Showing papers on "Pyruvate kinase published in 2005"

Structural basis for tumor pyruvate kinase M2 allosteric regulation and catalysis.

Abstract: Four isozymes of pyruvate kinase are differentially expressed in human tissue. Human pyruvate kinase isozyme M2 (hPKM2) is expressed in early fetal tissues and is progressively replaced by the other three isozymes, M1, R, and L, immediately after birth. In most cancer cells, hPKM2 is once again expressed to promote tumor cell proliferation. Because of its almost ubiquitous presence in cancer cells, hPKM2 has been designated as tumor specific PK-M2, and its presence in human plasma is currently being used as a molecular marker for the diagnosis of various cancers. The X-ray structure of human hPKM2 complexed with Mg(2+), K(+), the inhibitor oxalate, and the allosteric activator fructose 1,6-bisphosphate (FBP) has been determined to a resolution of 2.82 A. The active site of hPKM2 is in a partially closed conformation most likely resulting from a ligand-induced domain closure promoted by the binding of FBP. In all four subunits of the enzyme tetramer, a conserved water molecule is observed on the 2-si face of the prospective enolate and supports the hypothesis that a proton-relay system is acting as the proton donor of the reaction (1). Significant structural differences among the human M2, rabbit muscle M1, and the human R isozymes are observed, especially in the orientation of the FBP-activating loop, which is in a closed conformation when FBP is bound. The structural differences observed between the PK isozymes could potentially be exploited as unique structural templates for the design of allosteric drugs against the disease states associated with the various PK isozymes, especially cancer and nonspherocytic hemolytic anemia.

Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation

Abstract: Dietary polyunsaturated fatty acids (PUFAs) are potent inhibitors of hepatic glycolysis and lipogenesis. Recently, carbohydrate-responsive element-binding protein (ChREBP) was implicated in the regulation by glucose of glycolytic and lipogenic genes, including those encoding L-pyruvate kinase (L-PK) and fatty acid synthase (FAS). The aim of our study was to assess the role of ChREBP in the control of L-PK and FAS gene expression by PUFAs. We demonstrated in mice, both in vivo and in vitro, that PUFAs [linoleate (C18:2), eicosapentanoic acid (C20:5), and docosahexaenoic acid (C22:6)] suppressed ChREBP activity by increasing ChREBP mRNA decay and by altering ChREBP translocation from the cytosol to the nucleus, independently of an activation of the AMP-activated protein kinase, previously shown to regulate ChREBP activity. In contrast, saturated [stearate (C18)] and monounsaturated fatty acids [oleate (C18:1)] had no effect. Since glucose metabolism via the pentose phosphate pathway is determinant for ChREBP nuclear translocation, the decrease in xylulose 5-phosphate concentrations caused by a PUFA diet favors a PUFA-mediated inhibition of ChREBP translocation. In addition, overexpression of a constitutive nuclear ChREBP isoform in cultured hepatocytes significantly reduced the PUFA inhibition of both L-PK and FAS gene expression. Our results demonstrate that the suppressive effect of PUFAs on these genes is primarily caused by an alteration of ChREBP nuclear translocation. In conclusion, we describe a novel mechanism to explain the inhibitory effect of PUFAs on the genes encoding L-PK and FAS and demonstrate that ChREBP is a pivotal transcription factor responsible for coordinating the PUFA suppression of glycolytic and lipogenic genes.

In vivo respiratory metabolism of illuminated leaves.

