A malate-oxaloacetate shuttle linking cytosolic fatty acid α-oxidation to the mitochondrial electron transport chain in uncoupled fresh potato slices

The possible existence of a malonate-sensitive dicarboxylate-mediated electron shuttle between microsomal NAD-linked fatty acid α-oxidation and the mitochondrial electron transport chain in uncoupled fresh potato slices was investigated. Uncoupled slice respiration is inhibited by benzylmalonate and butylmalonate, inhibitors of dicarboxylate transport into mitochondria. Uncoupled slice respiration is also inhibited by rotenone, an indication of intramitochondrial NADH oxidation. Since fatty acid α-oxidation per se is rotenone insensitive, rotenone and benzylmalonate inhibition of the oxidation of carboxyl-labeled myristate in slices points to a dicarboxylic acid shuttle linking microsomal fatty acid a-oxidation with intramitochondrial NADH dehydrogenase.
Malonute inhibits both respiration and ¹⁴CO², release from carboxyl-labeled myristate in fresh uncoupled slices, as do inhibitors of dicarboxylate transport. Mitochondrial studies show that malonate inhibits malate oxidation but not malate dehydrogenase per se. Furthermore, malonate inhibits malate transport more severely than malate oxidation. Accordingly, mulonate inhibition of uncoupled slice respiration in the absence of tricarboxylic acid cycle activity is attributed to its interference with mitochondrial malate transport, and its consequent curtailment of a putative malate-OAA shuttle linked to cytosolic NAD-mediated fatty acid α-oxidation.     

Entamoeba histolytica. I. Aerobic metabolism

The respiration of intact trophozoites harvested from axenic cultures of Entamoeba histolytica was studied with the polarographic technique utilizing the Clark oxygen electrode. A typical Q_o2 value for the freshly harvested amebae was 1 μatom oxygen/mg protein/hr.It was conclusively demonstrated that this parasite, a putative anaerobe, not only consumes oxygen when provided, but has a high affinity for the gas.Added glucose, galactose, and ethanol increased the respiratory rates, whereas other carbohydrates were without effect on the endogenous respiration. Intermediates of the tricarboxylic acid cycle, amino and fatty acids did not stimulate the respiration of E. histolytica.Inhibitors of the mammalian respiratory chain (cyanide, antimycin) as well as agents that inhibit enzymes catalyzing the tricarboxylic acid cycle (malonate, fluoropyruvate, fluoroacetate, fluorocitrate) had little effect on the endogenous or glucose-supported respiration. Alkylating agents (iodoacetamide, iodoacetate), cinnamate, and N-ethylymaleimide strongly inhibited the oxygen consumption of E. histolytica. The chemotherapeutic agents, Paromomycin, Emetine and Metronidazole, at concentrations that inhibit growth in vitro, did not restrict the respiration.Storage of the trophozoites at 4 C led to progressive deterioraion of the parasites and loss of endogenous and glucose-supported respiration. The deterioration was paralled by loss of SH-materials from the amebae. Likewise, sonication or lysis with detergents abolished both the endogenous respiration and response to glucose.Exogenous NADH or NADPH evoked only marginal increases in oxygen consumption of the freshly harvested amebae, but were effective respiratory substrates with stored or sonicated organisms. Addition of vitamin K₃ greatly enhanced the endogenous and glucose-supported respiration of the intact amebae, as well as enhancing the response of stored or sonicated amebae to reduced pyridine nucleotides.     

Dark Respiration Protects Photosynthesis Against Photoinhibition in Mesophyll Protoplasts of Pea (Pisum sativum)

The optimal light intensity required for photosynthesis by mesophyll protoplasts of pea (Pisum sativum) is about 1250 microeinsteins per square meter per second. On exposure to supra-optimal light intensity (2500 microeinsteins per square meter per second) for 10 min, the protoplasts lost 30 to 40% of their photosynthetic capacity. Illumination with normal light intensity (1250 microeinsteins per square meter per second) for 10 min enhanced the rate of dark respiration in protoplasts. On the other hand, when protoplasts were exposed to photoinhibitory light, their dark respiration also was markedly reduced along with photosynthesis. The extent of photoinhibition was increased when protoplasts were incubated with even low concentrations of classic respiratory inhibitors: 1 micromolar antimycin A, 1 micromolar sodium azide, and 1 microgram per milliliter oligomycin. At these concentrations, the test inhibitors had very little or no effect directly on the process of photosynthetic oxygen evolution. The promotion of photoinhibition by inhibitors of oxidative electron transport (antimycin A, sodium azide) and phosphorylation (oligomycin) was much more pronounced than that by inhibitors of glycolysis and tricarboxylic acid cycle (sodium fluoride and sodium malonate, respectively). We suggest that the oxidative electron transport and phosphorylation in mitochondria play an important role in protecting the protoplasts against photoinhibition of photosynthesis. Our results also demonstrate that protoplasts offer an additional experimental system for studies on photoinhibition.     

