Activities of phosphopyruvate carboxylase and citrate synthase in rat and cow liver after glucocorticoid administration.
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Animals↗
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Results for “Pyruvate Synthase”
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Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli.
The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.
Acetolactate Synthase↗
Identification of an Arabidopsis thaliana mutant accumulating threonine resulting from mutation in a new dihydrodipicolinate synthase gene.
A novel Arabidopsis DHDPS gene named DHDPS2 was found through identification of a mutant by promoter trapping. The mutation promotes a reduction of growth resulting from combination of a defect in lysine biosynthesis and accumulation of a toxic level of threonine or derived products. The mutant also modifies the amino acid composition issuing from the pyruvate and aspartate pathways, affecting mainly the root compartment. These data are in accordance with the expression of DHDPS2 in the root apex as visualized by expression of the GUS reporter gene. This suggests that a large proportion of the amino acids derived from pyruvate and aspartate are synthesized in this organ.
Amino Acid Sequence↗
Changes in mitochondrial activity during avian myoblast differentiation: influence of triiodothyronine or v-erb A expression.
Numerous data suggest that mitochondrial activity is involved in the regulation of cell growth and differentiation. Therefore, we have studied the changes in mitochondrial activity in avian myoblast cultures (QM7 line) undergoing differentiation or in BrdU-treated, differentiation-deficient cells. As we have previously shown that triiodothyronine and v-erb A expression stimulate myogenic differentiation, we have also observed their influence upon mitochondrial activity. Comparison of control and BrdU-treated myoblasts indicated that precocious differentiation events were associated with a stimulation of citrate synthase and cytochrome oxidase activities. They also induced a transient decrease in mitochondrial membrane potential assessed by rhodamine 123 uptake. In control myoblasts, a general stimulation of mitochondrial activity was recorded at cell confluence, prior to terminal differentiation. These events did not occur in BrdU-treated myoblasts, thus indicating that they were tightly linked to myoblast commitment. Whereas no significant triiodothyronine influence could be detected upon mitochondrial activity, we observed that v-erb A expression significantly depresses the mitochondrial membrane potential in control myoblasts. This action was not observed in BrdU-treated myoblasts, thus suggesting that it involves an indirect pathway linked to differentiation. Moreover, the oncoprotein abrogated the decrease in E2-PDH subunit level observed at cell confluence. These data underline that changes in mitochondrial activity occurred prior to myoblast terminal differentiation and could be involved in the processes regulating myogenesis. In addition, they provide the first evidence that the v-erb A oncoprotein influences mitochondrial activity.
Animals↗
Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry.
The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.
Animals↗
High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme.
Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 10(6) cells) by cytokine- or ionophore-stimulated human fibroblasts is known. However, we here show that these cells also contain an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate superoxide (2.19 +/- 0.14 nmol/min per 10(6) cells) at levels similar to phorbol ester-stimulated monocytes on exogenous NADH addition. Superoxide generation was temperature-dependent, insensitive to chelation (desferal), and had a K(m) (app)(NADH) of 11.5 microM. Inhibitor studies showed that there was no involvement of NADPH oxidase (diphenylene iodonium, diphenyl iodonium), prostaglandin H synthase (indomethacin), xanthine oxidase (allopurinol), cytochrome P-450 (metyrapone) or mitochondrial respiration (rotenone, antimycin A). NAD+ was a competitive inhibitor, whereas NADPH supported 40% of the rate seen with NADH. No luminescence was observed after the addition of lactate, malate, pyruvate, GSH or L-cysteine. NADH-stimulated superoxide generation was enhanced by the addition of (3-30 microM) arachidonic acid, linoleic acid or (5S)-hydroxyeicosatetraenoic acid [(5S)-HETE] but not palmitic acid, (15S)-hydroperoxyeicosatetraenoic acid [(15S)-HPETE], (15S)-HETE or (12S)-HETE. Several features suggest involvement of an enzyme related to 15-lipoxygenase, and, in support of this, we show superoxide generation and NADH oxidation by recombinant rabbit reticulocyte 15-lipoxygenase. The large amounts of superoxide measured suggest that the fibroblast extracellular enzyme could be a major source of reactive oxygen species after tissue damage.
