Glyoxylate Cycle Enzyme Activities Research Articles

Magnesium deprivation affects cellular circuitry involved in drug resistance and virulence in Candida albicans.

Candida albicans has to struggle for the limited micronutrients present in the hostile host niche, including magnesium (Mg). The aim of this study was to examine the effect of Mg deprivation on drug resistance mechanisms and virulence traits of C. albicans. The drug susceptibility of C. albicans strain SC5314 was determined by broth microdilution and spot assay. Efflux pump activity was measured using the substrate rhodamine 6G. Membrane intactness was studied by propidium iodide influx, and ergosterol levels were determined by the alcoholic KOH method. Metabolic flexibility was examined by studying the activity of glyoxylate cycle enzymes. Virulence factors were assessed by yeast-to-hyphae transition, biofilm formation and cell adherence. An in vivo study was also performed in a Caenorhabditis elegans infection model. Mg chelation leads to potentiation of membrane-targeting antifungals. The role of Mg on membrane homeostasis was explored and significant differences in ergosterol levels were found. Interestingly, it was also observed that Mg deprivation impedes the metabolic flexibility of C. albicans SC5314 by inhibiting glyoxylate cycle enzymes. Furthermore, Mg deprivation inhibited potential virulence traits, including morphological transition, biofilm formation and buccal epithelial cell adherence. All of the disrupted gene targets were validated by reverse transcription PCR. Lastly, enhanced survival of C. elegans infected with C. albicans SC5314 under Mg deprivation was observed. In view of the restricted growth of C. albicans in a Mg-deficient environment, approaches could be utilised to boost the effectiveness of existing antifungals thereby improving the management of fungal infections.

The glyoxylate cycle enzyme activities in the pathogenic isolates of Candida albicans obtained from HIV/AIDS, diabetic and burn patients

Recently it has been found that Candida albicans harbours enzymes involved in the glyoxylate cycle (GC), which have a role in its virulence, especially the two key enzymes, isocitrate lyase (ICL) and malate synthase (MS). There are however, few studies on the GC enzyme activities isolated in the clinical isolates. Samples were collected from three groups of patients namely, HIV/AIDS, diabetic and burn patients suffering from candidiasis at different body locations. Isolation, identification and the antifungal susceptibility test of all the isolates of C. albicans were followed by the standard techniques. Measurements of all the GC enzyme activities were also carried out by the standard methods. Levels of the principal GC enzymes showed significant changes when calculated and compared taking control strains of C. albicans. The activity of the two key enzymes of the GC, ICL and MS were significantly higher in the isolates from diabetic patients. No significant relationship between the drug susceptibility and the level of enzymes of the GC was observed. As GC activity is absent in mammalian cells, a specific inhibitor for the GC could be developed and these enzymes therefore can be used as a new antifungal target.

Soybean ( Glycine max. L.) and bacteroid glyoxylate cycle activities during nodular senescence

Soybean (Glycine max. L.) nodular senescence results in the dismantling of the peribacteroid membrane (PBM) and in an increase of soybean isocitrate lyase (ICL; EC 4.1.3.1) and malate synthase (MS; EC 4.1.3.2) mRNA and protein levels. This suggests that in senescing soybean nodular cells, the specific glyoxylate cycle enzyme activities might be induced to reallocate carbon obtained from the PBM degradation. In order to evaluate as well the carbon metabolism of the nitrogen-fixing Bradyrhizobium japonicum endosymbiotic bacteroids during nodular senescence, their glyoxylate cycle activities were also investigated. To this end, partial DNA sequences were isolated from their icl and ms genes, but the corresponding mRNAs were not detected in the microorganisms. It was also observed that the bacteroid ICL and MS activities were negligible during nodular senescence. This suggests that glyoxylate cycle activities are not reinitiated in the bacteroids under these physiological conditions. In case the microorganisms nevertheless feed on the PBM degradation products, this might occur via the citric acid cycle exclusively.

The induction of glyoxylate cycle enzymes in tissues of starving rats.

The induction of glyoxylate cycle enzyme activities was revealed in the liver and other organs of starving rats. A five day deprivation of food was followed by the appearance of isocitrate lyase (ICL) and malate synthase activities and the increase of malate dehydrogenase (MDH) and citrate synthase activities. The induction of MDH was associated with the appearance of its new isoform with Rf 0.52. ICL activity was revealed in the liver, blood, pancreas, kidney, lungs, heart, and skeletal muscles of starving rats, reaching a peak on day 5 of food deprivation. No significant changes of blood glucose level in starving rats were revealed until day 9. A homogeneous ICL preparation with a specific activity of 12.4 IU per mg protein was obtained as the result of a five-stage purification procedure.

Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions.

Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165-170 nmol min-1 (mg protein)-1] were found in cells grown on acetate or beta-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50-80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent Km of isocitrate lyase for D,L-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 microM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the Vmax nor the D,L-isocitrate Km of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum.

Involvement of cyanide-resistant and rotenone-insensitive pathways of mitochondrial electron transport during oxidation of glycine in higher plants

Metabolism of glycine in isolated mitochondria and protoplasts was investigated in photosynthetic, etiolated (barley and pea leaves) and fat-storing (maize scutellum) tissues using methods of [1- 14C]glycine incorporation and counting of 14CO 2 evolved, oxymetric measurement of glycine oxidation and rapid fractionation of protoplasts incubated in photorespiratory conditions with consequent determination of ATP/ADP ratios in different cell compartments. The involvement of different paths of electron transport in mitochondria during operation of glycine decarboxylase complex (GDC) was tested in different conditions, using aminoacetonitrile (AAN), the inhibitor of glycine oxidation in mitochondria, rotenone, the inhibitor of Complex I of mitochondrial electron transport, and inhibitors of cytochrome oxidase and alternative oxidase. It was shown that glycine has a preference to other substrates oxidized in mitochondria only in photosynthetic tissue where succinate and malate even stimulated its oxidation. Rotenone had no or small effect on glycine oxidation, whereas the role of cyanide-resistant path increased in the presence of ATP. Glycine oxidation increased ATP/ADP ratio in cytosol of barley protoplasts incubated in the presence of CO 2, but not in the CO 2-free medium indicating that in conditions of high photorespiratory flux oxidation of NADH formed in the GDC reaction passes via the non-coupled paths. Activity of GDC in fat-storing tissue correlated with the activity of glyoxylate-cycle enzymes, glycine oxidation did not reveal preference to other substrates and the involvement of paths non-connected with proton translocation was not pronounced. It is suggested that the preference of glycine to other substrates oxidized in mitochondria is achieved in photosynthetic tissue by switching to rotenone-insensitive intramitochrondrial NADH oxidation and by increasing of alternative oxidase involvement in the presence of glycine.

Evidences of glyoxylate cycle in peroxisomes of senescent cotyledons

The metabolic pathway of the glyoxylate cycle has been investigated in peroxisomes isolated from senescent pumpkin ( Cucurbita sp.) cotyledons. β-oxidation activity, as well as activities of glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1), malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 4.1.3.7) were detected. In order to establish if there is a channelling of acetyl CoA into the glyoxylate cycle, peroxisomes have been incubated with various substrates. The incubations show acetyl CoA utilization by the glyoxylate cycle. When the incubation medium for citrate formation is used, all the label from [1- 14C]acetyl CoA is recovered in citrate, whereas only 18% of the added radioactivity is recovered in malic acid (by isocitrate lyase and malate synthase) after a long incubation time (3 h). Only by feeding peroxisomes with [1,5- 14C]citric acid and exogenous aconitase (EC 4.2.1.3) can a weak formation of other organic acids (glyoxylate and succinate) be note. The requirement for exogenous aconitase to carry out the peroxisomal glyoxylate cycle points towards the isocitric acid step as a crucial factor for the operation of the global cycle.

β-oxidation of fatty acids is linked to the glyoxylate cycle in the aleurone but not in the embryo of germinating barley

An assay for the measurement of overall β-oxidation was optimized for use with extracts from germinating barley. The most important adaptation is the addition of commercially available acyl-CoA synthetase to the incubation medium in order to reactivate the palmitate formed by the high thioesterase activity of the extracts. The β-oxidation activity determined by the overall assay originated exclusively from microbodies, since inhibitors of the electron-transport chain and alternative respiration in mitochondria did not affect the total β-oxidation activity. Activity patterns of β-oxidation upon germination were determined both for barley aleurone and embryo. In the aleurone, β-oxidation activity was already present in the quiescent grain and increased during germination, reaching an optimum 4 days after germination. In addition, in the embryo β-oxidation activity could be demonstrated in the quiescent grain, and it increased upon germination. Within the growing embryo, β-oxidation activity was present in leaves, roots and scutellum. The maximal activity levels reached in the various embryo parts were significantly lower than those in the aleurone. Organelle separation by density gradient centrifugation of homogenates from scutellum suggests that, in this tissue, microbodies constitute the only subcellular compartment carrying out β-oxidation. In the aleurone, the developmental activity profiles of glyoxylate cycle enzymes closely resemble those of β-oxidation indicating that, in this tissue, β-oxidation is localized in so-called glyoxysomes. Inability to detect glyoxylate cycle enzyme activities in the embryo indicates different metabolic functions of the β-oxidation route in aleurone and embryo.

