Evidence for the occurrence of the malate-citrate shuttle in intact Ehrlich ascites tumor cells

A possible activity of the malate-citrate shuttle has been investigated in Ehrlich ascites cells by testing the effects of 1,2,3-benzenetricarboxylic acid, an inhibitor of the malate-citrate exchange, and (?)-hydroxycitrate, an inhibitor of the citrate cleavage enzyme, on the glucose-dependent oxidation-reduction rates of pyridine nucleotides and cytochrome b as well as on ATP levels of glycolyzing cells. Moreover, to quantitate such an activity, the effects of these two inhibitors have been compared with those induced under the same experimental conditions by aminooxyacetate, an inhibitor of the malate-aspartate shuttle which is known to operate in this strain of ascites tumor. Both benzenetricarboxylic acid and hydroxycitrate are able to increase the reduction of pyridine nucleotides, which follows glucose addition to whole cells, to about the same extent. A much more pronounced effect is elicited by aminooxyacetate under the same condition. When n-butylmalonate is added to slow down the flux of glycolytic reducing equivalents to the respiratory chain via the malate-aspartate shuttle, benzenetricar-boxylic acid or hydroxycitrate promotes an ATP-driven reversal of electron transfer. Indeed, the glucose-induced reduction of cytochrome b becomes sensitive to oligomycin and the ATP level is raised significantly with respect to the value of uninhibited cells. It is concluded that the malate-citrate shuttle operates in Ehrlich ascites cells, although with a substantially lower activity with respect to the malate-aspartate shuttle.     

Control of the pyridine nucleotide-linked Ca release from mitochondria by respiratory substrates

Oxidation of mitochondrial pyridine nucleotides followed by their hydrolysis promotes Ca²⁺ release from intact liver mitochondria. In most of the previous studies oxidation was achieved with pro-oxidants which were added to mitochondria respiring on succinate in the presence of rotenone, a site I-specific inhibitor of the respiratory chain. Here we investigate pro-oxidant dependent and independent Ca²⁺ release from mitochondria when respiration is supported either by the NAD⁺-linked substrate β-hydroxybutyrate, or by succinate. In the presence, as well as in the absence, of the pro-oxidant t-butylhydroperoxide mitochondria retain Ca²⁺ much better with succinate than with β-hydroxybutyrate, as respiratory substrate. When Ca²⁺ release is induced by t-butylhydroperoxide succinate-supported Ca²⁺ retention is impeded by rotenone. Ca²⁺ release (pro-oxidant dependent or independent) is paralleled by oxidation and hydrolysis of intramitochondrial pyridine nucleotides, and Ca²⁺ retention is paralleled by reduction of pyridine nucleotides. It is concluded that the pyridine nucleotide-linked Ca²⁺ release from mitochondria can be controlled by respiratory substrates which regulate the intramitochondrial hydrolysis of oxidized pyridine nucleotides.     

Fluorometric studies of oxidative metabolism in isolated papillary muscle of the rabbit

The fluorometric technique for measuring the levels of reduced pyridine nucleotides was used to study oxidative metabolism in isolated rabbit papillary muscle at 23°C. The 100% standard level of tissue fluorescence was defined as that measured for muscles resting in oxygenated 10 mM pyruvate solution. This level increased 15% with anoxia and decreased 45% with stimulation in substrate-free solution. Thus, about one-half of the standard tissue fluorescence was metabolically labile and this labile fraction is suggested to be mitochondrial in origin. Decreased tissue fluorescence following mechanical activity was identified with increased oxidation of mitochondrial reduced nicotinamide adenine dinucleotide (NADH) owing to stimulation by adenosine diphosphate (ADP), released during activity, of mitochondrial respiration. The kinetics of the fluorescence transients were slowed fourfold by removal of pyruvate. This effect was not significantly reversed by addition of 10 mM glucose. The time integrals of the fluorescence transients were linearly related to the amounts of mechanical activity in the presence, but not in the absence, of pyruvate. A positive correlation was observed between the steady-state peak tension at constant stimulus rate and the resting level of reduction of pyridine nucleotides in various media. The fluorometric results are interpreted to be indicative of the steady and transient states established by the substrate dehydrogenases and the respiratory chain during oxidative phosphorylation in mitochondria.     

