. frigidimarina NCIMB400 was found 8 kb away from the TnphoA-flanking genomic DNA of CMTn-3. PCR analysis demonstrated that a large deletion (>or=9.2 kb and 相似文献   

NADH content and lactate production in the perfused rabbit heart

The influence of oxygen availability and absence of contractile activity on the NADH content and lactate production were investigated in the rabbit heart. Isolated hearts were perfused according to Langendorff with a modified Tyrode solution, saturated with a gas mixture containing either 95% O2:5% CO2 (control), 50% O2:5% CO2 in N2 (hypoxia), or 5% CO2 in N2 (anoxia). In another series of hearts cardiac arrest was induced by perfusion with Tyrode solution (95% O2:5% CO2) where the KCl concentration was increased to 15 mmol l-1 (hyperkalemia). Oxygen uptake (VO2) was similar in hypoxic and control hearts (P greater than 0.05), whereas lactate production was four-fold higher during hypoxia vs. control (P less than 0.01). Hyperkalemia resulted in a 60% decrease in VO2 (P less than 0.05), and no significant change in lactate production vs. control (P greater than 0.05). Both PCr and ATP were substantially decreased only during anoxia. Muscle NADH, whose changes reflect those within the mitochondria, averaged (+/- SE) 0.074 +/- 0.010, 0.153 +/- 0.016, 0.486 +/- 0.162 and 1.771 +/- 0.091 mmol kg-1 dry wt during control, hyperkalemia, hypoxia and anoxia, respectively. It is concluded that: muscle contraction during conditions of adequate oxygen supply results in an oxidation of mitochondrial NADH (presumably due to ADP stimulation of respiration), and a decreased oxygen availability results in an increase in NADH and an accelerated lactate production, although the VO2 is not affected.     

Effect of decreased oxygen availability on NADH and lactate contents in human skeletal muscle during exercise

Eight men cycled for 5 min at 120 +/- 6 W (mean +/- SE) at which O2 uptake was 50% of its maximal normoxic value, breathing room air (21% O2; normoxia) on one occasion and 11% O2 in N2 (respiratory hypoxia/hypoxic--Resp. Hx.) on the other. Biopsies were taken from the quadriceps femoris muscle. Oxygen uptake during exercise was not significantly different between Resp. Hx (1.59 +/- 0.08 1 min-1) and normoxia (1.55 +/- 0.08 1 min-1). At rest, muscle lactate was the same under both conditions but was four times higher after Resp. Hx (33.2 +/- 5.2 mmol kg-1 dry wt) than normoxic cycling (8.6 +/- 1.0 mmol kg-1 dry wt; P less than 0.01). The muscle lactate/pyruvate (which is proportional to cytosolic NADH/NAD) was significantly higher after Resp. Hx.(76 +/- 19) than after normoxic cycling (26 +/- 2; P less than 0.05). At rest, analytically determined NADH averaged 0.14 +/- 0.02 mmol kg-1 dry wt under both conditions. However, exercise during Resp. Hx. resulted in a significantly higher NADH content (0.17 +/- 0.01) than exercise during normoxia (0.12 +/- 0.01; P less than 0.01). Indirect evidence indicates that the difference in muscle NADH reflects a difference in the mitochondrial redox state (Sahlin & Katz 1986). The increased muscle NADH during Resp. Hx. therefore indicates a relative lack of O2 at the cellular level (muscle hypoxia). It is suggested that the increased lactate production during Resp. Hx. is a consequence of the cellular adaptation to muscle hypoxia (i.e. increases in cytosolic ADP, AMP, Pi and NADH).     

Endogenous nitric oxide in the control of skeletal muscle oxygen extraction during exercise

Our previous studies uncovered an inhibitory effect of nitric oxide (NO) on leg skeletal muscle respiration in dogs at rest. The role of NO in the modulation of O2 consumption and O2 extraction in hindlimb muscle during elevated metabolic states was investigated in chronically instrumented dogs while walking and at three exercise intensities which markedly increased hindlimb blood flow. Walking resulted in increased O2 consumption by 17 +/- 4 mL min-1 and O2 extraction from 24 +/- 1 to 37 +/- 8%, with no alteration in hindlimb blood flow (BFLeg) and vascular resistance (VRLeg). Running at the highest speed (9.1 mph) resulted in an increase in BFLeg from 0.67 +/- 0.05 to 2.2 +/- 0.1 L min-1, a reduction of VRLeg and elevation of hindlimb O2 consumption from 33 +/- 3 to 226 +/- 21 mL min-1 and O2 extraction from 29 +/- 2 to 61 +/- 5%, with a decrease in leg venous PO2 from 38 +/- 1 to 25 +/- 1 mmHg. After nitro-L-arginine (NLA) (35 mg kg-1, i.v.) to inhibit endogenous NO synthesis, walking caused greater increases in hindlimb O2 consumption (29 +/- 5 mL min-1) and O2 extraction (43 +/- 1 to 60 +/- 3%) (both P < 0.05), with no significant change in BFLeg. During running at the highest speed, BFLeg was 1.9 +/- 0.1 L min-1 (P < 0. 05) and VRLeg was higher, accompanied by increases in hindlimb O2 consumption from 49 +/- 7 to 318 +/- 24 mL min-1 and O2 extraction from 41 +/- 2 to 79 +/- 4% (both P < 0.05), with a greater decrease in leg venous PO2 from 33 +/- 1 to 20 +/- 1 mmHg (P < 0.05). Similar results were found for intermediate levels of exercise. Our results indicate that NO modulates hindlimb skeletal muscle O2 extraction and O2 usage whether blood flow increased or not during exercise.     