Mitochondrial and peroxisomal fractions derived from human white adipocytes

1.1. A procedure is described for the separation of intact peroxisomes from human white adipocytes using a linear metrizamide gradient (20–50% w/v).2.2. Peroxisomes were found in the high density region of the gradient in an intact form.3.3. Mitochondria were distributed in the high density and low density regions of the gradient.4.4. Lysosomes separated well from the peroxisomes, occurring only in the low density region of the gradient.5.5. Low levels of glyoxylate cycle enzyme activities (isocitrate lyase and malate synthase) were detected within the light and heavy mitochondrial pellet fractions.

Effect of Leaf Senescence on Glyoxylate Cycle Enzyme Activities

In order to elucidate the metabolism of the peroxisomes during foliar senescence of leaf beet (Beta vulgaris L., var. cicla), peroxisomal activities have been determined at various stages of senescence. Catalase and hydroxypyruvate reductase activities decreased whereas those of the β-oxidation pathway and glyoxylate cycle enzymes increased at the same time. The increased activities of malate synthase, isocitrate lyase, malate dehydrogenase and citrate synthase indicate that the glyoxylate cycle might be activated during the foliar senescence of leaf beet.

Tricarboxylic acid and glyoxylate cycle enzyme activities in Thiobacillus versutus, an isocitrate lyase negative organism

An analysis was made of the specific enzyme activities of the TCA and glyoxylate cycle in Thiobacillus versutus cells grown in a thiosulphate- or acetate-limited chemostat. Activities of all enzymes of the TCA cycle were detected, irrespective of the growth substrate and they were invariably lower in the thiosulphate-grown cells. Of the glyoxylate cycle enzymes, isocitrate lyase was absent but malate synthase activity was increased from 15 nmol·min-1·mg-1 protein in thiosulphate-grown cells to 58 nmol·min-1·mg-1 protein in acetate-grown cells. Suspensions of cells grown on thiosulphate were able to oxidize acetate, although the rate was 3 times lower than that observed with acetate-grown cells. The respiration of acetate was completely inhibited by 10 mM fluoroacetate or 5 mM arsenite. Partially purified citrate synthase from both thiosulphate- and acetate-grown cells was completely inhibited by 0.5 mM NADH and was insensitive to inhibition by 1 mM 2-oxoglutarate or 1 mM ATP. The specific enzyme activities of the TCA and glyoxylate cycle in T. versutus were compared with those of Pseudomonas fluorescens, an isocitrate lyase positive organism, after growth in a chemostat limited by acetate, glutarate, succinate or glutamate. The response of the various enzyme activities to a change in substrate was similar in both organisms, with the exception of isocitrate lyase.

Mobilisation of storage reserves during fern spore germination

SynopsisWe have studied morphological and biochemical aspects of storage reserves and their degradation in fern spores during the germination process. The results presented here are concerned with the fate of lipid, protein and phytic acid. Depletion of lipid reserves and breakdown of lipid bodies was directly correlated with increased activity of glyoxylate cycle enzymes during early stages of germination. Degradation of protein reserves coincided with the depletion of salt soluble proteins (globulins) from the spores and was related to the time when high activity of aminopeptidase and carboxypeptidase was observed. Lipid and protein appeared to play an important role in germination. Phytic acid reserves were present in the spore and were hydrolysed by phytase after germination occurred. This storage material was implicated in growth of the prothallus.

Derepression of glyoxylate cycle enzyme activities in anise suspension culture cells

In cells of suspension cultures of anise ( Pimpinella anisum L.) the key enzymes for the glyoxylate cycle, isocitrate lyase and malate synthase, are active only when the growth medium lacks any source of carbon. They appear after a lag phase of about 20 h during which over 60% of the cell's starch is degraded and reach maximum activity after about 48 h. Addition of 20 mmol/l acetate induces doubling of the level of enzyme activities but only when sucrose is absent from the medium. The effect of acetate is, over the range of 1–20 mmol/l, essentially independent of the dose added. Also, derepression is not affected by the phase of growth of the cultures used. It is concluded that similar to fungal cells the level of glycolytic metabolites is involved in enzyme derepression through yet unknown mechanisms.