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.     

Regulation of respiration and nitrogen fixation in different types of Azotobacter vinelandii.

The levels of the adenine nucleotides, pyridine nucleotides and the kinetical parameters of the enzymes of the Entner-Doudoroff pathway (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined in Azotobacter vinelandii cells, grown under O2- or N2-limiting conditions. It was concluced that the levels of both the adenine nucleotides and pyridine nucleotides do not limit the rate of sucrose oxidation. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation of Azotobacter cells is associated with an increase in the rate of sucrose uptake. The sites of oxidative phosphorylation and the composition of the respiratory membranes with respect to cytochromes c4 + c5, b and d differ in cells growth either O2- or N2-limited. It was possible to show that the respiration protection of the nitrogen-fixing system in Azotobacter is mainly independent of the oxidation capacity of the cells. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted. The maximum activity of the nitrogenase in Azotobacter depends on the type of substrate oxidized. Although the level of energy charge is somewhat dependent on the type of substrate used, no obvious relation can be derived between changes in energy charge and nitrogenase activity. An alternative proposal is given.     

Modification of ferredoxin-NADP+ reductase from the alga Bumilleriopsis with butanedione and dansyl chloride

Modification of ferredoxin-NADP⁺ reductase from the alga Bumilleriopsis with butanedione (diacetyl) and dansyl chloride results in loss of enzymatic activity. Under pseudo-first order conditions the rate of inactivation by butanedione is directly proportional to the concentration of the modifying reagent with a slope of unity. The protective effect of pyridine nucleotides, as well as their analogs against inactivation by butanedione indicates involvement of arginine in the binding of pyridine nucleotides at the active site. Inactivation by dansyl chloride suggests that a further amino acid is involved, possibly lysine. Amino acid analyses of the butanedione-treated reductase show that the degree of inactivation correlates well with the decrease in arginine. Furthermore, two arginine residues are modified concomitant with complete inactivation of the enzyme, although this does not imply that both residues participate necessarily in the binding of pyridine nucleotides. Fingerprint analysis of the carboxymethylated, trypsin-digested enzyme indicates loss of one arginine-containing peptide when the protein had been modified by butanedione. There was no change in cysteine-containing peptides.     

Studies of interaction of intracellular signalling and metabolic pathways under inhibition of mitochondrial aconitase with fluoroacetate

Mitochondrial aconitase has been shown to be inactivated by a spectrum of substances or critical states. Fluoroacetate (FA) is the most known toxic agent inhibiting aconitase. The biochemistry of toxic action of FA is rather well understood, though no effective therapy has been proposed for the past six decades. In order to reveal novel approaches for possible antidotes to be developed, experiments were performed with rat liver mitochondria, Ehrlich ascite tumor cells and cardiomyocytes, exposed to FA or fluorocitrate in vitro. The effect of FA developed at much higher concentrations in comparison with fluorocitrate and was dependent upon respiratory substrates in experiments with mitochondria: with pyruvate, FA induced a slow oxidation and/or leak of pyridine nucleotides and inhibition of respiration. Oxidation of pyridine nucleotides was prevented by incubation of mitochondria with cyclosporin A. Studies of the pyridine nucleotides level and calcium response generated in Ehrlich ascite tumor cells under activation with ATP also revealed a loss of pyridine nucleotides from mitochondria resulting in a shift in the balance of mitochondrial and cytosolic NAD(P)H under exposure to FA. An increase of cytosolic [Ca2+] was observed in the cell lines exposed to FA and is explained by activation of plasma membrane calcium channels; this mechanism, could have an impact on amplitude and rate of Ca2+ waves in cardiomyocytes. Highlighting the reciprocal relationship between intracellular pyridine nucleotides and calcium balance, we discuss metabolic pathway modulation in the context of probable development of an effective therapy for FA poisoning and other inhibitors of aconitase.     