Animals↗
Plasmodium falciparum possesses organelle-specific alpha-keto acid dehydrogenase complexes and lipoylation pathways.
The human malaria parasite Plasmodium falciparum possesses a single mitochondrion and a plastid-like organelle called the apicoplast. Both organelles contain members of the KADH (alpha-keto acid dehydrogenase) complexes--multienzyme complexes that are involved in intermediate metabolism. In the asexual blood stage forms of the parasites, the alpha-ketoglutarate dehydrogenase and branched chain KADH complexes are both located in the mitochondrion, whereas the pyruvate dehydrogenase is exclusively found in the apicoplast. In agreement with this distribution, Plasmodium parasites have two separate and organelle-specific pathways that guarantee lipoylation of the KADH complexes in both organelles. A biosynthetic pathway comprised of lipoic acid synthase and lipoyl (octanoyl)-ACP:protein Nepsilon-lipoyltransferase B is present in the apicoplast, whereas the mitochondrion is supplied with exogenous lipoic acid, and ligation of the metabolite to the KADH complexes is accomplished by a lipoate protein ligase A similar to that of bacteria and plants. Both pathways are excellent potential targets for the design of new antimalarial drugs.
3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
The role of liver X receptor-alpha in the fatty acid regulation of hepatic gene expression.
Liver X receptors (LXR) alpha and beta play an important role in regulating the expression of genes involved in hepatic bile and fatty acid synthesis, glucose metabolism, as well as sterol efflux. Studies with human embryonic kidney 293 cells indicate that unsaturated fatty acids interfere with oxysterols binding to LXR and antagonize oxysterol-induced LXRalpha activity. In this report, we evaluated the effects of unsaturated fatty acids on LXR-regulated hepatic gene expression. The LXR agonist, T1317, induced mRNAs encoding sterol regulatory element-binding protein 1c (SREBP-1c) and two SREBP-1c-regulated lipogenic genes, e.g. fatty-acid synthase and the S14 protein in primary hepatocytes. Treatment of hepatocytes with eicosapentaenoic acid (20:5n-3) suppressed these mRNAs in the absence and presence of T1317. The cis-regulatory elements targeted by T1317 were not required for fatty-acid suppression of FAS or S14 promoter activity. In contrast to SREBP-1-regulated lipogenic genes, 20:5n-3 had no effect on the T1317 induction of ABCG5 or ABCG8 in the rat hepatoma cell line, FTO-2B. These two genes require LXR but not SREBP-1c for their expression. Feeding rats a diet supplemented with fish oil suppressed hepatic SREBP-1c-regulated genes and induced PPARalpha-regulated genes but had no effect on the LXR-regulated transcripts, CYP7A1, ABCG5, or ABCG8. Transfection studies, using either full-length hLXRalpha or a chimera containing only the LXRalpha ligand binding domain, indicate that a wide array of unsaturated fatty acids had little effect on LXRalpha activity in primary hepatocytes or FTO-2B. These studies suggest that LXRalpha is not a target for unsaturated fatty acid regulation in primary rat hepatocytes or in liver. Thus, oxysterol/LXR-mediated regulation of transcripts involved in bile acid synthesis or sterol efflux appear insensitive to dietary unsaturated fatty acids. The unsaturated fatty acid suppression of SREBP-1 and its targeted lipogenic genes is independent of LXRalpha
Animals↗
Inhibition of enzymes which interact with citrate by (--)hydroxycitrate and 1,2,3,-tricarboxybenzene.
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ATP Citrate (pro-S)-Lyase↗
Effects of dietary fatty acids on insulin sensitivity and secretion.