Nitrate and Ammonium Induced Photosynthetic Suppression in N-Limited Selenastrum minutum

Nitrate-limited chemostat cultures of Selenastrum minutum Naeg. Collins (Chlorophyta) were used to determine the effects of nitrogen addition on photosynthesis, dark respiration, and dark carbon fixation. Addition of NO₃⁻ or NH₄⁺ induced a transient suppression of photosynthetic carbon fixation (70 and 40% respectively). Intracellular ribulose bisphosphate levels decreased during suppression and recovered in parallel with photosynthesis. Photosynthetic oxygen evolution was decreased by N-pulsing under saturating light (650 microeinsteins per square meter per second). Under subsaturating light intensities (<165 microeinsteins per square meter per second) NH₄⁺ addition resulted in O₂ consumption in the light which was alleviated by the presence of the tricarboxylic acid cycle inhibitor fluoroacetate. Addition of NO₃⁻ or NH₄⁺ resulted in a large stimulation of dark respiration (67 and 129%, respectively) and dark carbon fixation (360 and 2080%, respectively). The duration of N-induced perturbations was dependent on the concentration of added N. Inhibition of glutamine 2-oxoglutarate aminotransferase by azaserine alleviated all these effects. It is proposed that suppression of photosynthetic carbon fixation in response to N pulsing was the result of a competition for metabolites between the Calvin cycle and nitrogen assimilation. Carbon skeletons required for nitrogen assimilation would be derived from tricarboxylic acid cycle intermediates. To maintain tricarboxylic acid cycle activity triose phosphates would be exported from the chloroplast. This would decrease the rate of ribulose bisphosphate regeneration and consequently decrease net photosynthetic carbon accumulation. Stoichiometric calculations indicate that the Calvin cycle is one source of triose phosphates for N assimilation; however, during transient N resupply the major demand for triose phosphates must be met by starch or sucrose breakdown. The effects of N-pulsing on O₂ evolution, dark respiration, and dark C-fixation are shown to be consistent with this model.     

Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.     

Dark Respiration during Photosynthesis in Wheat Leaf Slices

The metabolism of [¹⁴C]succinate and acetate was examined in leaf slices of winter wheat (Triticum aestivum L. cv Frederick) in the dark and in the light (1000 micromoles per second per square meter photosynthetically active radiation). In the dark [1,4-¹⁴C]succinate was rapidly taken up and metabolized into other organic acids, amino acids, and CO₂. An accumulation of radioactivity in the tricarboxylic acid cycle intermediates after ¹⁴CO₂ production became constant indicates that organic acid pools outside of the mitochondria were involved in the buildup of radioactivity. The continuous production of ¹⁴CO₂ over 2 hours indicates that, in the dark, the tricarboxylic acid cycle was the major route for succinate metabolism with CO₂ as the chief end product. In the light, under conditions that supported photorespiration, succinate uptake was 80% of the dark rate and large amounts of the label entered the organic and amino acids. While carbon dioxide contained much less radioactivity than in the dark, other products such as sugars, starch, glycerate, glycine, and serine were much more heavily labeled than in darkness. The fact that the same tricarboxylic acid cycle intermediates became labeled in the light in addition to other products which can acquire label by carboxylation reactions indicates that the tricarboxylic acid cycle operated in the light and that CO₂ was being released from the mitochondria and efficiently refixed. The amount of radioactivity accumulating in carboxylation products in the light was about 80% of the ¹⁴CO₂ release in the dark. This indicates that under these conditions, the tricarboxylic acid cycle in wheat leaf slices operates in the light at 80% of the rate occurring in the dark.     

