Fumarase Activity Research Articles

Middle age aggravates myocardial ischemia through surprising upholding of complex II activity, oxidative stress, and reduced coronary perfusion

Aging compromises restoration of the cardiac mechanical function during reperfusion. We hypothesized that this was due to an ampler release of mitochondrial reactive oxygen species (ROS). This study aimed at characterising ex vivo the mitochondrial ROS release during reperfusion in isolated perfused hearts of middle-aged rats. Causes and consequences on myocardial function of the observed changes were then evaluated. The hearts of rats aged 10- or 52-week old were subjected to global ischemia followed by reperfusion. Mechanical function was monitored throughout the entire procedure. Activities of the respiratory chain complexes and the ratio of aconitase to fumarase activities were determined before ischemia and at the end of reperfusion. H(2)O(2) release was also evaluated in isolated mitochondria. During ischemia, middle-aged hearts displayed a delayed contracture, suggesting a maintained ATP production but also an increased metabolic proton production. Restoration of the mechanical function during reperfusion was however reduced in the middle-aged hearts, due to lower recovery of the coronary flow associated with higher mitochondrial oxidative stress indicated by the aconitase to fumarase ratio in the cardiac tissues. Surprisingly, activity of the respiratory chain complex II was better maintained in the hearts of middle-aged animals, probably because of an enhanced preservation of its membrane lipid environment. This can explain the higher mitochondrial oxidative stress observed in these conditions, since cardiac mitochondria produce much more H(2)O(2) when they oxidize FADH(2)-linked substrates than when they use NADH-linked substrates. In conclusion, the lower restoration of the cardiac mechanical activity during reperfusion in the middle-aged hearts was due to an impaired recovery of the coronary flow and an insufficient oxygen supply. The deterioration of the coronary perfusion was explained by an increased mitochondrial ROS release related to the preservation of complex II activity during reperfusion.

The Discovery of Glyoxysomes: the Work of Harry Beevers

Sucrose Synthesis from Acetate in the Germinating Castor Bean: Kinetics and Pathway (Canvin, D. T., and Beevers, H. (1961) J. Biol Chem. 236, 988–995) Mitochondria and Glyoxysomes from Castor Bean Endosperm. Enzyme Constituents and Catalytic Capacity (Cooper, T. G., and Beevers, H. (1969) J. Biol. Chem. 244, 3507–3513)

Утворення глікогену в клітинах Chlorobium limicola за умов порушення функціонування окремих ланок циклу Арнона

Nitrates are not utilized by photosynthetic green sulfur bacteria Chlorobium limicola as the nitrogen source. Addition of these compounds into the medium inhibits the growth of bacteria but stimulates the glycogen accumulation. Nitrates are rapidly converted by the cells of C. limicola to the nitrites. Maximal nitrate reductase activity in the cells of C. limicola was detected after one day cultivation. Nitrates and nitrites inhibit activity of fumarase – the key enzyme of the reductive tricarboxylic acid cycle. Nitrites have more potential action as compared to the nitrate. The glycogen content in the cells of C. limicola cultivated in the medium with nitrates, but not acetate and pyruvate, show no significant change. Addition of acetate and pyruvate to the medium stimulate the glycogen synthesis in the presence of nitrate. Iodine acetate decreases the glycogen level under these conditions. Keywords: Chlorobium limicola , glycogen, Arnon’ cycle, nitrates.

Fumarase Activity in Human Ejaculate Relationship to Sperm-Motility or -Penetration of Zona-Free Hamster Eggs

Summary Fumarase activity, sperm count, sperm motility and in vitro fertilizing ability of spermatozoa were determined in semen samples of 69 men attending an infertility clinic. There existed a significant correlation between sperm count and fumarase activity (r = 0.52). No significant relationship was observed between sperm motility or hamster egg fertilizing ability of the spermatozoa and fumarase activity. From these results it is concluded that fumarase activity in human ejaculates can probably not be used to define the fertilizing ability of spermatozoa. Fumarase-Aktivitat im menschlichen Ejakulat Beziehungen zwischen Spermatozoen-Motilitat oder -Penetration in Zona-freie-Hamster-Eier Zusammenfassung An den Spermaproben von 69 Mannern wurde die Fumarase-Aktivitat, die Spermatozoendichte, die Spermatozoenmotilitat und die in-vitro-Fertilisierungs-Fahigkeit von Spermatozoen untersucht. Dabei konnte eine signifikante Beziehung zwischen der Spermatozoendichte und der Fumarase-Aktivitat (r = 0,52) festgestellt werden. Zwischen der Spermatozoenmotilitat oder der Fertilisierungs-Fahigkeit menschlicher Spermatozoen fur Hamster-Eier lies sich dagegen keine Signifikanz zur Fumarase-Aktivitat herstellen. Aus den Ergebnissen wird die Schlusfolgerung gezogen, das die Fumarase-Aktivitat im menschlichen Ejakulat im allgemeinen nicht fur die Beurteilung der Fertilisierungs-Fahigkeit der Spermatozoen herangezogen werden kann.