Activation of the de novo pathway for pyridine nucleotide biosynthesis prior to ricinine biosynthesis in castor beans

The ricinine content of etiolated seedlings of Ricinus communis increased nearly 12-fold over a 4-day period. In plants quinolinic acid is an intermediate in the de novo pathway for the synthesis of pyridine nucleotides. The only known enzyme in the de novo pathway for pyridine nucleotide biosynthesis, quinolinic acid phosphoribosyltransferase, increased 6-fold in activity over a 4-day period which preceded the onset of ricinine biosynthesis by 1 day. The activity of the remainder of the pyridine nucleotide cycle enzymes in the seedlings, as monitored by the specific activity of nicotinic acid phosphoribosyltransferase and nicotinamide deamidase, was similar to that found in the mature green plant. In the roots of Nicotiana rustica, where the pyridine alkaloid nicotine is synthesized, the level of quinolinic acid phosphoribosyltransferase was 38-fold higher than the level of nicotinic acid phosphoribosyltransferase, whereas in most other plants examined, the specific activity of quinolinic acid phosphoribosyltransferase was similar to the level of activity of enzymes in the pyridine nucleotide cycle itself. A positive correlation therefore exists between the specific activity of a de novo pathway enzyme catalyzing pyridine nucleotide biosynthesis in Ricinus communis and Nicotiana rustica and the biosynthesis of ricinine and nicotine, respectively.     

Control of oxidative phosphorylation in rat heart mitochondria: The role of the adenine nucleotide carrier

1. Inhibitor titration experiments carried out with carboxyatractyloside, oligomycin and rotenone show that in the case of heart mitochondria the membrane-bound ATPase and the respiratory chain are the major factors controlling the rate of oxidative phosphorylation whereas the adenine nucleotide carrier exhibits no control strength. 2. As shown by carboxyatractyloside titration curves under different conditions, the relative importance of the adenine nucleotide carrier depends on the mode of regeneration (F₁-ATPase or glucose plus hexokinase) of ADP from ATP exported outside mitochondria, on the total concentration of adenine nucleotides present in the medium and on the mode of limitation of the rate of respiration (cyanide, rotenone, oligomycin or mersalyl). 3. Concomitantly with the inhibition of O₂ consumption, carboxyatractyloside brings about a rise in membrane potential. The inverse relationship between the two processes is observed for carboxyatractyloside concentrations ranging between 0.7 and 1.5 nmol per mg protein. Carboxyatractyloside concentrations below and above this range increase the membrane potential without affecting significantly the rate of respiration. 4. Titration experiments aimed at comparing the effects of ADP, carboxyatractyloside and the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, corroborate the conclusion that in heart mitochondria a major limiting factor in oxidative phosphorylation is the capacity of the respiratory chain.     

Veränderungen im oxidativen Stoffwechsel der Tomatenpflanze (Solanum lycopersicum L.) bei Befall mit der Blattrollkrankheit der Kartoffel

Metabolic changes in tomato plants infected with potato leafroll virus were followed. The virus was transferred by grafts taken from diseased tomato plants. Sharp changes in the respiration rate and in the activities of the investigated enzymes observed before the concrescence of the graft with the stock are obviously connected with the defence reactions of the plant. In the roots of the experimental plants a nearly complete correspondence of the course ofo-diphenol oxidase and ascorbic acid oxidase activities with the respiration rate occurs. In the shoots the respiratory chain with ascorbic acid oxidase as terminal oxidase is involved, whereas both chains probably take part in the respiration in the roots by which a link with sugar degradation is achieved. The rate of glycolysis and that of pentose phosphate cycle in the roots of experimental plants are nearly identical. The comparison of glucose-6-phosphate dehydrogenase activity with ascorbic acid oxidase activity reveals that both curves show the same course, which indicates the presence and action of a respiratory thain with ascorbic acid oxidase functioning as terminal oxidase.     

Death Through Respiratory Failure of a Fraction of Ultraviolet-Irradiated Escherichia coli B/r Cells

Escherichia coli B/r cells grown on a glycerol-containing medium and ultraviolet (UV)-irradiated to about 0.5% survival respire for about 1 hr and then cease for several hours. The cells that have completed repair and recovery processes begin to divide about 120 min after UV treatment, but this division is completely inhibited in liquid medium by caffeine, which delays repair of the irradiated deoxyribonucleic acid (DNA). When 5-fluorouracil (FU) is used to maintain respiration, the number of cells which form colonies when plated increases about 60-fold within 1 hr after irradiation. At least part of this increase does not involve repair while the cells are in the liquid medium because when caffeine is present there is still a 20-fold increase in colony formation. We conclude that many irradiated cells, although capable of carrying out complete and accurate repair of their DNA, die of respiratory failure; only when continuance of respiration is favored by FU treatment is their colony-forming potential realized. After an early increase, the number of cells able to form colonies in medium that contains FU remains constant while the completion of repair and recovery occurs. After these processes are completed, the number of cells able to form colonies increases slowly, except in the presence of caffeine, presumably because the late increase requires that repair steps take place while the cells are in liquid medium prior to cell division.     