Abstract: 13 C-enriched compounds. Using different positional 13 C-enrichments, it is shown that the Krebs cycle is reduced by 95% in the light and that the pyruvate dehydrogenase reaction is much less reduced, by 27% or less. Glucose molecules are scarcely metabolized to liberate CO 2 in the light, simply suggesting that they can rarely enter glycolysis. Nuclear magnetic resonance analysis confirmed this view; when leaves are fed with 13 C-glucose, leaf sucrose and glucose represent nearly 90% of the leaf 13 C content, demonstrating that glucose is mainly directed to sucrose synthesis. Taken together, these data indicate that several metabolic down-regulations (glycolysis, Krebs cycle) accompany the light/dark transition and emphasize the decrease of the Krebs cycle decarboxylations as a metabolic basis of the light-dependent inhibition of mitochondrial respiration. Illuminated leaves simultaneously assimilate CO 2 through the photosynthetic carbon reduction cycle and lose CO 2 through photorespiration and day respiration. In darkness, leaves no longer assimilate CO 2 via the photosynthetic carbon reduction cycle but produce CO 2 through dark respiration. Although dark respiration is known to involve glycolysis and CO 2 production through pyruvate dehydrogenation and the degradative Krebs cycle (Trethewey and ap Rees, 1994; Plaxton, 1996), the carbon metabolism that is responsible for the CO 2 respiratory release in the light is almost unknown. This is so because the day respiratory CO 2 flux is very low and masked by the photosynthetic carbon fixation and the photorespiratory CO2 production in the light, and is thus difficult to study. Nevertheless, it has been repeatedly shown, using either the Laisk’s (Laisk, 1977) or Kok’s method (Kok, 1948), that the rate of day respiration (Rd) is less than that of dark respiration (Rn; for review, see Atkin et al., 2000) so that light is known to inhibit respiration, with a R d /R n value (usually denoted as m) ranging from 30% to 100% (for a recent study, see Peisker and Apel, 2001). Pioneering gas exchange measurements on mustard suggested that some enzymatic activities are inhibited in the light so that substrates accumulate (Cornic, 1973), explaining the respiratory burst when leaves are darkened: the light enhanced dark respiration. More recently, it has been shown in the unicellular alga Selenastrum minutum that pyruvate kinase (Lin et al., 1989) is inhibited by light. It is also the case of the pyruvate dehydrogenase complex that is partly inactivated by (reversible) phosphorylation in extracts from illuminated leaves (Budde and Randall, 1990;

Red cell pyruvate kinase deficiency: molecular and clinical aspects

Abstract: Red cell pyruvate kinase (PK) deficiency is the most frequent enzyme abnormality of the glycolytic pathway causing hereditary non-spherocytic haemolytic anaemia. The degree of haemolysis varies widely, ranging from very mild or fully compensated forms, to life-threatening neonatal anaemia and jaundice necessitating exchange transfusions. Erythrocyte PK is synthesized under the control of the PK-LR gene located on chromosome 1. To date, more than 150 different mutations in the PK-LR gene have been associated with PK deficiency. First attempts to delineate the biochemical and clinical consequences of the molecular defect were mainly based on the observation of the few homozygous patients and on the analysis of the three-dimensional structure of the enzyme. More recently, the comparison of the recombinant mutants of human red cell PK with the wild-type enzyme has enabled the effects of amino acid replacements on the enzyme molecular properties to be determined and help to correlate genotype to clinical phenotype.

The pyruvate requirement of some members of the Mycobacterium tuberculosis complex is due to an inactive pyruvate kinase: implications for in vivo growth

Abstract: Through examination of one of the fundamental in vitro characteristics of Mycobacterium bovis--its requirement for pyruvate in glycerol medium--we have revealed a lesion in central metabolism that has profound implications for in vivo growth and nutrition. Not only is M. bovis unable to use glycerol as a sole carbon source but the lack of a functioning pyruvate kinase (PK) means that carbohydrates cannot be used to generate energy. This disruption in sugar catabolism is caused by a single nucleotide polymorphism in pykA, the gene which encodes PK, that substitutes glutamic acid residue 220 with an aspartic acid residue. Substitution of this highly conserved amino acid residue renders PK inactive and thus blocks the ATP generating roles of glycolysis and the pentose phosphate pathway. This mutation was found to occur in other members of the M. tuberculosis complex, namely M. microti and M. africanum. With carbohydrates unable to act as carbon sources, the importance of lipids and gluconeogenesis for growth in vivo becomes apparent. Complementation of M. bovis with the pykA gene from M. tuberculosis H37Rv restored growth on glycerol. Additionally, the presence of a functioning PK caused the colony morphology of the complemented strain to change from the characteristic dysgonic growth of M. bovis to eugonic growth, an appearance normally associated with M. tuberculosis. We also suggest that the glycerol-soaked potato slices used for the derivation of the M. bovis bacillus Calmette and Guerin (BCG) vaccine strain selected for an M. bovis PK+ mutant, a finding that explains the alteration in colony morphology noted during the derivation of BCG. In summary, the disruption of a key step in glycolysis divides the M. tuberculosis complex into two groups with distinct carbon source utilization.