Tirandamycin: inhibition of oxidative phosphorylation in rat liver mitochondria

Tirandamycin inhibits respiration and phosphorylation in rat liver mitochondria. An investigation of individual reaction sequences occurring within the respiratory chain showed that the antibiotic stimulates reduced nicotinamide adenine dinucleotide (NADH)- and succinate-linked coenzyme Q reductase. NADH-linked reduction of tetrazolium salts remains unaffected by tirandamycin. Succinotetrazolium salt reductase is inhibited significantly. Reduction of cytochrome c by succinate is blocked by the antibiotic; NADH-cytochrome c reductase is inhibited but not completely blocked. Cytochrome c oxidase remains unaffected. Mitochondrial difference spectra prepared in the presence of tirandamycin indicate that the reduction of cytochrome b is not impaired but no reduction of cytochromes c or a is apparent. These results indicate that tirandamycin interferes with the respiratory chain at a point beyond the cytochrome b and prior to the cytochrome c reduction site. Tirandamycin acts also as a potent inhibitor of ribonucleic acid polymerase as discussed in the foregoing paper.     

Effect of prolonged undernutrition on rat diaphragm mitochondrial respiration.

Previous studies have shown that undernutrition induces an impairment of the respiratory muscle function in patients with chronic lung disease. To explain this, we hypothesized that undernutrition could decrease oxidative metabolism in the diaphragm. We therefore examined the effect of prolonged undernutrition on diaphragm mitochondrial oxygen uptake with pyruvate and palmitate as substrates in adult rats. Ten rats served as controls (CTL). Ten nutritionally deprived rats (ND) received 40% of their estimated daily nutrition. Five weeks of undernutrition induced a 33% decrease in state 3 respiration with pyruvate plus malate as substrate (993 +/- 171 versus 1488 +/- 167 nmol atomic O/mg/min, P < 0.01) and a 39% decrease with palmitate plus malate (516 +/- 89 versus 850 +/- 165 nmol atomic O/mg/min, P < 0.05). With succinate plus rotenone, there was no significant difference in the respiratory rate between groups. In the ND group, we found a significant decrease in citrate synthase activity (P < 0.01), and also in reduced nicotinamine adenine dinucleotide (NADH) dehydrogenase activity (P < 0.05), which cannot alone induce such a state 3 respiratory decrease. This showed that undernutrition in rat diaphragm does not induce an alteration in protein complexes I, II, III, and IV, or the F complex containing the mitochondrial ATPase of the electron transport chain. In conclusion, the main result of this study was that prolonged undernutrition induced a decrease in mitochondrial respiration secondary to a significant reduction in NADH generation by the Krebs cycle, which may affect respiratory muscle function with implications for patient care.     

Succinate-dependent energy generation in Ascaris suum mitochondria

Phosphorylation in isolated Ascaris suum mitochondria was much greater in the presence of malate than succinate, but, in the absence of added adenine nucleotides, incubations in succinate resulted in substantial elevations in intramitochondrial ATP levels. Succinate-dependent phosphorylation was stimulated aerobically and this stimulation was due almost entirely to a site I, rotenone-sensitive, phosphorylation. Increased substrate level phosphorylation, coupled to propionate formation, or additional sites of electron-transport associated ATP synthesis were not significant. Under aerobic conditions, 14CO2 evolution from 1,4-[14C]succinate was stimulated and NADH/NAD+ ratios were elevated, but the formation of [14C]propionate was unchanged. It appears that succinate was metabolized to pyruvate and acetate, and NADH, generated from the decarboxylations of malate and pyruvate, was the primary source of reducing power fueling electron-transport. The terminal oxidase and final electron-acceptor are still not clearly defined. However, ferricyanide, H2O2, and 100% oxygen all stimulated succinate-dependent phosphorylation. A possible role for cytochrome c peroxidase in A. suum mitochondrial metabolism is discussed.     