Expression of the yggE gene protects Escherichia coli from potassium tellurite-generated oxidative stress

Potassium tellurite is highly toxic to most forms of life and specific bacterial tellurite defense mechanisms are not fully understood to date. Recent evidence suggests that tellurite would exert its toxic effects, at least in part, through the generation of superoxide anion that occurs concomitantly with intracellular tellurite (Te(4+)) reduction to elemental tellurium (Te(o)). In this work the putative antioxidant role of YggE from Escherichia coli, a highly conserved protein in several bacterial species and whose function is still a matter of speculation, was studied. When exposed to tellurite, E. coli lacking yggE exhibited increased activity of superoxide dismutase and fumarase C, augmented levels of reactive oxygen species and high concentration of carbonyl groups in proteins. Upon genetic complementation with the homologous yggE gene these values were restored to those observed in the parental, isogenic, wild type strain, suggesting a direct participation of YggE in E. coli tolerance to tellurite.

Cadmium toxicity in tomato ( Lycopersicon esculentum) plants grown in hydroponics

The effects of Cd have been investigated in tomato ( Lycopersicon esculentum) plants grown in a controlled environment in hydroponics, using Cd concentrations of 10 and 100 μM. Cadmium treatment led to major effects in shoots and roots of tomato. Plant growth was reduced in both Cd treatments, leaves showed chlorosis symptoms when grown at 10 μM Cd and necrotic spots when grown at 100 μM Cd, and root browning was observed in both treatments. An increase in the activity of phospho enolpyruvate carboxylase, involved in anaplerotic fixation of CO 2 into organic acids, was measured in root extracts of Cd-exposed plants. Also, significant increases in the activities of several enzymes from the Krebs cycle were measured in root extracts of tomato plants grown with Cd. In leaf extracts, significant increases in citrate synthase, isocitrate dehydrogenase and malate dehydrogenase activities were also found at 100 μM Cd, whereas fumarase activity decreased. These data suggest that at low Cd supply (10 μM) tomato plants accumulate Cd in roots and this mechanism may be associated to an increased activity in the PEPC–MDH–CS metabolic pathway involved in citric acid synthesis in roots. Also, at low Cd supply some symptoms associated with a moderate Fe deficiency could be observed, whereas at high Cd supply (100 μM) effects on growth overrule any nutrient interaction caused by excess Cd. Cadmium excess also caused alterations on photosynthetic rates, photosynthetic pigment concentrations and chlorophyll fluorescence, as well as in nutrient homeostasis.

Decreased mitochondrial activities of malate dehydrogenase and fumarase in tomato lead to altered root growth and architecture via diverse mechanisms.

Transgenic tomato (Solanum lycopersicum) plants in which either mitochondrial malate dehydrogenase or fumarase was antisense inhibited have previously been characterized to exhibit altered photosynthetic metabolism. Here, we demonstrate that these manipulations also resulted in differences in root growth, with both transgenics being characterized by a dramatic reduction of root dry matter deposition and respiratory activity but opposite changes with respect to root area. A range of physiological, molecular, and biochemical experiments were carried out in order to determine whether changes in root morphology were due to altered metabolism within the root itself, alterations in the nature of the transformants' root exudation, consequences of alteration in the efficiency of photoassimilate delivery to the root, or a combination of these factors. Grafting experiments in which the transformants were reciprocally grafted to wild-type controls suggested that root length and area were determined by the aerial part of the plant but that biomass was not. Despite the transgenic roots displaying alteration in the expression of phytohormone-associated genes, evaluation of the levels of the hormones themselves revealed that, with the exception of gibberellins, they were largely unaltered. When taken together, these combined experiments suggest that root biomass and growth are retarded by root-specific alterations in metabolism and gibberellin contents. These data are discussed in the context of current models of root growth and biomass partitioning.