Serum autoantibodies do not differentiate PANDAS and Tourette syndrome from controls

Abstract: We read the article by Singer et al. with interest and would like to raise a number of methodologic issues and concerns.1 The authors found no autoantibody binding against brain homogenates and candidate neuronal autoantigens in PANDAS and Tourette syndrome sera. Our group has demonstrated that autoantibodies in PANDAS and Tourette syndrome sera bind to autoantigens of molecular weight 40, 45 (doublet) and 60 kDa.2,3 We have identified these autoantigens as neuronal isoforms of the glycolytic enzymes aldolase C, neuron-specific enolase and pyruvate kinase M1, and the ubiquitous glycolytic enzyme non-neuronal enolase. We found autoantibodies against these neuronal glycolytic enzymes (NGE) more commonly in patients compared to controls.4 We would like to point out the following methodologic issues. Singer et al. claim to use the human brain autoantigens pyruvate kinase M1 and aldolase C from Santa Cruz Biotechnology Inc.1 The antigens produced by this company are rabbit muscle pyruvate kinase (not the human brain M1 isoform), and …

Reperfusion-Induced Translocation of δPKC to Cardiac Mitochondria Prevents Pyruvate Dehydrogenase Reactivation

Abstract: Cardiac ischemia and reperfusion are associated with loss in the activity of the mitochondrial enzyme pyruvate dehydrogenase (PDH). Pharmacological stimulation of PDH activity improves recovery in contractile function during reperfusion. Signaling mechanisms that control inhibition and reactivation of PDH during reperfusion were therefore investigated. Using an isolated rat heart model, we observed ischemia-induced PDH inhibition with only partial recovery evident on reperfusion. Translocation of the redox-sensitive delta-isoform of protein kinase C (PKC) to the mitochondria occurred during reperfusion. Inhibition of this process resulted in full recovery of PDH activity. Infusion of the deltaPKC activator H2O2 during normoxic perfusion, to mimic one aspect of cardiac reperfusion, resulted in loss in PDH activity that was largely attributable to translocation of deltaPKC to the mitochondria. Evidence indicates that reperfusion-induced translocation of deltaPKC is associated with phosphorylation of the alphaE1 subunit of PDH. A potential mechanism is provided by in vitro data demonstrating that deltaPKC specifically interacts with and phosphorylates pyruvate dehydrogenase kinase (PDK)2. Importantly, this results in activation of PDK2, an enzyme capable of phosphorylating and inhibiting PDH. Thus, translocation of deltaPKC to the mitochondria during reperfusion likely results in activation of PDK2 and phosphorylation-dependent inhibition of PDH.

ATP Production in Chlamydomonas reinhardtii Flagella by Glycolytic Enzymes

Abstract: Eukaryotic cilia and flagella are long, thin organelles, and diffusion from the cytoplasm may not be able to support the high ATP concentrations needed for dynein motor activity. We discovered enzyme activities in the Chlamydomonas reinhardtii flagellum that catalyze three steps of the lower half of glycolysis (phosphoglycerate mutase, enolase, and pyruvate kinase). These enzymes can generate one ATP molecule for every substrate molecule consumed. Flagellar fractionation shows that enolase is at least partially associated with the axoneme, whereas phosphoglycerate mutase and pyruvate kinase primarily reside in the detergent-soluble (membrane + matrix) compartments. We further show that axonemal enolase is a subunit of the CPC1 central pair complex and that reduced flagellar enolase levels in the cpc1 mutant correlate with the reduced flagellar ATP concentrations and reduced in vivo beat frequencies reported previously in the cpc1 strain. We conclude that in situ ATP synthesis throughout the flagellar compartment is essential for normal flagellar motility.