Arbuscular Mycorrhizal Symbiosis with Arundo donax Decreases Root Respiration and Increases Both Photosynthesis and Plant Biomass Accumulation

The effect of arbuscular mycorrhiza (AM) symbiosis on plant growth is associated with the balance between costs and benefits. A feedback regulation loop has been described in which the higher carbohydrate cost to plants for AM symbiosis is compensated by increases in their photosynthetic rates. Nevertheless, plant carbon balance depends both on photosynthetic carbon uptake and respiratory carbon consumption. The hypothesis behind this research was that the role of respiration in plant growth under AM symbiosis may be as important as that of photosynthesis. This hypothesis was tested in Arundo donax L. plantlets inoculated with Rhizophagus irregularis and Funneliformis mosseae. We tested the effects of AM inoculation on both photosynthetic capacity and in vivo leaf and root respiration. Additionally, analyses of the primary metabolism and ion content were performed in both leaves and roots. AM inoculation increased photosynthesis through increased CO₂ diffusion and electron transport in the chloroplast. Moreover, respiration decreased only in AM roots via the cytochrome oxidase pathway (COP) as measured by the oxygen isotope technique. This decline in the COP can be related to the reduced respiratory metabolism and substrates (sugars and tricarboxylic acid cycle intermediates) observed in roots.     

Use of β-Methylene-D,L-Aspartate to Assess the Role of Aspartate Aminotransferase in Cerebral Oxidative Metabolism

Several inhibitors of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. beta-Methylene-D,L-aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L-vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of beta-methyleneaspartate, a one-to-one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-methyl-D,L-didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of beta-methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-methyleneaspartate. At 1 h the concentration of lactate and the lactate/pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate-aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways.(ABSTRACT TRUNCATED AT 400 WORDS)     

Participation of the cyanide-resistant pathway in respiration of winter rape leaves as affected by plant cold acclimation

Leaf slices sampled from winter rape plants ( Brassica napus L., var. oleifera L., cv. GórczaánAski), grown in cold (5°C), showed an increase in the dark respiration rate (measured at 25°C) as compared to slices cut from control plants (grown at 20/15°C). The effect of low temperature was most pronounced after 4 days of plant growth in the cold. Oxygen uptake by control slices was 60% inhibited by 1 m M KCN and was insensitive to 2.5 m M salicylhydroxamic acid (SHAM). On the contrary, respiration of leaf slices from cold-pretreated plants was more resistant to cyanide (35% inhibition after 4 days of cold treatment) and was 30% inhibited by SHAM. The patterns of cold-induced changes in total respiratory activity and in the estimated activity of alternative pathway were similar. It seems that in leaf slices from plants grown in the cold, the cyanide-resistant, alternative pathway participates in oxygen uptake. Cold treatment of plants also brought about a 4-fold increase in the level of soluble sugars, which reached a maximum on day 4 of exposure to cold. Addition of sucrose to the incubation medium resulted in an immediate increase in oxygen uptake by slices with low endogenous sugar level. The respiration stimulated by sucrose addition was more resistant to cyanide than the basal respiration and it was inhibited by SHAM. It is concluded that the operation of the alternative pathway is responsible for the increased oxygen uptake by the cold-grown winter rape leaves and it may be induced by an increased sugar supply for respiratory processes.     

Regulation of respiration in plants: a role for alternative metabolic pathways

Respiratory metabolism includes the reactions of glycolysis, the tricarboxylic acid cycle and the mitochondrial electron transport chain, but is also directly linked with many other metabolic pathways such as protein and lipid biosynthesis and photosynthesis via photorespiration. Furthermore, any change in respiratory activity can impact the redox status of the cell and the production of reactive oxygen species. In this review, it is discussed how respiration is regulated and what alternative pathways are known that increase the metabolic flexibility of this vital metabolic process. By looking at the adaptive responses of respiration to hypoxia or changes in the oxygen availability of a cell, the integration of regulatory responses of various pathways is illustrated.     

Germination of Echinochloa crus-galli (Barnyard Grass) Seeds under Anaerobic Conditions : Respiration and Response to Metabolic Inhibitors

Echinochloa crus-galli L. Beauv., a rice-field weed, can germinate and grow for extended periods of time in an anaerobic environment. Compared to pea, which does not germinate under anaerobiosis, the evolution of CO₂ in Echinochloa and rice is lower and the peak rate of CO₂ evolution is delayed when germinated without oxygen. The plants studied also differ with respect to their respiration ratio ([CO₂] N₂/[CO₂] air) and metabolism used during the early stages of germination. Echinochloa does not increase its glycolytic rate under anaerobiosis, whereas pentose phosphate pathway activity appears to increase during the first 40 to 50 hours of germination.