Ancestral roles of eukaryotic frataxin: mitochondrial frataxin function and heterologous expression of hydrogenosomal Trichomonas homologues in trypanosomes

Frataxin is a small conserved mitochondrial protein; in humans, mutations affecting frataxin expression or function result in Friedreich's ataxia. Much of the current understanding of frataxin function comes from informative studies with yeast models, but considerable debates remain with regard to the primary functions of this ubiquitous protein. We exploit the tractable reverse genetics of Trypanosoma brucei in order to specifically consider the importance of frataxin in an early branching lineage. Using inducible RNAi, we show that frataxin is essential in T. brucei and that its loss results in reduced activity of the marker Fe-S cluster-containing enzyme aconitase in both the mitochondrion and cytosol. Activities of mitochondrial succinate dehydrogenase and fumarase also decreased, but the concentration of reactive oxygen species increased. Trypanosomes lacking frataxin also exhibited a low mitochondrial membrane potential and reduced oxygen consumption. Crucially, however, iron did not accumulate in frataxin-depleted mitochondria, and as T. brucei frataxin does not form large complexes, it suggests that it plays no role in iron storage. Interestingly, RNAi phenotypes were ameliorated by expression of frataxin homologues from hydrogenosomes of another divergent protist Trichomonas vaginalis. Collectively, the data suggest trypanosome frataxin functions primarily only in Fe-S cluster biogenesis and protection from reactive oxygen species.

Effect of antifungal agents on the physiological activities of towo plant pathogenic fungi: <i>Rhizoctonia solani </i> & <i>Fusarium oxysporum</i>

Each of the antifungal compounds extracted from each of the antagonistic microorganisms was found to has an inhibitory effect on Rhizoctonia solani and Fusarium oxysporum. Four antifungal compounds were found to be the most inhibitory than the other compounds. The first one from Eupenicillium senticosum, the second from Streptomyces aurantiacus, the third from Curtobacterium pusillum and the last one from Saccharomyces unispora respectively. The first three compounds were active against R. solani, while the last one was active against F. oxysporum. All the four compounds reduce or inhibit the growth of Rhizoctonia solani and Fusarium oxysporum. They also reduced the carbohydrate concentrations, as well as reducing the nitrogen content (total nitrogen, soluble nitrogen and protein nitrogen). Regarding their effects on the enzyme activity, the active compounds increased the activity of fumarase, malic dehydrogenase and succinic dehydrogenase, the activity of catalase enzyme of both R. solani and F. oxysporum was sharply decreased by such compounds as compared to control.Egyptian Journal of Biotechnology Vol. 25 (1) 2007: pp.159-168

Deficiency of mitochondrial fumarase activity in tomato plants impairs photosynthesis via an effect on stomatal function

Transgenic tomato (Solanum lycopersicum) plants expressing a fragment of a fumarate hydratase (fumarase) gene in the antisense orientation and exhibiting considerable reductions in the mitochondrial activity of this enzyme show impaired photosynthesis. The rate of the tricarboxylic acid cycle was reduced in the transformants relative to the other major pathways of carbohydrate oxidation and the plants were characterized by a restricted rate of dark respiration. However, biochemical analyses revealed relatively little alteration in leaf metabolism as a consequence of reducing the fumarase activity. That said, in comparison to wild-type plants, CO(2) assimilation was reduced by up to 50% under atmospheric conditions and plants were characterized by a reduced biomass on a whole plant basis. Analysis of further photosynthetic parameters revealed that there was little difference in pigment content in the transformants but that the rate of transpiration and stomatal conductance was markedly reduced. Analysis of the response of the rate of photosynthesis to variation in the concentration of CO(2) confirmed that this restriction was due to a deficiency in stomatal function.

Human Epidermal Keratinocytes Accumulate Superoxide Due to Low Activity of Mn-SOD, Leading to Mitochondrial Functional Impairment

The energy metabolism of the epidermis has been the subject of controversy; thus we characterized the mitochondrial phenotype of human primary keratinocytes and fibroblasts, in cell culture and in human skin sections. We found that keratinocytes respire as much as fibroblasts, however, maximal activities of the respiratory chain (RC) complexes were 2- to 5-fold lower, whereas expression levels of RC proteins were similar. Maximal activities of aconitase and isocitrate dehydrogenase, two mitochondrial enzymes especially vulnerable to superoxide, were lower than in fibroblasts. Indeed, superoxide anion levels were much higher in keratinocytes, and keratinocytes displayed higher lipid peroxidation levels and a lower reduced glutathione/oxidized glutathione ratio, indicating enhanced oxidative stress. Although superoxide dismutase activity and especially expression of the mitochondrial superoxide dismutase, Mn-SOD, were drastically lower in keratinocytes, explaining the high superoxide levels, glutathione peroxidase activity and protein were almost undetectable in fibroblasts. Catalase activity and hydrogen peroxide levels were similar. In summary, we could show that keratinocytes actively use the mitochondrial RC not only for adenosine 5' triphosphate synthesis but also for the accumulation of superoxide anions, even at the expense of mitochondrial functional capacity, indicating that superoxide-driven mitochondrial impairment might be a prerequisite for keratinocyte differentiation.

