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PubMed1980

Interaction between the pyruvate dehydrogenase complex and citrate synthase.

Kinetic studies of the individual reaction of pig heart pyruvate dehydrogenase complex (pyruvate dehydrogenase (pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1); dihydrolipoamide reductase(NAD+) (NADH:lipoamide oxidoreductase, EC 1.6.4.3); dihydrolipoamide acetyltransferase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12)), citrate synthase (citrate oxaloacetate-lyase (pro-3S-CH2COO- leads to acetyl-CoA), EC 4.1.3.7) and the pyruvate dehydrogenase complex-citrate synthase coupled system show that the KmCoA value of pyruvate dehydrogenase complex and KmCoASAc value of citrate synthase decrease in the coupled system when compared to those in the individual enzyme reactions. The explanation for this interaction may be an association between the two enzymes. When it was centrifuged with 150 000 x g for 140 min, 30% of the citrate synthase sedimented in the presence of the pyruvate dehydrogenase complex, while no sedimentation was observed in the absence of the pyruvate dehydrogenase complex. Sedimentation of cytoplasmic malate dehydrogenase, phosphotransacetylase, hemoglobin and Blue albumin were negligible under the same condition. In gel chromatography experiments a significant peak of citrate synthase activity co-migrated with the pyruvate dehydrogenase complex peak. This observation also suggests the possible association of two enzymes.

Animals↗
PubMed1999

A novel factor binding to the glucose response elements of liver pyruvate kinase and fatty acid synthase genes.

Transcription of the liver type pyruvate kinase and lipogenesis enzyme genes is induced by high carbohydrate in liver. We have found a novel protein factor in rat liver nuclei that binds to the glucose response element (CACGTG motifs) of the pyruvate kinase gene (Liu, Z. , Thompson, K. S., and Towle, H. C. (1993) J. Biol. Chem. 268, 12787-12795) and the "insulin response element" of fatty acid synthase gene. The amounts of this DNA-binding protein, termed "glucose response element binding protein" (GRBP) in the nuclear extract, were increased in liver by a high carbohydrate diet and decreased by starvation, high fat, and high protein diet. GRBP also occurs in cytosols of liver and is dependent on carbohydrate. Both the nuclear and the cytosolic GRBP showed similar properties, except the former was more resistant to thermal inactivation than the latter. Kinetics of glucose activation of the cytosolic GRBP in a primary culture of hepatocytes indicated that a half-maximum activation was achieved after 6 h, and glucose concentration required for the maximum activation of the GRBP was approximately 12 mM. Dibutyryl-cAMP, okadaic acid, and forskolin inhibited glucose activation of both GRBP and liver pyruvate kinase transcription. These results suggested that GRBP may be a factor that recognizes the glucose response motif site and may be involved in mediating carbohydrate response of the pyruvate kinase gene.

Animals↗
PubMed1987

Effects of insulin infusion on human skeletal muscle pyruvate dehydrogenase, phosphofructokinase, and glycogen synthase. Evidence for their role in oxidative and nonoxidative glucose metabolism.

To determine whether activation by insulin of glycogen synthase (GS), phosphofructokinase (PFK), or pyruvate dehydrogenase (PDH) in skeletal muscle regulates intracellular glucose metabolism, subjects were studied basally and during euglycemic insulin infusions of 12, 30, and 240 mU/m2 X min. Glucose disposal, oxidative and nonoxidative glucose metabolism were determined. GS, PFK, and PDH were assayed in skeletal muscle under each condition. Glucose disposal rates were 2.37 +/- 0.11, 3.15 +/- 0.19, 6.71 +/- 0.44, and 11.7 +/- 1.73 mg/kg X min; glucose oxidation rates were 1.96 +/- 0.18, 2.81 +/- 0.28, 4.43 +/- 0.32, and 5.22 +/- 0.52. Nonoxidative glucose metabolism was 0.39 +/- 0.13, 0.34 +/- 0.26, 2.28 +/- 0.40, and 6.52 +/- 1.21 mg/kg X min. Both the proportion of active GS and the proportion of active PDH were increased by hyperinsulinemia. PFK activity was unaffected. Activation of GS was correlated with nonoxidative glucose metabolism, while activation of PDH was correlated with glucose oxidation. Sensitivity to insulin of GS was similar to that of nonoxidative glucose metabolism, while the sensitivity to insulin of PDH was similar to that of glucose oxidation. Therefore, the activation of these enzymes in muscle may regulate nonoxidative and oxidative glucose metabolism.