Measuring whole plant CO2 exchange with the environment reveals opposing effects of the gin2–1 mutation in shoots and roots of Arabidopsis thaliana

Using a cuvette for simultaneous measurement of net photosynthesis in above ground plant organs and root respiration we investigated the effect of reduced leaf glucokinase activity on plant carbon balance. The gin2–1 mutant of Arabidopsis thaliana is characterized by a 50% reduction of glucokinase activity in the shoot, while activity in roots is about fivefold higher and similar to wild type plants. High levels of sucrose accumulating in leaves during the light period correlated with elevated root respiration in gin2–1. Despite substantial respiratory losses in roots, growth retardation was moderate, probably because photosynthetic carbon fixation was simultaneously elevated in gin2–1. Our data indicate that futile cycling of sucrose in shoots exerts a reduction on net CO₂ gain, but this is over-compensated by the prevention of exaggerated root respiration resulting from high sucrose concentration in leaf tissue.     

Development and activation of cyanide-resistant respiration in the yeast Yarrowia lipolytica.

Changes in respiratory activity and in the contents of adenine nucleotides (ATP, ADP, AMP) were studied in cells of the yeast Yarrowia lipolytica during the development of cyanide-resistant respiration. The transition of the yeast from the logarithmic to the stationary growth phase due to exhaustion of glucose was associated with decreased endogenous respiration and with the activation of a cyanide-resistant oxidase. Cyanide activated cell respiration during the stationary growth phase. The cyanide-resistant respiration was inhibited by benzohydroxamic acid (BHA), an inhibitor of the alternative oxidase. In the absence of cyanide, BHA had no effect on the cells which had the cyanide-resistant oxidase. This indicates that the cells do not use the alternative pathway in vivo. The decreased endogenous respiration of the cells was accompanied by decreased contents of adenine nucleotides. Addition of cyanide resulted in a sharp decrease in the content of ATP, in a twofold increase in the content of ADP, and in a fivefold increase in the content of AMP. In the absence of cyanide, BHA had virtually no effect on the contents of adenine nucleotides. The decreased rate of oxygen consumption during the transition of the cells to the stationary growth phase was caused by the decreased activity of the main cytochrome-containing respiratory chain (2,4-dinitrophenol (DNP) stimulated respiration). The alternative oxidase was synthesized in the cell but was inactive. Cyanide stimulated respiration due to activation of the alternative oxidase via the AMP produced. The decrease in the cell content of ATP is suggested to be a factor inducing the synthesis of the alternative oxidase.     

Energy metabolism of isolated rat thymus cells

The energy metabolism of rat thymus cells has been investigated using preparations of isolated cells obtained by mechanical treatment of whole organs. The addition of glycolytic substrates such as glucose, pyruvate and lactate stimulates the endogenous respiration of these cells by 50%. On the other hand, succinate, glutamate and malate do not produce any effect. Oligomycin (10 mug/ml) inhibits both endogenous and glucose stimulated respiration by about 40%; 2, 4-DNP (50 muM) increases by 100% glucose induced respiration. The results obtained by using mitochondrial and glycolytic inhibitors as well as aminoxyacetic acid (AOA) and following pyridine nucleotides redox changes, support the idea that in thymus cells glucose is able to induce a great enhancement of O2 consumption both by raising the level of endogenous pyruvate and feeding the mitochondrial respiratory chain with cytosolic reducing equivalents, through an active malate-aspartate shuttle. Thymus cells exhibit a high Pasteur effect (74%). Both AOA and 2,4 DNP are able to stimulate aerobic lactate accumulation by 200% and 100% respectively, indicating that either the redox or phosphate potential do influence the rate of aerobic glycolysis in isolated thymus cells. Similar experiments are also reported on other cells with well known biochemical characteristics.     