Escherichia coli Di-iron YtfE Protein Is Necessary for the Repair of Stress-damaged Iron-Sulfur Clusters

DNA microarray experiments showed that the expression of the Escherichia coli ytfE gene is highly increased upon exposure to nitric oxide. We also reported that deletion of ytfE significantly alters the phenotype of E. coli, generating a strain with enhanced susceptibility to nitrosative stress and defective in the activity of several iron-sulfur-containing proteins. In this work, it is shown that the E. coli ytfE confers protection against oxidative stress. Furthermore, we found that the damage of the [4Fe-4S](2+) clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild type E. coli once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to the mutant cell extracts, the enzymatic activities of fumarase and aconitase are fully recovered and at rates similar to the wild type strain. We concluded that YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions.

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [ 3H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40–50% of values in parietal cortex. In contrast, formation of aspartate from [U- 14C]glucose in awake rats (which reflects the passage of 14C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, α-ketoglutarate dehydrogenase, and fumarase in white structures were 10–23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex (∼3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20–40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.

Proteomic analysis of the renal medulla of Dahl salt‐sensitive rats and consomic SS‐13BN rats

The consomic SS-13BN rat, genetically identical to the Dahl salt-sensitive (SS) rat except chromosome 13, exhibited a substantial reduction of blood pressure salt-sensitivity. The present study utilized a proteomic approach to identify proteins that might contribute to the phenotypic differences between SS and SS-13BN. Differential in-gel electrophoresis analysis of the renal medulla indicated that of approximately 2200 spots analyzed, 58 spots exhibited differential expression between 6-week old SS and SS-13BN rats on a 0.4% NaCl diet (n=4, P<0.05, ratio >1.2 or <−1.2). MALDI-TOF-MS analysis successfully identified 42 spots, representing 28 unique proteins, 4 of which are encoded by genes located on chromosome 13. Western blot analysis confirmed the differential expression of 3 proteins (n=4, P<0.05) including fumarase (1.65 ± 0.12 fold higher in SS), heterogeneous nuclear ribonucleoprotein K (2.40 ± 0.32 fold higher in SS), and superoxide dismutase 1 (1.1 ± 0.03 fold higher in SS-13BN). Moreover, fumarase, which catalyzes the inter-conversion of fumarate and malate, exhibited a significant shift of isoelectric point toward the basic side in SS. The specific activity of fumarase was 1.89 ±0.14 fold lower in the renal medulla of SS (n=4, P<0.01). The present study provided new insights into the molecular basis for the phenotypic differences between SS and SS-13BN. (NIH R01HL077263 and N01-HV-28182)

Fumarate Is an Essential Intermediary Metabolite Produced by the Procyclic Trypanosoma brucei

The procyclic stage of Trypanosoma brucei, a parasitic protist responsible for sleeping sickness in humans, converts most of the consumed glucose into excreted succinate, by succinic fermentation. Succinate is produced by the glycosomal and mitochondrial NADH-dependent fumarate reductases, which are not essential for parasite viability. To further explore the role of the succinic fermentation pathways, we studied the trypanosome fumarases, the enzymes providing fumarate to fumarate reductases. The T. brucei genome contains two class I fumarase genes encoding cytosolic (FHc) and mitochondrial (FHm) enzymes, which account for total cellular fumarase activity as shown by RNA interference. The growth arrest of a double RNA interference mutant cell line showing no fumarase activity indicates that fumarases are essential for the parasite. Interestingly, addition of fumarate to the medium rescues the growth phenotype, indicating that fumarate is an essential intermediary metabolite of the insect stage trypanosomes. We propose that trypanosomes use fumarate as an essential electron acceptor, as exemplified by the fumarate dependence previously reported for an enzyme of the essential de novo pyrimidine synthesis (Takashima, E., Inaoka, D. K., Osanai, A., Nara, T., Odaka, M., Aoki, T., Inaka, K., Harada, S., and Kita, K. (2002) Mol. Biochem. Parasitol. 122, 189-200).