Glucose↗
PubMed1989

Pyruvate dehydrogenase and glycogen synthase activity at transition from fasted to fed state.

The aim of the present study was to evaluate whether the PDC and GS activities at the transition from fasted into fed state are consistent with indirect pathway for glycogen synthesis, as suggested previously. Refeeding of glucose given to rats after 72 hr of starvation did not reactivate PDC in the liver; however, the PDC activity in the muscle was increased. In comparison to PDC, glucose refeeding leads to an opposite effect on GS in both liver and muscle as evidenced by the immediate increase in the active form of GS. The low activity of liver PDC restricts 3-carbon flux through the Krebs cycle and enables their transfer to the gluconeogenic pathway for glycogen synthesis. In contrast, an immediate activation of muscle PDC following refeeding indicates that 3-carbon flux will be oxidized in the citric acid cycle, which thereby eliminates the indirect pathway for glycogen synthesis in this tissue. Glucose infusion increased plasma lactate, insulin, and glycogen content in the liver and muscle to the same extent as observed in the fed rats. The results are in agreement with the suggestion that at the transition from fasted to fed state, liver glycogen synthesis occurs mainly from 3-carbon precursors.

Animals↗
PubMed1976

Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms.

The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 1.2.7.1 and EC 1.2.7.2) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively.

Acetoin↗
PubMedDate not supplied

Phylogenetic analysis of the putative phosphorylation domain in the pyruvate kinase of Bacillus stearothermophilus.

All sequenced phosphoenolpyruvate synthases (PPS), pyruvate:phosphate dikinases (PPDK) and enzymes I (EI) of the phosphoenolpyruvate:sugar phosphotransferase system comprise the PEP family. Linked to the C terminus of the sequenced pyruvate kinase from Bacillus stearothermophilus (PKBst) is a domain that is homologous to the putative phosphorylation domains of PEP family enzymes. We report sequence and phylogenetic analyses that lead to the following conclusions: (1) the phosphorylation domain of PKBst was derived from a PPS, late in the evolutionary process, after the divergence of PPSs from PPDKs and EIs; (2) this domain is probably functional in phosphoryl transfer; (3) the C-terminal phosphorylation domain in PKBst probably defines a compact domain in all PEP family proteins that is linked to other domains in these proteins via flexible linkers.

Amino Acid Sequence↗
PubMed1987

"Stable" effects of insulin and isoproterenol on adipocyte pyruvate dehydrogenase.

Insulin, at a concentration of 1 mU/ml, stimulated glycogen synthase and pyruvate dehydrogenase about threefold in isolated rat adipocytes. Upon the removal of insulin, glycogen synthase activity remained in the activated state for 10 min and thereafter rapidly returned to basal level. On the other hand, insulin-stimulated pyruvate dehydrogenase activity remained elevated for at least 30 min. Isoproterenol (10(-8) M) stimulated phosphorylase and inhibited pyruvate dehydrogenase through the activation of beta-adrenergic receptors. Addition of the beta-antagonist, propranolol (10(-5) M), after isoproterenol reversed the action of isoproterenol on phosphorylase but not its action on pyruvate dehydrogenase. Dibutyryl cyclic AMP, when added to intact adipocytes, produced an effect on pyruvate dehydrogenase similar to that induced by isoproterenol. Our results indicate that both insulin and the beta-agonist have a unique action on pyruvate dehydrogenase which is different from their effects on other enzymes such as glycogen synthase and phosphorylase.