Changes in the metabolome of Saccharomyces cerevisiae associated with evolution in aerobic glucose-limited chemostats.

Abstract: The effect of culture age on intra- and extracellular metabolite levels as well as on in vitro determined specific activities of enzymes of central carbon metabolism was investigated during evolution for over 90 generations of Saccharomyces cerevisiae CEN.PK 113-7D in an aerobic glucose/ethanol-limited chemostat at a specific dilution rate of 0.052 h−1. It was found that the fluxes of consumed (O2, glucose/ethanol) and secreted compounds (CO2) did not change significantly during the entire cultivation period. However, morphological changes were observed, leading to an increased cellular surface area. During 90 generations of chemostat growth not only the residual glucose concentration decreased, also the intracellular concentrations of trehalose, glycolytic intermediates, TCA cycle intermediates and amino acids were found to have decreased with a factor 5–10. The only exception was glyoxylate which showed a fivefold increase in concentration. In addition to this the specific activities of most glycolytic enzymes also decreased by a factor 5–10 during long-term cultivation. Exceptions to this were hexokinase, phosphofructokinase, pyruvate kinase and 6-phosphogluconate dehydrogenase of which the activities remained unchanged. Furthermore, the concentrations of the adenylate nucleotides as well as the energy charge of the cells did not change in a significant manner. Surprisingly, the specific activities of glucose-6-phosphate dehydrogenase (G6PDH), malate synthase (MS) and isocitrate lyase (ICL) increased significantly during 90 generations of chemostat cultivation. These changes seem to indicate a pattern where metabolic overcapacities (for reversible reactions) and storage pools (trehalose, high levels of amino acids and excess protein in enzymes) are lost during the evolution period. The driving force is proposed to be a growth advantage in the absence of these metabolic overcapacities.

Induction in Primary Culture of ‘Gluconeogenic’ and ‘Glycolytic’ Hepatocytes Resembling Periportal and Perivenous Cells

Abstract: Adult rat hepatocytes were kept in primary culture for 48 h under different hormonal conditions to induce an enzyme pattern which with respect to carbohydrate metabolism approximated that of periportal and perivenous hepatocytes in vivo. 1 Glucagon-treated cells compared with control cells possessed a lower activity of glucokinase, a 4.5-fold higher activity of phosphoenolpyruvate carboxykinase and unchanged levels of glucose-6-phosphatase, phosphofructokinase, fructose-bisphosphatase and pyruvate kinase; they resembled in a first approximation the periportal cell type and are called for simplicity ‘periportal’. Inversely, insulin-treated cells compared with control cells contained a 2.2-fold higher activity of glucokinase, a slightly decreased activity of phosphoenolpyruvate carboxykinase, increased activities of phosphofructokinase and pyruvate kinase and unaltered levels of glucose-6-phosphatase and fructose-bisphosphatase; they resembled perivenous cells and are called simply ‘perivenous’. Gluconeogenesis and glycolysis were studied under various substrate and hormone concentrations. 2 Physiological concentrations of glucose (5 mM) and lactate (2 mM) gave about 80% saturation of gluconeogenesis from lactate and less than 15% saturation of glycolysis at a simultaneous 40% inhibition of the glycolytic rate by lactate. 3 Comparison of the two cell types showed that under identical assay conditions (5 mM glucose, 2 mM lactate, 0.5 nM insulin, 0.1 μM dexamethasone) gluconeogenesis was 1.5-fold faster in the ‘periportal’ cells and glycolysis was 2.4-fold faster in the ‘perivenous’ cells. 4 Metabolic rates were under short-term hormonal control. Insulin increased glycolysis three fold in both cell types with a half-maximal effect at about 0.4 nM, but did not influence the gluconeogenic rate. Glucagon inhibited glycolysis by 70% with a half-maximal effect at about 0.1 nM. Gluconeogenesis was stimulated by glucagon (half-maximal dose: 0.5 nM) 1.8-fold only in ‘periportal’ cells containing high phosphoenolpyruvate carboxykinase activity, not in the ‘perivenous’ cells with a low level of this enzyme. 5 A comparison of the two cell types showed that with maximally stimulating hormone concentrations gluconeogenesis was threefold faster in ‘periportal’ cells and glycolysis was eightfold faster in ‘perivenous’ cells. The results support the view that periportal and perivenous hepatocytes in vivo catalyse gluconeogenesis and glycolysis at inverse rates.