Globalization and global market have contributed to increased consumption of high-fat, energy-dense diets, particularly rich in saturated fatty acids( SFAs). Polyunsaturated fatty acids (PUFAs) regulate fuel partitioning within the cells by inducing their own oxidation through the reduction of lipogenic gene expression and the enhancement of the expression of those genes controlling lipid oxidation and thermogenesis. Moreover, PUFAs prevent insulin resistance by increasing membrane fluidity and GLUT4 transport. In contrast, SFAs are stored in non-adipocyte cells as triglycerides (TG) leading to cellular damage as a sequence of their lipotoxicity. Triglyceride accumulation in skeletal muscle cells (IMTG) derives from increased FA uptake coupled with deficient FA oxidation. High levels of circulating FAs enhance the expression of FA translocase the FA transport proteins within the myocites. The biochemical mechanisms responsible for lower fatty acid oxidation involve reduced carnitine palmitoyl transferase (CPT) activity, as a likely consequence of increased intracellular concentrations of malonyl-CoA; reduced glycogen synthase activity; and impairment of insulin signalling and glucose transport. The depletion of IMTG depots is strictly associated with an improvement of insulin sensitivity, via a reduced acetyl-CoA carboxylase (ACC) mRNA expression and an increased GLUT4 expression and pyruvate dehydrogenase (PDH) activity. In pancreatic islets, TG accumulation causes impairment of insulin secretion. In rat models, beta-cell dysfunction is related to increased triacylglycerol content in islets, increased production of nitric oxide, ceramide synthesis and beta-cell apoptosis. The decreased insulin gene promoter activity and binding of the pancreas-duodenum homeobox-1 (PDX-1) transcription factor to the insulin gene seem to mediate TG effect in islets. In humans, acute and prolonged effects of FAs on glucose-stimulated insulin secretion have been widely investigated as well as the effect of high-fat diets on insulin sensitivity and secretion and on the development of type 2 diabetes.
Animals↗
Skeletal muscle respiratory capacity, endurance, and glycogen utilization.
This study was undertaken to evaluate the relationship between physical performance capacity and the mitochondrial content of skeletal muscle. Four groups of rats were trained by means of treadmill running 5 days/wk for 13 wk. One group ran 10 min/day, a second group ran 30 min/day, a third group ran 60 min/day, and a fourth group ran 120 min/day. The magnitude of the exercise-induced adaptive increase in gastrocnemius muscle respiratory capacity varied over a twofold range in the four groups. There were significant correlations between the levels of three mitochondrial markers (cytochrome c, citrate synthase, respiratory capacity) in the animals' gastrocnemius muscles and the duration of a run to exhaustion. There was also a significant correlation between the amounts of glycogen remaining in liver and skeletal muscle after a 30-min-long exercise test and the respiratory capacity of the animal's leg muscles. These findings are compatible with the interpretation that a close relationshiop exists between skeletal muscle mitochondrial content and the capacity to perform endurance exercise.
Adenosine Diphosphate↗
Effect of epinephrine on glucose metabolism and hydrogen peroxide content in incubated rat macrophages.
The effects of epinephrine on glucose metabolism and hydrogen peroxide content were examined in incubated rat macrophages. An increase in the activities of hexokinase and citrate synthase and a reduction in that of glucose-6-phosphate dehydrogenase was found in resident, inflammatory and activated macrophages incubated for 1 hr in the presence of epinephrine. Glucose utilization by incubated resident, inflammatory and activated macrophages was augmented markedly by the addition of epinephrine, whereas lactate formation was depressed. Under the same conditions, there was a 2.6-fold increment of hydrogen peroxide content and of [U-14C]glucose decarboxylation in activated macrophages incubated for 40 min. Similar results were obtained when pyruvate and oxoglutarate was substituted for glucose. These findings suggest that epinephrine may increase hydrogen peroxide production in activated macrophages possibly through a mitochondrial mechanism other than the pentose-phosphate pathway. Between 40 and 90 min of incubation, the content of hydrogen peroxide decreased markedly, and there was no detectable glucose utilization in the presence of epinephrine. These observations are consistent with the idea that this catecholamine stimulates both hydrogen peroxide production and metabolism, the first process being dependent on mitochondrial fuels.