Adipose Tissue↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed1987

Insulin insensitivity and skeletal muscle enzyme activities in response to overinsulinization in the rat.

Periods of overinsulinization with low blood glucose levels are a recognized feature of intensive insulin injection therapy. The relationship of these features to insulin insensitivity is controversial, and the mechanisms underlying any such changes are unclear. Normal rats have therefore been overinsulinized for 6 weeks before measurement of in vivo insulin sensitivity by the glucose clamp technique. Skeletal muscle glycogen synthase and pyruvate dehydrogenase activities were measured at the end of the clamp. Sensitivity to insulin as measured by the glucose clamp technique at euglycemic levels was decreased in the insulin overtreated animals (glucose requirements, 108 +/- 2 mumol/min/kg v 170 +/- 10 mumol/min/kg, P less than 0.001). Total skeletal muscle glycogen synthase activity was increased in the experimental group (2.83 +/- 0.12 v 1.96 +/- 0.14 U/g wet weight, P less than 0.001), and as a result the active fraction was higher at the end of the clamp (0.79 +/- 0.04 v 0.66 +/- 0.04 U/g wet weight, P less than 0.05). Skeletal muscle glycogen content was consistent with the glycogen synthase activity. Pyruvate dehydrogenase in the same tissue showed increased activation prior to the clamp (6.6 +/- 0.6 v 4.7 +/- 0.6%, P less than 0.05), but neither active nor total activity was abnormal at the end of the clamp.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Comparative development of the pyruvate dehydrogenase complex and citrate synthase in rat brain mitochondria.

The enzyme activity of the pyruvate dehydrogenase complex (PDHC) was measured in mitochondria prepared from developing rat brain, before and after steady-state dephosphorylation of the E1 alpha subunit. A marked increase in dephosphorylated (fully activated) PDHC activity occurred between days 10 and 15 post partum, which represented approx. 60% of the difference in fully activated PDHC activity measured in foetal and adult rat brain mitochondria. There was no detectable change in the active proportion of the enzyme during mitochondrial preparation nor any qualitative alteration in the detectable catalytic and regulatory components of the complex, which might account for developmental changes in PDHC activity. The PDHC protein content of developing rat brain mitochondria and homogenates was measured by an enzyme-linked immunoadsorbent assay. The development of PDHC protein in both fractions agreed closely with the development of the PDHC activity. The results suggest that the developmental increase in PDHC activity is due to increased synthesis of PDHC protein, which is partly a consequence of an increase in mitochondrial numbers. However, the marked increase in PDHC activity measured between days 10 and 15 post partum is mainly due to an increase in the amount of PDHC per mitochondrion. The development of citrate synthase enzyme activity and protein was measured in rat brain homogenates and mitochondria. As only a small increase in citrate synthase activity and protein was detected in mitochondria between days 10 and 15 post partum, the marked increase in PDHC protein and enzyme activity may represent specific PDHC synthesis. As several indicators of acquired neurological competence become apparent during this period, it is proposed that preferential synthesis of PDHC may be crucial to this process. The results are discussed with respect to the possible roles played by PDHC in changes of respiratory-substrate utilization and the acquisition of neurological competence occurring during the development of the brain of a non-precocial species such as the rat.

Animals↗
PubMed1992

Inactivation of 5-enolpyruvylshikimate 3-phosphate synthase by its substrate analogue pyruvate in the presence of sodium cyanoborohydride.

Incubation of Escherichia coli 5-enolpyruvylshikimate 3-phosphate synthase with its substrate analogue pyruvate in the presence of sodium cyanoborohydride resulted in a significant decrease in its enzyme activity. The inactivation followed pseudo-first order and saturation kinetics with a Kinact of 16.12 mM and a maximum rate of constant of 0.046 min-1. The inactivation was specifically prevented by preincubation of the enzyme with a combination of the substrate shikimate 3-phosphate plus competitive inhibitor glyphosate. Upon 90% inactivation, approximately 1 mole of [14C]-label from [14C]-pyruvate was incorporated per mole of enzyme. Tryptic mapping of the modified enzyme as well as analysis of an isolated radioactive peptide indicated that Lys22 was the modified site. The results suggested that pyruvate inactivated the enzyme by forming a Schiff-base with Lys22 at the enzyme's active site.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2001

Mitochondrial biogenesis during germination in maize embryos.