Regulation of rat liver L-type pyruvate kinase mRNA by insulin and by fructose.

Abstract: The effects have been studied of streptozotocin-induced diabetes and subsequent insulin administration or feeding of a high fructose diet on the amount of enzyme protein and mRNA activity of L-type pyruvate kinase in rat liver. Diabetes markedly decreased the L-type enzyme activity in rat liver and insulin treatment resulted in restoration of the enzyme activity to normal. A high fructose diet also increased the enzyme activity in diabetic rats but to a lesser extent. Immunochemical analysis showed that these alterations in the enzyme activity were due to changes in the amount of immunoreactive enzyme protein. The mechanism of the changes was studied further by assaying the level of functional mRNA coding for this enzyme in a nuclease-treated reticulocyte lysate system with total RNA isolated from rat liver. L-type pyruvate kinase mRNA, expressed as a percentage of the total protein synthesized, was greatly decreased in diabetic rats. Insulin administration resulted in recovery of the mRNA activity to the normal level within 24 h. The lag period before accumulation of translatable mRNA of the L-type enzyme was about 4-5 h. The mRNA activity was also increased in diabetic rats fed a high fructose diet. This fructose effect, which was much smaller than the insulin effect, was maximal after feeding fructose diet for one day. These changes were approximately comparable to the changes in enzyme activity. Thus, it is suggested that regulations of rat liver L-type pyruvate kinase by insulin and by fructose are primarily due to changes in the level of translatable mRNA of this enzyme.

Gene expression profiling of hypoxia signaling in human hepatocellular carcinoma cells.

Abstract: CELLS, TISSUES, AND ORGANISMS are said to be hypoxic when they receive less than normal levels of oxygen. Given the central role of oxygen in the production of ATP through oxidative phosphorylation, it is critical for cells and tissues to respond rapidly to hypoxia. The importance of hypoxia signaling is further highlighted by its essential role in mammalian development and several pathological conditions such as cardiovascular disease and cancer (4). The primary response to hypoxia within the cell is the upregulation of proteins and pathways such as glycolytic enzymes and angiogenic factors that ultimately lead to alternative routes of ATP generation and an increased oxygen availability (10). Glycolytic enzymes that are targets for such upregulation include glyceraldehydes-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and phophofructokinase (27). At the tissue level, there is a stimulation of angiogenesis through the upregulation of growth factors such as vascular endothelial growth factor (VEGF) (19). These responses and others are regulated by a family of transcription

Enzymatic regulation of glucose disposal in human skeletal muscle after a high-fat, low-carbohydrate diet

Abstract: Whole body glucose disposal and skeletal muscle hexokinase, glycogen synthase (GS), pyruvate dehydrogenase (PDH), and PDH kinase (PDK) activities were measured in aerobically trained men after a st...

Dynamics of intracellular metabolites of glycolysis and TCA cycle during cell-cycle-related oscillation in Saccharomyces cerevisiae.