Animals↗
Quantification of compartmented metabolic fluxes in maize root tips using isotope distribution from 13C- or 14C-labeled glucose.
Metabolic pathways of the intermediate metabolism of maize root tips were identified and quantified after labeling to isotopic and metabolic steady state using glucose labeled on carbon-1, -2, or -6 with 14C or 13C. The specific radioactivity of amino acids and the 13C-specific enrichment of specific carbons of free glucose, sucrose, alanine and glutamate were measured and used to calculate metabolic fluxes. The non-triose pathways, including synthesis of polysaccharides, accumulation of free hexoses, and to a lesser extent starch synthesis, were found to consume 75% of the glucose entering the root tips. The cycle of synthesis and hydrolysis of sucrose was found to consume about 70% of the ATP produced by respiration. The comparison of the specific radioactivities of amino acids and phospholipid glycerol phosphate after labeling with [1-(14)C] or [6-(14)C]glucose revealed the operation of the pentose phosphate pathway. The transfer of label from [2-(14)C]glucose to carbon-1 of starch glucosyl units confirmed the operation of this pathway and indicated that it is located in plastids. It was found to consume 32% of the hexose phosphates entering the triose pathways. The remaining 68% were consumed by glycolysis. The determination of the specific enrichment of carbohydrate carbons -1 and -6 after labeling with [1-(13)C]glucose indicated that both the conversion of triose phosphates back to hexose phosphates and the transaldolase exchange contributed to this randomization. Of the triose phosphates produced by glycolysis and the pentose phosphate pathway, about 60% were found to be recycled to hexose phosphates, and 28% were directed to the tricarboxylic acid cycle. Of this 28%, two-thirds were found to be directed through the pyruvate kinase branch and one-third through the phosphoenolpyruvate branch. The latter essentially has an anaplerotic function since little malate was found to be converted to pyruvate (malic enzyme reaction).
Biological Transport↗
An improved assay for pyruvate dehydrogenase in liver and heart.
A radiochemical assay was developed to measure pyruvate dehydrogenase complex (PDC) activity in liver and heart without interference by branched-chain 2-oxo acid dehydrogenase (BCODH). Decarboxylation of pyruvate by BCODH was eliminated by using low pyruvate concentration (0.5 mM), a preferred substrate for BCODH (3-methyl-2-oxopentanoate) that is not used by PDC, and a competitive inhibitor of BCODH, dichloroacetate. This method was validated by assaying a combination of both purified enzymes and tissue homogenates with known amounts of added BCODH. The actual percentage of active PDC decreased after 48 h starvation from 13.6 to 3.1 in liver and from 77.1 to 9.0 in heart. Total PDC activity (munits of PDC/units of citrate synthase) in starved rats was increased by 34% in liver and decreased by 23% in heart. Total PDC activity (munits/g wet wt.) in fed- and starved-rat liver was 0.8 and 1.3, and in heart was 6.6 and 5.8, respectively.
Animals↗
Cystathionine gamma-synthase from Salmonella. Spectral changes in the presence of substrates.
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Amino Acids↗
Poly- -hydroxybutyrate biosynthesis and the regulation of glucose metabolism in Azotobacter beijerinckii.