Mitochondrial biogenesis and metabolism were investigated during maize (Zea mays) seed germination. Mitochondria from dry and imbibed seed exhibited NADH-dependent O(2) uptake that was completely inhibited by KCN and antimycin A. Mitochondria in the dry seed had a lower rate of succinate-dependent O(2) uptake relative to that measured in imbibed and germinated seed. The activities of the tricarboxylic acid (TCA) cycle enzymes, pyruvate dehydrogenase complex, 2-oxoglutarate dehydrogenase complex, NAD-malic enzyme, and citrate synthase, are similarly low in mitochondria from dry seed and this correlates with a lower relative abundance of the mitochondrial matrix-located citrate synthase and pyruvate dehydrogenase complex E1alpha-subunit polypeptides. Electron microscopy revealed that mitochondria in the dry seed have a poorly developed internal membrane structure with few cristae; following 24 h of germination the mitochondria developed a more normal structure with more developed cristae. The mitochondria from maize embryos could be fractionated into two subpopulations by Suc density gradient centrifugation: one subpopulation of buoyant density equivalent to 22% to 28% (w/w) Suc; the other equivalent to 37% to 42% (w/w) Suc. These two subpopulations had different activities of specific mitochondrial enzymes and contained different amounts of specific mitochondrial proteins as revealed by western-blot analysis. Both subpopulations from the dry embryo were comprised of poorly developed mitochondria. However, during imbibition mitochondria in the heavy fraction (37%-42% [w/w] Suc) progressively acquired characteristics of fully functional mitochondria found in the germinated seedling in terms of structure, enzymic activity, and protein complement. In contrast, mitochondria in the light fraction (22% to 28% [w/w] Suc) show no significant structural change during imbibition and the amounts of specific mitochondrial proteins decreased significantly during germination.

Gene Expression Regulation, Developmental↗
PubMed2004

Ethyl pyruvate inhibits nuclear factor-kappaB-dependent signaling by directly targeting p65.

Ethyl pyruvate has been shown to have anti-inflammatory properties in numerous cell culture and animal studies. In this series of experiments, we tested the hypothesis that ethyl pyruvate inhibits signaling by the pro-inflammatory transcription factor, NF-kappaB. Ethyl pyruvate inhibited luciferase expression in lipopolysaccharide-stimulated murine macrophage-like RAW 264.7 cells transfected with an NF-kappaB-dependent luciferase reporter vector. Ethyl pyruvate also decreased NF-kappaB DNA-binding activity in lipopolysaccharide-stimulated RAW 264.7 cells and decreased lipopolysaccharide-induced expression of an NF-kappaB-dependent gene, inducible nitric oxide synthase. Ethyl pyruvate had no effect on the degradation of IkappaBalpha or IkappaBbeta in lipopolysaccharide-stimulated RAW 264.7 cells, suggesting that ethyl pyruvate acts distally to this step in the activation of NF-kappaB. In a cell-free system, binding of p50 homodimers to an NF-kappaB consensus oligonucleotide sequence was unaffected by ethyl pyruvate over a wide range of concentrations, indicating that ethyl pyruvate probably does not modify or interact with the p50 subunit of NF-kappaB. In contrast, ethyl pyruvate inhibited DNA binding by ectopically overexpressed wild-type p65 homodimers. However, ethyl pyruvate failed to inhibit the DNA-binding activity of homodimers of an overexpressed mutant form of a p65 with substitution of serine for cysteine 38. Taken together, these results suggest that ethyl pyruvate inhibits DNA-binding by covalently modifying p65 at Cys(38). We conclude that some of the beneficial anti-inflammatory effects of ethyl pyruvate may be due to modification of p65, thereby inhibiting signaling via the NF-kappaB pathway.