Abstract: In the present work LC-MS/MS was applied to measure the concentrations of intermediates of glycolysis and TCA cycle during autonomous, cell-cycle synchronized oscillations in aerobic, glucose-limited chemostat cultures of Saccharomyces cerevisiae. This study complements previously reported oscillations in carbon dioxide production rate, intracellular concentrations of trehalose and various free amino acids, and extracellular acetate and pyruvate in the same culture. Of the glycolytic intermediates, fructose 1,6-bisphosphate, 2- and 3-phosphoglycerate, and phosphoenolpyruvate show the most pronounced oscillatory behavior, the latter three compounds oscillating out of phase with the former. This agrees with previously observed metabolic control by phosphofructokinase and pyruvate kinase. Although individually not clearly oscillating, several intermediates of the TCA cycle, i.e., alpha-ketoglutarate, succinate, fumarate, and malate, exhibited increasing concentration during the cell cycle phase with high carbon flux through glycolysis and TCA cycle. The average mass action ratios of beta-phosphoglucomutase and fumarase agreed well with previously determined in vitro equilibrium constants. Minor differences resulted for phosphoglucose isomerase and enolase. Together with the observed close correlation of the pool sizes of the involved metabolites, this might indicate that, in vivo, these reactions are operating close to equilibrium, whereby care must be taken due to possible differences between in vivo and in vitro conditions. Combining the data with previously determined intracellular amino acid levels from the same culture, a few clear correlations between catabolism and anabolism could be identified: phosphoglycerate/serine and alpha-ketoglutarate/lysine exhibited correlated oscillatory behavior, albeit with different phase shifts. Oscillations in intracellular amino acids might therefore be, at least partly, following oscillations of their anabolic precursors.

Quantitative Proteomic Analysis of Mitochondria from Primary Neuron Cultures Treated with Amyloid Beta Peptide

Abstract: Increasing evidence supports a role for altered mitochondrial function in the pathogenesis of neuron degeneration in Alzheimer’s disease (AD). Although several studies have examined the effect of amyloid beta peptide (As), on activities of individual proteins in primary neuron cultures, there have been no studies of the effects of As on the mitochondrial proteome. Here, we quantitatively measured changes in mitochondrial proteins of primary rat cortical neuron cultures exposed to 25 μM As25–35 for 16 h using isotope coded affinity tag (ICAT) labeling and 2-dimensional liquid chromatography/tandem mass spectrometry (2D-LC/MS/MS) which allows simultaneous identification and quantification of cysteine-containing proteins. The analysis of enriched mitochondrial fractions identified 10 proteins including sodium/potassium-transporting ATPase, cofilin, dihydropyrimidinase, pyruvate kinase and voltage dependent anion channel 1 that were statistically significantly (P < 0.05) altered in As-treated cultures. Elevations of proteins associated with energy production suggest that cells undergoing As-mediated apoptosis increase synthesis of proteins essential for ATP production and efflux in an attempt to maintain metabolic function.

Role of AMP-activated protein kinase in the glycolysis of postmortem muscle

Abstract: AMP-activated protein kinase (AMPK) is a newly identified kinase controlling energy metabolism in vivo. The objective of this study was to show the role of AMPK in postmortem glycolysis. Rapid and excessive postmortem glycolysis is directly related to the incidence of PSE (pale, soft and exudative) meat in pork, chicken and turkey, while insufficient glycolysis leads to dark cutters in beef and lamb, which causes significant loss to the meat industry. A total of 24 two-month-old C57BL/6J mice were assigned to three treatments: (1) wild-type mice without pre-slaughter treatment; (2) wild-type mice with a 2 min swim before slaughter; and (3) wild-type mice intraperitoneally injected with AICAr (50 mg kg−1), a specific activator of AMPK, to stimulate the activity of AMPK. In addition, 16 two-month-old C57BL/6J mice with AMPK knockout were assigned to two treatments: (4) AMPK knockout mice without pre-slaughter treatment; and (5) AMPK knockout mice with a 2 min swim before slaughter. The longissimus dorsi muscle was sampled at 0, 1 and 24 h postmortem for pH and enzyme activity measurements. Results showed that AMPK activity had a major role in determining the ultimate muscle pH. Pre-slaughter stress induced by swimming significantly accelerated the glycogenolysis in postmortem muscle through activating glycogen phosphorylase. AMPK is important for maintaining the activity of glycogen phosphorylase and pyruvate kinase, and glycogenolysis/glycolysis in postmortem muscle. Thus, AMPK has an important role in the control of postmortem glycolysis and is crucial for a lower ultimate pH in postmortem muscle. However, the activation of AMPK cannot fully account for the initial rapid glycogenolysis/glycolysis induced by stress and another mechanism must exist for the accelerated glycolysis induced by pre-slaughter stress. Copyright © 2005 Society of Chemical Industry