Azotobacter beijerinckii possesses the enzymes of both the Entner-Doudoroff and the oxidative pentose phosphate cycle pathways of glucose catabolism and both pathways are subject to feedback inhibition by products of glucose oxidation. The allosteric glucose 6-phosphate dehydrogenase utilizes both NADP(+) and NAD(+) as electron acceptors and is inhibited by ATP, ADP, NADH and NADPH. 6-Phosphogluconate dehydrogenase (NADP-specific) is unaffected by adenosine nucleotides but is strongly inhibited by NADH and NADPH. The formation of pyruvate and glyceraldehyde 3-phosphate from 6-phosphogluconate by the action of the Entner-Doudoroff enzymes is inhibited by ATP, citrate, isocitrate and cis-aconitate. Glyceraldehyde 3-phosphate dehydrogenase is unaffected by adenosine and nicotinamide nucleotides but the enzyme is non-specific with respect to NADP and NAD. Citrate synthase is strongly inhibited by NADH and the inhibition is reversed by the addition of AMP. Isocitrate dehydrogenase, a highly active NADP-specific enzyme, is inhibited by NADPH, NADH, ATP and by high concentrations of NADP(+). These findings are discussed in relation to the massive synthesis of poly-beta-hydroxybutyrate that occurs under certain nutritional conditions. We propose that synthesis of this reserve material, to the extent of 70% of the dry weight of the organism, serves as an electron and carbon ;sink' when conditions prevail that would otherwise inhibit nitrogen fixation and growth.
Aconitum↗
The effect of high-intensity exhaustive exercise studied in isolated mitochondria from human skeletal muscle.
Six young men performed five 1-min bicycle exercise bouts to exhaustion. Muscle lactate increased to congruent with 114 mmol x kg(-1) dwt and pH decreased to congruent with 6.6. Mitochondria were prepared from a needle biopsy sample taken from m. vastus lateralis immediately after the last exercise bout. No significant effect of exhaustion on the proton permeability and amount of cytochromes c and aa3 in isolated mitochondria was detected. The activities of the following enzymes and systems were not altered either: citrate synthase, succinate dehydrogenase, cytochrome oxidase, succinate + glutamate respiration, malate + glutamate respiration, the respiratory chain, and the reactions involved in ATP synthesis. Thus, the mitochondria did not appear globally altered upon exhaustion. However, the following NAD-linked activities were significantly lowered: pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, glutamate dehydrogenase and fatty acid beta-oxidation. The activities of alpha-glycerophosphate dehydrogenase and exo-NADH oxidase, enzymes that might catalyze the oxidation of sarcoplasmic NADH, were increased. These changes may be due to the action of reactive oxygen species, protons and Ca2+. Transient opening of the permeability transition pore may also be involved. Some effects may have been reversed during isolation of the mitochondria and the changes in mitochondrial function in situ upon exhaustion may have been more extensive than observed.
Acyltransferases↗
Carbon, Nitrogen, and Nutrient Interactions in Beta vulgaris L. as Influenced by Nitrogen Source, NO3- versus NH4+
Sugar beets (Beta vulgaris L. cv F58-554H1) were grown hydroponically in a 16-h light, 8-h dark period (photosynthetic photon flux density of 0.5 mmol m-2 s-1) for 4 weeks from sowing in half-strength Hoagland nutrient solution containing 7.5 mM nitrate. Half of the plants were then transferred to 7.5 mM ammonium N; the rest remained in solution with 7.5 mM nitrate N. Upon transfer from nitrate to ammonium, the total N concentration decreased sharply in the fibrous roots and petiole/midribs and increased substantially in the leaf blades. This was because of the decreased nitrate concentrations in fibrous roots and petioles and a concomitant increase in amino acid/amide-N and protein N in leaf blades. Sugar beets acclimated to ammonium partly by a 2.5-fold increase in glutamine synthase activity in fibrous roots and a 1.7-fold increase in leaf blades. Rapid ammonium assimilation into glutamine consumed carbon skeletons, leading to a depletion of foliar starch, sucrose, and maltose. Ammonium treatment stimulated activities of some glycolytic/Krebs cycle enzymes, e.g. pyruvate dehydrogenase. Nitrate-fed leaf blades contained substantially larger concentrations of osmolytes (i.e. nitrate, cations, and sucrose), which may have contributed to the faster rates of leaf expansion in nitrate-fed compared to ammonium-fed plants.
Journal Article↗