Based on its response to metabolic inhibitors (NaF, dinitrophenol, and malonate), anaerobic metabolism in Echinochloa proceeds primarily through glycolysis, with partial operation of the tricarboxylic acid cycle and little or no oxidative phosphorylation. Also, Echinochloa is sensitive to CN during aerobic germination, whereas rice appears to be able to shift to CN-insensitive electron transport. Finally, the effectiveness of cyanide and azide on inhibiting germination of Echinochloa in N₂, but not CO, suggests that cytochrome oxidase is not used to reoxidize pyridine nucleotides in the absence of oxygen. The possible existence of an alternate electron acceptor is discussed.

     

Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.     

Relative Contribution of Cytochrome-mediated and Cyanide-resistant Electron Transport in Fresh and Aged Potato Slices

The respiration of fresh potato (Solanum tuberosum, var. Russet Burbank) slices is predominantly cyanide-sensitive whether in the presence or absence of uncoupler. By contrast, the wound-induced respiration which develops in thin slices with aging is cyanide-resistant, and in the presence of cyanide, sensitive to chlorobenzhydroxamic acid, a selective inhibitor of the cyanide-resistant respiration. Titration of the alternate path in coupled slices with chlorobenzhydroxamic acid, in the presence and absence of cyanide, shows that the contribution of the cyanide-resistant pathway to the wound-induced respiration is zero. Similar titrations with uncoupled slices reveal that the alternate path is engaged and utilized extensively.

The maximal capacity of the cytochrome path (V_cyt) has been estimated in fresh and aged slices in the presence of the uncoupler carbonyl-cyanide m-chlorophenyl hydrazone. It has been found that V_cyt of aged slices is but 30 to 40% higher than that of fresh slices. The results suggest that the bulk of the wound-induced respiration is mediated through the cytochrome pathway which exists in fresh slices in suppressed form, and which is fully expressed by slice aging. The engagement of the alternate path by uncouplers in aged slices is attributed to an increase in substrate mobilization, with the result that the electron transport capacity of the cytochrome chain is exceeded.

     

A defect in menadione biosynthesis induces global changes in gene expression in Staphylococcus aureus

Both the high-resolution two-dimensional protein gel electrophoresis technique and full-genome DNA microarrays were used for identification of Staphylococcus aureus genes whose expression was changed by a mutation in menD. Because the electron transport chain is interrupted, the mutant should be unable to use oxygen and nitrate as terminal electron acceptors. Consistent with this, a mutation in menD was found to cause a gene expression pattern typically detected under anaerobic conditions in wild-type cells: proteins involved in glycolytic as well as in fermentation pathways were upregulated, whereas tricarboxylic acid (TCA) cycle enzymes were significantly downregulated. Moreover, the expression of genes encoding enzymes for nitrate respiration and the arginine deiminase pathway was strongly increased in the mutant strain. These results indicate that the menD mutant, just as the site-directed S. aureus hemB mutant, generates ATP from glucose or fructose mainly by substrate phosphorylation and might be defective in utilizing a variety of carbon sources, including TCA cycle intermediates and compounds that generate ATP only via electron transport phosphorylation. Of particular interest is that there are also differences in the gene expression patterns between hemB and menD mutants. While some anaerobically active enzymes were present in equal amounts in both strains (Ldh1, SACOL2535), other classically anaerobic enzymes seem to be present in higher amounts either in the hemB mutant (e.g., PflB, Ald1, IlvA1) or in the menD mutant (arc operon). Only genes involved in nitrate respiration and the ald1 operon seem to be additionally regulated by a depletion of oxygen in the hemB and/or menD mutant.     

COMPARATIVE EFFECTS OF ORGANIC AND INORGANIC MERCURY ON BRAIN SLICE RESPIRATION AND METABOLISM

—Respiration and carbohydrate metabolism were measured in guinea-pig brain slices exposed to organic and inorganic mercury. Organic mercury decreased oxygen uptake and ¹⁴CO₂ production at consistently lower concentrations than inorganic mercury. Organic mercury also caused a striking elevation of pyruvate and lactate at low doses where inorganic mercury had no effect on respiration or metabolism. A unique inhibition of tricarboxylic acid cycle function is suggested and might partially explain the distinctive neurotoxicity of organic mercury.     