Organic acids: old metabolites, new themes

AbstractFumaric, L‐malic and citric acids are intermediates of the oxidative tricarboxylic acid (TCA) cycle which in eukaryotes is localized in mitochondria. These organic acids are synthesized and accumulated in the medium to very high concentrations by filamentous fungi such as Aspergillus spp. and Rhizopus sp. This article reviews basic research on the unusual acid production capability and the associated metabolic pathways operating under defined stress conditions in these specific fungi. In particular, we describe and discuss the importance of the cytosolic reductive TCA pathway, which includes the cytosolic activities of pyruvate carboxylase, malate dehydrogenase and fumarase, for production of fumaric and L‐malic acids. This article also describes the differences between fumaric acid, L‐malic acid and citric acid production by different organisms (filamentous fungi, yeast, and higher eukaryotes), and the possible application of novel technologies (genetic engineering and bioinformatics) to fungal systems which may offer new industrial potential of filamentous fungi for the production of valuable metabolites. Copyright © 2006 Society of Chemical Industry

Modulation of D-Serine Levels via Ubiquitin-dependent Proteasomal Degradation of Serine Racemase

Mammalian serine racemase is a brain-enriched enzyme that converts L- into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain d-serine levels and NMDA receptor activity.

Effect Of Antifungals On Physiological Activities Of Some Plant Pathogenic Fungi

Each of the antifungal compounds extracted from each of the antagonistic microorganisms was found to has an inhibitory effect on Rhizoctonia solani and Fusarium oxysporum. Four antifungal compounds were found to be the most inhibitory than the other compounds. The first one from Eupenicillium senticosum, the second from Streptomyces aurantiacus, the third from Curtobacterium pusillum and the last one from Saccharomyces unispora. The first three compounds were active against R. solani, while the last one was active against F. oxysporum. All the four compounds were found to reduce or inhibit the growth of R. solani and F. oxysporum. They also reduce the carbohydrate concentrations, as well as reducing the nitrogen content (total nitrogen, soluble nitrogen and protein nitrogen). Regarding their effect on the enzyme activity, they increase the activity of fumarase, malic dehydrogenase and succinic dehydrogenase, while they reduce the activity of catalase enzyme of both R. solani and F. oxysporum as compared to control.

Production of L-Malic Acid by Permeabilized Cells of Commercial Saccharomyces Sp. Strains

Of various yeasts tested in the conversion of fumaric to L-malic acid, Saccharomyces bayanus had the highest activity of fumarase. Cells permeabilized with 0.2% (w/v) CTAB for 5 min gave maximum enzyme activity. Under non-growth conditions, fumarase activity in the permeabilized cells was four times higher (271 U/g) than that of the intact cells (67 U/g). The proposed mathematical model for the batch production of L-malic acid was validated at different initial fumaric acid concentrations. The average conversion of fumaric acid was up to 82% and gave 21, 40, 83 and 175 mM L-malic acid from respectively, 25, 50, 100 and 210 mM: fumaric acid.

Responses of spinach leaf mitochondria to low N availability

Low N availability induces carbohydrate accumulation in leaf cells, which often causes suppression of photosynthesis. Under low N supply, excess carbohydrates would be preferentially respired by the non-phosphorylating pathways, such as the alternative oxidase (AOX) and uncoupling protein (UCP), which would suppress the excessive increase in the ratio of C to N (C/N ratio). In leaves, however, responses of these pathways to the low N stress are still unknown. We examined the mitochondrial respiratory pathways in spinach leaves grown at three different N availabilities to clarify whether the respiratory pathways change depending on the N availabilities. With the decrease in N availability, leaf respiratory rates per leaf area decreased, but the rates on the leaf N basis were comparable. Using fumarase activities of whole leaf extracts and isolated mitochondria, we estimated mitochondrial protein contents per leaf N. The contents increased with the decrease in the N availability, that is, at the low N availability, N was preferentially invested into mitochondria. On the mitochondrial protein basis, capacities of cytochrome pathway (CP) and cytochrome c oxidase (COX) were comparable regardless of the N availabilities, whereas both AOX capacity and the amounts of AOX protein increased with the decrease in the N availability. Some enzymes of tricarboxylic acid (TCA) cycle, especially NAD-dependent malic enzyme (NAD-ME), showed higher capacities under lower N. On the other hand, amounts of UCP did not differ amongst the N availabilities. These results indicated that, under low N stress, AOX will be preferentially up-regulated and will efficiently consume excess carbohydrates, which leads to suppressing the rise in the C/N ratio to a moderate level.