Mitochondrial function in the yeast form of the pathogenic fungus <Emphasis Type="Italic">Paracoccidioides brasiliensis</Emphasis>

Differences between the respiratory chain of the fungus Paracoccidioides brasiliensis and its mammalian host are reported. Respiration, membrane potential, and oxidative phosphorylation in mitochondria from P. brasiliensis spheroplasts were evaluated in situ, and the presence of a complete (Complex I–V) functional respiratory chain was demonstrated. In succinate-energized mitochondria, ADP induced a transition from resting to phosphorylating respiration. The presence of an alternative NADH–ubiquinone oxidoreductase was indicated by: (i) the ability to oxidize exogenous NADH and (ii) the lack of sensitivity to rotenone and presence of sensitivity to flavone. Malate/NAD⁺-supported respiration suggested the presence of either a mitochondrial pyridine transporter or a glyoxylate pathway contributing to NADH and/or succinate production. Partial sensitivity of NADH/succinate-supported respiration to antimycin A and cyanide, as well as sensitivity to benzohydroxamic acids, suggested the presence of an alternative oxidase in the yeast form of the fungus. An increase in activity and gene expression of the alternative NADH dehydrogenase throughout the yeast’s exponential growth phase was observed. This increase was coupled with a decrease in Complex I activity and gene expression of its subunit 6. These results support the existence of alternative respiratory chain pathways in addition to Complex I, as well as the utilization of NADH-linked substrates by P. brasiliensis. These specific components of the respiratory chain could be useful for further research and development of pharmacological agents against the fungus.     

Preparation of algal extracts for bacterial luciferase assay of pyridine nucleotides

Pyridine nucleotides in the cyanobacterium Synechococcus leopoliensis (Racib) Komárek and in the green algae Scenedesmus obtusiusulus Chod. and Ankistrodesmus braunii (Naegeli) Brunnth. were extracted with either hot NaOH, hot ethanol, hot acidic or alkaline methanol, perchloric acid (PCA) or trichloroacetic acid (TCA). Reduced pyridine nucleotides were determined with NADH- and NADPH-dependent bacterial luciferase. Oxidized nucleotides were determined after enzymatic conversion to the reduced forms. The yields of pyridine nucleotides in the extracts were compared to the relative extraction efficiency for ATP. Extraction with hot NaOH appeared satisfactory for reduced nucleotides in the green algae but less so in Synechococcus. Extraction with PCA seemed preferable for the oxidized nucleotides. The sensitivity of the bacterial luciferase assay was lowered by all extractants. Criteria for assessment of optimal extraction procedures for pyridine nucleotides are discussed.     

Deterioration of rat liver mitochondria under conditions of metabolite deprivation.

In a previous study [Parce, Cunningham & Waite (1978) Biochemistry 17, 1634-1639] changes in mitochondrial phospholipid metabolism and energy-linked functions were monitored as coupled mitochondria were aged in iso-osmotic sucrose solution at 18 degrees C. The sequence of events that occur in mitochondrial deterioration under the above conditions have been established more completely. Total adenine nucleotides are depleted early in the aging process, and their loss parallels the decline in respiratory control. Related to the loss of total adenine nucleotides is a dramatic decrease in ADP and ATP translocation (uptake). The decline of respiratory control is due primarily to a decrease in State-3 respiration; loss of this respiratory activity can be related to the decline in ADP translocation. Mitochondrial ATPase activity does not increase significantly until State-4 respiration has increased appreciably. At the time of loss of respiratory control the ATPase activity increases to equal the uncoupler-stimulated activity. The H+/O ratio and P/O ratios do not decrease appreciably until respiratory control is lost. Similarly, permeability of the membrane to the passive diffusion of protons increases only after respiratory control is lost. There observations reinforce our earlier conclusion that there are two main phases in mitochondrial aging. The first phase is characterized by loss of the ability to translocate adenine nucleotides. The second phase is characterized by a decline in the ability of the mitochondrion to conserve energy (i.e. maintain a respiration-driven proton gradient) and to synthesize ATP.