Phenolic metabolism in root slices of selected carrot cultivars

Carrot root slices, stored for 4 days at 20 °C reacted with a strong accumulation of total phenols, especially chlorogenic acid. A significant accumulation of isocoumarin content within the peel was observed in stored slices. Synthesis of phenols was accompanied by an increase in phenylalanine ammonia lyase activity, wound induced respiration and ethylene production. The great variability among the studied four cultivars was found concerning isocoumarin synthesis, PAL activity, respiration rate and ethylene evolution, but less distinct in the case of chlorogenic acid accumulation. The carrot slices obtained from freshly harvested roots were more sensitive to mechanical damage and short-term storage than those prepared from roots previously stored.     

Titles of related papers in other sections

Culture of Trypanosoma cruzi (Tulahuen strain) in the presence of ethidium bromide (1–20 μg/ml) resulted in dyskinetoplasty and inhibition of growth, to an extent depending on the dye concentration and the medium composition. The ethidium bromide-induced dyskinetoplasty caused a decrease of (a) the cytochrome content of epimastigotes (a,a₃ and b species); (b) the rate of respiration (endogenous or supported by D-glucose); and (c) the rate of production of ¹⁴CO₂ from [2-¹⁴C]acetate and [1-¹⁴C]glucose. [2-¹⁴C]Acetate oxidation to ¹⁴CO₂ was affected by dyskinetoplasty more than [1-¹⁴C]glucose oxidation, particularly at the exponential growth phase. With dyskinetoplastic epimastigotes, diminution of ¹⁴CO₂ production from [2-¹⁴C]acetate largely exceeded that of oxygen uptake, while with [1-¹⁴C]glucose, ¹⁴CO₂production and respiration were affected to about the same extent. Dyskinetoplasty also decreased the incorporation of [2-¹⁴C]acetate carbon into intermediates of the tricarboxylic acid cycle and related amino acids, and modified the distribution pattern of ¹⁴C in accordance with the decrease of respiration. Reduction of cytochrome content of epimastigotes by restriction of heme compounds during growth decreased ¹⁴CO₂ production from [2-¹⁴C]acetate, like the ethidium-induced dyskinetoplasty. The same occurred after inhibition of electron transfer by antimycin and cyanide, though to a much more significant extent, thus confirming the functional association of electron transport at the mitochondrial cytochrome system of T. cruzi and the enzymatic reactions of the tricarboxylic acid cycle.     

Metabolic fate of fumarate, a side product of the purine salvage pathway in the intraerythrocytic stages of Plasmodium falciparum

In aerobic respiration, the tricarboxylic acid cycle is pivotal to the complete oxidation of carbohydrates, proteins, and lipids to carbon dioxide and water. Plasmodium falciparum, the causative agent of human malaria, lacks a conventional tricarboxylic acid cycle and depends exclusively on glycolysis for ATP production. However, all of the constituent enzymes of the tricarboxylic acid cycle are annotated in the genome of P. falciparum, which implies that the pathway might have important, yet unidentified biosynthetic functions. Here we show that fumarate, a side product of the purine salvage pathway and a metabolic intermediate of the tricarboxylic acid cycle, is not a metabolic waste but is converted to aspartate through malate and oxaloacetate. P. falciparum-infected erythrocytes and free parasites incorporated [2,3-(14)C]fumarate into the nucleic acid and protein fractions. (13)C NMR of parasites incubated with [2,3-(13)C]fumarate showed the formation of malate, pyruvate, lactate, and aspartate but not citrate or succinate. Further, treatment of free parasites with atovaquone inhibited the conversion of fumarate to aspartate, thereby indicating this pathway as an electron transport chain-dependent process. This study, therefore, provides a biosynthetic function for fumarate hydratase, malate quinone oxidoreductase, and aspartate aminotransferase of P. falciparum.     

Measurements of the Engagement of Cyanide-Resistant Respiration in the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana with the Use of On-Line Oxygen Isotope Discrimination

Discrimination against ¹⁸O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%_o for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%_o) compared with nongreen tissues (25.3-25.9%_o). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.