Fatty acid ?-oxidation and glyoxylate cycle enzyme activities of induced glyoxysomes from anise suspension cultures

Homogenates of dedifferentiated anise (Pimpinella anisum L.) suspension cultures grown in B-5 medium with sucrose as source of carbon show all but 3 glyoxysomal enzyme activities: NAD-dependent oxidation of palmitoyl-CoA, isocitrate lyase, and malate synthase are lacking. Substitution of 20 mmol/l acetate for sucrose leads to the appearance of these enzyme activities. Only then glyoxysomes with a buoyant density of 1.23 kg/l in sucrose gradients are formed showing the enzyme activities for both ß-oxidation of fatty acids and glyoxylate cycle. Quantitatively and qualitatively they resemble glyoxysomes isolated from endosperm of 4 d old anise seedlings. Therefore, the suspension cultures constitute a valuable system for the study of both mechanisms and regulation of glyoxysome formation in anise.

Microbodies and glyoxylate-cycle enzyme activities in filamentous fungi

Microbodies were observed in the hyphal tips of all 14 fungi investigated. Their morphology varied among the fungi and their numbers were influenced by the growth medium. Microbodies were closely associated with mitochondria in one fungus and with the endoplasmic reticulum in several fungi. Catalase was not detected in microbodies with the diaminobenzidine cytochemical procedure even though catalase activity was present in extracts of these fungi. The activities of the glyoxylate-cycle enzymes isocitrate lyase and malate synthase were affected by the growth medium and were particulate in the two fungi studied by differential centrifugation. Microbodies are abundant, and they are ubiquitous among the fungi and in some cases they may contain glyoxylate-cycle enzymes.

Changes in activity of glyoxylate cycle enzymes during myxospore development in Myxococcus xanthus.

Activities of the glyoxylate cycle enzymes isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) were assayed in extracts prepared at different stages of myxospore formation in liquid cultures of Myxococcus xanthus. Activities of both enzymes attained peak values during conversion of rods to spheres. Isocitrate lyase activity decreased after reaching its peak value. Malate synthase activity also declined but at a much slower rate. The loss of isocitrate lyase activity could be prevented by the addition of chloramphenicol to cultures early in myxospore formation (during the initial rise in enzyme activity), but not by such addition at later stages of myxospore formation. The increase in glyoxylate cycle enzymes was not observed in a mutant unable to form myxospores in liquid culture under conditions suitable for morphological conversion of the wild type, or in wild-type cells incubated in the absence of an inducer for myxospore formation. It is concluded that the changes in the glyoxylate cycle enzymes represent regulatory phenomena associated with the development of the myxospore.

Microbodies (Glyoxysomes and Peroxisomes) in Cucumber Cotyledons

The changes in activities of glyoxysomal and peroxisomal enzymes have been correlated with the fine structure of microbodies in cotyledons of the cucumber (Cucumis sativus L.) during the transition from fat degradation to photosynthesis in light-grown plants, and in plants grown in the dark and then exposed to light. During early periods of development in the light (days 2 through 4), the microbodies (glyoxysomes) are interspersed among lipid bodies and contain relatively high activities of glyoxylate cycle enzymes involved in lipid degradation. Thereafter, these activities decrease rapidly as the cotyledons expand and become photosynthetic, and the activity of glycolate oxidase rises to a peak (day 7); concomitantly the microbodies (peroxisomes) become preferentially associated with chloroplasts.In seedlings grown in the dark for 10 days, the reserve lipid and the glyoxylate cycle enzyme activities persist for a longer time than in the light; correlated with this, there is a continued association of the microbodies with the lipid bodies. When these dark-grown seedlings are then exposed to 51 hours of the light-dark cycle, peroxisomal marker enzymes increase rapidly in activity, and the microbodies become appressed to chloroplasts. We conclude that the characteristic association observed between glyoxysomes and lipid bodies reflects their mutual involvement in net gluconeogenesis through the conversion of fatty acids to carbohydrate, while the close spatial relationship observed between peroxisomes and chloroplasts at later stages of development reflects their mutual involvement in glycolate metabolism.Although glyoxysomal enzyme activities are dropping rapidly while peroxisomal enzyme activities are increasing rapidly during the transition period in the light, the electron microscopic evidence does not indicate that glyoxysomes are being degraded or peroxisomes are being formed. Since in the dark-grown seedlings the activities of peroxisomal enzymes remain low and do not increase as they do in the light, an opportunity is afforded to compare quantitatively any changes in numbers of microbodies per cell with the changes in activities of glyoxysomal enzymes. It is found that the magnitude of the decrease in numbers of microbodies is considerably less than that of the decrease in glyoxysomal enzyme activities between days 4 and 10. When the cotyledons are exposed to light, peroxisomal enzyme activities increase greatly, but again there is no ultrastructural evidence for the synthesis of a new population of microbodies to accommodate this increase. These results allow us to conclude that the developmental transition from glyoxysomal to peroxisomal function almost certainly does not involve the actual replacement of one population of microbodies by another. Rather, the transition probably occurs within existing particles, either by a sequential functioning of two different kinds of microbodies or by a change in enzyme complement within a single population. Our findings with both light- and dark-grown cotyledons favor the latter possibility. The cytoplasmic invaginations into microbodies seen during greening of both light-grown cotyledons and etiolated cotyledons exposed to light may be morphological manifestations of the mechanism by which the microbodies lose or gain enzymes.