Changes in some liver enzymes in streptozotocin-induced diabetic rats fed sapogenin extract from bitter yam (Dioscorea polygonoides) or commercial diosgenin.

Abstract: The effects of steroidal sapogenin extract from bitter yam or commercial diosgenin on liver enzyme changes were investigated. Diabetic male Wistar rats were fed diets supplemented with 1% steroidal sapogenin extract or commercial diosgenin for three weeks. Plasma glucose levels and the activities of hepatic glucose-6-phosphatase, pyruvate kinase and glucose-6-phosphate dehydrogenase were assessed. Liver total cholesterol, HDL-cholesterol and total phospholipid were also measured. Plasma glucose decreased significantly (p < 0.05) in diabetic rats fed the three test diets compared to the diabetic control. The three test diets significantly decreased glucose-6-phosphatase activity compared to the diabetic control. The activities of ATP-citrate lyase, pyruvate kinase and glucose-6-phosphate dehydrogenase were significantly reduced in the liver of diabetic rats compared to normal control. Supplementation of the diet with bitter yam steroidal sapogenin extract or commercial diosgenin did not significantly alter ATP citrate lyase and pyruvate kinase activities but significantly increased glucose6-phosphate dehydrogenase activity in the liver compared to diabetic rats. This study shows that the feeding of the two test diets to diabetic rats results in alterations in the metabolism of glucose with subsequent reduction in plasma glucose concentration.

Glycolysis and Perinatal Hypoxic-Ischemic Brain Damage

Abstract: To ascertain the regulation of glycolysis during perinatal hypoxia-ischemia, 7-day postnatal rats were subjected to unilateral common carotid artery ligation followed by hypoxia with 8% oxygen for up to 90 min. Brain concentrations of glucose, lactate, and key glycolytic intermediates were determined at specific intervals of hypoxia. During hypoxia-ischemia, anaerobic glycolysis increased to approximately 62% of its maximal capacity, which equates to a 135% stimulation of the glycolytic flux. The key regulatory enzymes, hexokinase, phosphofructokinase and pyruvate kinase, were all stimulated during hypoxia-ischemia, and there were no enzymatic rate limitations. The major rate-limiting step for glycolysis was the transport of glucose across the blood-brain barrier into the brain.

Cytosolic pyruvate kinase: subunit composition, activity, and amount in developing castor and soybean seeds, and biochemical characterization of the purified castor seed enzyme