Effect of inhibitors of electron transport and oxidative phosphorylation on trypanosoma cruzi respiration and growth

Antimycin A and 2-heptyl-4-hydroxyquinoline N-oxide, two specific inhibitors of the b-c₁ segment of the respiratory chain, affected the respiration of Trypanosoma cruzi epimastigote forms. The half-maximum inhibitory concentration were about 0.05 and 0.04 μg/mg cells (dry wt.), respectively. The maximum effect of antimycin (about 80% inhibition of respiration) was at about 0.1 μg antimycin/mg cells. Differential spectrophotometry of T. cruzi epimastigotes in the presence of antimycin, cyanide (or sulfide) and uncouplers, revealed the presence of functional cytochromes aa₃, b and c₅₅₈. In the stationary growth phase respiration by T. cruzi was completely inhibited by cyanide and effectively ihibited by sulfide, but in the expontential growth phase respiration was about 20% insensitive to 5 mM cynaide. Cyanide- and antimycin-insensitive respiration was completely inhibited by salicylhydroxamic acid (2 mM).Antimycin inhibited the operation of the tricarboxylic acids cycle in T. cruzi, as shown by the lesser production of ¹⁴CO₂ and by the modification of ¹⁴C distribution in epimastigotes incubated with [1-¹⁴C]glucose, [2-¹⁴C]acetate or NaH¹⁴CO₃. The inhibition of electron transport by antimycin increased the rate of the fumarate reductase reaction, an alternative electron pathway for the oxidation of reduced pyridine nucleotides.Addition of carbonyl cyanide 3-chlorophenylhydrazone to epimastigotes increased the rate of respiration and promoted the oxidation of reduced cytochrome b components, thus showing that these components are subjects to respiratory (acceptor) control. Pentachlorhenol similarly affected the cytochrome b redox level but did not modify the rate of respiration. The uncouplers released N,N′-dicyclohexycarbodiimide inhibition of respiration, and uncouplers and cyanide significantly decreased the ATP level in epimastigotes. The combined effects of the assayed inhibitors on respiration, cytochrome b redox level, ATP content and energy charge confirmed the operation of oxidative phosphorylation in T. cruzi epimastigotes. Antimycin, uncouplers and N,N′-dicyclohexylcarbodiimide inhibited growth of T. cruzi, thus proving the essential role of oxidative phosphorylation for the parasite.     

The effect of high-intensity exhaustive exercise studied in isolated mitochondria from human skeletal muscle

Six young men performed five 1-min bicycle exercise bouts to exhaustion. Muscle lactate increased to congruent with 114 mmol x kg(-1) dwt and pH decreased to congruent with 6.6. Mitochondria were prepared from a needle biopsy sample taken from m. vastus lateralis immediately after the last exercise bout. No significant effect of exhaustion on the proton permeability and amount of cytochromes c and aa3 in isolated mitochondria was detected. The activities of the following enzymes and systems were not altered either: citrate synthase, succinate dehydrogenase, cytochrome oxidase, succinate + glutamate respiration, malate + glutamate respiration, the respiratory chain, and the reactions involved in ATP synthesis. Thus, the mitochondria did not appear globally altered upon exhaustion. However, the following NAD-linked activities were significantly lowered: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, glutamate dehydrogenase and fatty acid beta-oxidation. The activities of alpha-glycerophosphate dehydrogenase and exo-NADH oxidase, enzymes that might catalyze the oxidation of sarcoplasmic NADH, were increased. These changes may be due to the action of reactive oxygen species, protons and Ca2+. Transient opening of the permeability transition pore may also be involved. Some effects may have been reversed during isolation of the mitochondria and the changes in mitochondrial function in situ upon exhaustion may have been more extensive than observed.     

Chemiluminescence of superoxide generated by Candida albicans: differential effects of the superoxide generator paraquat on a wild-type strain and a respiratory mutant.

We previously reported that a respiration-competent parent strain (K) of Candida albicans was more susceptible to the intracellular superoxide radical (O2-) generator paraquat (PQ) than was a respiration-deficient mutant (KRD-19), although both showed a similar sensitivity to extracellularly generated O2-. To clarify the cause of the differential PQ lethality, we developed a chemiluminescence method for measuring O2- generated by C. albicans cells by using the probe methyl-Cypridina-luciferin analogue (MCLA), and examined the effects of PQ on O2- generation in both parent and mutant strains. Endogenous O2- generation without stimulation by PQ was unexpectedly low in both strains. PQ-induced O2- generation in the parent strain was maximal in logarithmic phase cells and lowered in stationary phase cells. In contrast, O2- generation in the mutant remained low throughout the growth phase, even when stimulated by PQ. The extent of PQ-induced O2- generation in the parent strain depended on the carbon source added to the assay mixture; in decreasing order, glucose, glycerol, no carbon source. The inhibitor of the cytochrome respiratory chain, antimycin A, suppressed almost completely the PQ-induced O2- generation in the parent strain. It has been established that PQ is converted to its radical form (PQ+) by receiving a single electron in cells. PQ+ then reduces molecular oxygen to O2- by redox cycling. Thus, the high tolerance to PQ of the respiration-deficient mutant can be explained by minimal PQ+/O2- production due to the limited supply of electrons from the impaired respiratory system.