Aerobic metabolism of -amino-n-butyric acid by Pseudomonas putida.

The aerobic metabolism of β -amino- n -butyric acid was studied with cell-free extracts of a strain of Pseudomonas putida isolated in pure culture through enrichment from a liquid medium containing DL -β- amino -n- butyrate as the sole energy source. The extracts catalyze the decomposition of L -(+)-β- amino -n- butyrate in the presence of pyruvate. D -(−)-β- amino -n- butyrate is not decomposed and does not inhibit the deamination of the L-isomer . The products of the transamination reaction are aceto- acetate and alanine. Experiments on the substrate specificity of the transaminase activity showed that other β - and γ -amino acids can serve as efficient amino donors, whereas corresponding α -amino acids are poor substrates. The further metabolism of acetoacetate is discussed on the basis of the demonstration of acetoacetyl-CoA: succinyl-CoA transferase and acetoacetyl-CoA thiolase activities in the crude extracts as well as activities of glyoxylate cycle enzymes. A complete reaction scheme for the ultimate conversion of β -amino- n -butyric acid to succinate is presented and discussed.

Induction of the glyoxylate cycle in Tetrahymena

The activities of glyoxylate cycle enzymes and the incorporation of acetate-C 14 into metabolic intermediates during the induction of stationary phase in growing cultures of Tetrahymena pyriformis GL were determined. The specific activities of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, doubled in the stationary cells; at the same time, the activities of acetyl-coenzyme A synthase and malate dehydrogenase did not change significantly. The ratio of radioactivity in malate and aspartate to that in citrate and glutamate changed from 0.45 in log-phase suspensions to 1.5 in suspensions of induced stationary phase cells. The increased cellular capacity for glyconeogenesis from acetate could be accounted for by the increased metabolic role of the glyoxylate cycle.

The influence of growth substrates on metabolic pathways in micrococcus denitrificans

1. 1. Autotropically grown M. denitrificans incorporated 14C from 14CO 2 initially into phosphorylated compounds, the chief component of this fraction being 3-phosphoglyceric acid. 2. 2. Extracts of such cells catalysed the incorporation of isotope from NaH 14CO 3 into 3-phosphoglyceric acid, in the presence of ribulose 1,5-diphosphate, or of ribose 5-phosphate + ATP. 3. 3. These results show that autotrophic growth of the organism involves the Calvin cycle of assimilation of carbon dioxide. 4. 4. Extracts of the organisms grown on acetate did not catalyse the incorporations of isotope form NaH 14CO 3 described under 2 above, indicating that the Calvin cycle did not operate under these conditions. 5. 5. Extracts of M. denitrificans grown autotrophically or on a variety of carbon substrates differed in their ability to effect the component reactions of the glyoxylate cycle. Whilst all extracts were qualitatively shown to contain citrate condensing enzyme and malate synthetase, only those from acetate-grown cells contained high isocitratase activity. The activity of glyoxylate cycle enzymes in acetate-grown cells was sufficiently high to account for the growth of M. denitrificans on acetate as sole carbon source. 6. 6. These findings show that change in the mode of growth, from autotrophy to growth on acetate, was accompanied by an alteration of metabolic pathways from Calvin cycle to glyoxylate cycle, and suggest that isocitratase is under adaptive control. 7. 7. Studies on the effect of other carbon sources on the isocitratase formed by acetate-grown cells indicate that they control both the activity and formation of the enzyme by feed-back mechanisms. 8. 8. It is suggested that such feed-back mechanisms control the intracellular operation of the glyoxylate cycle.