Abstract: Antibodies against Brassica napus cytosolic pyruvate kinase (PKc) (EC 2.7.1.40) were employed to examine PKc subunit composition and developmental profiles in castor and soybean seeds. A 56-kDa immunoreactive polypeptide was uniformly detected on immunoblots of clarified extracts from developing castor endosperm or soybean embryos. Maximal PKc activities occurred early in castor oil seed (COS) and soybean development (7.1 and 5.5 (μmol of pyruvate produced/min) g−1 FW, respectively) and were up to 25-fold greater than those of fully mature seeds. Time-course studies revealed a close correlation between extractable PKc activity and the relative amount of the immunoreactive 56-kDa PKc polypeptide. PKc from developing COS was purified 1,874-fold to homogeneity and a final specific activity of 73.1 (μmol of pyruvate produced/min) mg−1 protein. Gel filtration and SDS-PAGE indicated that this PKc exists as a 230-kDa homotetramer composed of 56-kDa subunits. The mass fingerprint of tryptic peptides of the 56-kDa COS PKc subunit best matched three putative PKcs from Arabidopsis thaliana. The purified enzyme was relatively heat-stable and displayed a broad pH optimum of 6.4. However, more efficient substrate utilization (in terms of Vmax/Km for phosphoenolpyruvate or ADP) was observed at pH 7.4. Glutamate was the most effective inhibitor, whereas aspartate functioned as an activator by partially relieving glutamate inhibition. Together with our previous studies, the results: (1) allow a model to be formulated regarding the coordinate allosteric control of PKc and phosphoenolpyruvate carboxylase by aspartate and glutamate in developing COS, and (2) provide further biochemical evidence that castor plant PKc exists as tissue-specific isozymes that exhibit substantial differences in their respective physical and regulatory properties.

Fecal pyruvate kinase (M2-PK): A new predictor for inflammation and severity of pouchitis

Abstract: A dimeric isoform of pyruvate kinase (TuM2-PK) in stool has been suggested as a useful screening marker for gastrointestinal cancers with 73% sensitivity and 78% specificity [1]. Pyruvate kinase is...

Different involvement for aldolase isoenzymes in kidney glucose metabolism: Aldolase B but not aldolase A colocalizes and forms a complex with FBPase

Abstract: The expression of aldolase A and B isoenzyme transcripts was confirmed by RT-PCR in rat kidney and their cell distribution was compared with characteristic enzymes of the gluconeogenic and glycolytic metabolic pathway: fructose-1,6-bisphosphatase (FBPase), phosphoenol pyruvate carboxykinase (PEPCK), and pyruvate kinase (PK). We detected aldolase A isoenzyme in the thin limb and collecting ducts of the medulla and in the distal tubules and glomerula of the cortex. The same pattern of distribution was found for PK, but not for aldolase B, PEPCK, and FBPase. In addition, co-localization studies confirmed that aldolase B, FBPase, and PEPCK are expressed in the same proximal cells. This segregated cell distribution of aldolase A and B with key glycolytic and gluconeogenic enzymes, respectively, suggests that these aldolase isoenzymes participate in different metabolic pathways. In order to test if FBPase interacts with aldolase B, FBPase was immobilized on agarose and subjected to binding experiments. The results show that only aldolase B is specifically bound to FBPase and that this interaction was specifically disrupted by 60 μM Fru-1,6-P2. These data indicate the presence of a modulated enzyme–enzyme interaction between FBPase and isoenzyme B. They affirm that in kidney, aldolase B specifically participates, along the gluconeogenic pathway and aldolase A in glycolysis. © 2004 Wiley-Liss, Inc.

Engineering of Bacillus subtilis for enhanced total synthesis of folic acid.

Abstract: We investigated whether the yield of the B vitamin folic acid could be elevated in Bacillus subtilis. Strategies for increasing the folic acid yield were investigated by employing computer-aided flux analysis and mutation. Controlling the activity of the enzyme pyruvate kinase by placing it under inducible control was one strategy devised to elevate yield while insuring that a rapid growth rate results. Other single mutation strategies included amplifying the expression of the genes in the folate operon and overexpressing the Escherichia coli aroH gene, which encodes 2-dehydro-3-deoxyphosphoheptonate aldolase. The latter could conceivably elevate the abundance of the folic acid precursor, para-aminobenzoic acid. Strains that combined two or more mutations were also constructed. Overall, a strain possessing inducible pyruvate kinase, overexpressed aroH, and increased transcription and translation of genes from the folic operon exhibited the best yield. The yield was eightfold higher than that displayed by the parent B. subtilis 168 strain.