Animals↗
PubMed2002

Glucose deprivation decreases nitric oxide production via NADPH depletion in immunostimulated rat primary astrocytes.

We have previously reported that the production of nitric oxide (NO) in immunostimulated astrocytes was markedly decreased under glucose-deprived conditions. The present study was undertaken to find the contributing factor(s) for the decreased NO production in glucose-deprived immunostimulated astrocytes. NO production in rat primary astrocytes was stimulated for 24-48 h by cotreatment with lipopolysaccharides (1 microg/ml) and interferon-gamma (100 U/ml). Decreased NO production in immunostimulated astrocytes by glucose deprivation was mimicked by the glycolytic inhibitor 2-deoxyglucose and reversed by addition of pyruvate and lactate. Glucose deprivation did not alter the expression of inducible nitric oxide synthase (iNOS) in immunostimulated astrocytes. Addition of beta-NADPH, but not tetrahydrobiopterine, both of which are essential cofactors for NOS function, completely restored the NO production that was decreased in glucose-deprived immunostimulated astrocytes. Glucose deprivation and immunostimulation synergistically reduced intracellular NADPH level in astrocytes. The results indicate that glucose deprivation decreases NO production in immunostimulated astrocytes by depleting intracellular NADPH, a cofactor of iNOS.

Adjuvants, Immunologic↗
PubMed2001

Interaction of the herbicide glyphosate with its target enzyme 5-enolpyruvylshikimate 3-phosphate synthase in atomic detail.

Biosynthesis of aromatic amino acids in plants, many bacteria, and microbes relies on the enzyme 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, a prime target for drugs and herbicides. We have identified the interaction of EPSP synthase with one of its two substrates (shikimate 3-phosphate) and with the widely used herbicide glyphosate by x-ray crystallography. The two-domain enzyme closes on ligand binding, thereby forming the active site in the interdomain cleft. Glyphosate appears to occupy the binding site of the second substrate of EPSP synthase (phosphoenol pyruvate), mimicking an intermediate state of the ternary enzyme.substrates complex. The elucidation of the active site of EPSP synthase and especially of the binding pattern of glyphosate provides a valuable roadmap for engineering new herbicides and herbicide-resistant crops, as well as new antibiotic and antiparasitic drugs.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed1995

Insulin action and glucose metabolism in nondiabetic control and NIDDM subjects. Comparison using human skeletal muscle cell cultures.

Myoblasts from human skeletal muscle were isolated from needle biopsy samples of vastus lateralis and fused to differentiated multinucleated myotubes. Specific high-affinity insulin and insulin-like growth factor I (IGF-I) binding, glucose transporter proteins GLUT1 and GLUT4, glycogen synthase and pyruvate dehydrogenase proteins, and their specific mRNAs were identified in fused myotubes. Insulin and IGF-I stimulated 2-deoxyglucose uptake twofold with half-maximal stimulation by insulin at 0.98 +/- 0.12 nmol/l and maximal stimulation at 17.5 nmol/l. Acute insulin treatment (33 nmol/l) doubled glycogen synthase activity and glucose incorporation into glycogen while increasing pyruvate dehydrogenase approximately 30%. In cells cultured from NIDDM subjects, both basal (6.9 +/- 1.0 vs. 13.0 +/- 1.7 pmol.mg protein-1.min-1) and acute insulin-stimulated transport (13.5 +/- 2.0 vs. 22.4 +/- 1.3 pmol.mg protein-1.min-1) were significantly reduced compared with nondiabetic control subjects (both P < or = 0.005). GLUT1 protein content of total membranes from NIDDM subjects was decreased compared with control subjects, while GLUT4 levels were similar between groups. A significant correlation (r = 0.65, P < or = 0.05) was present when maximal rates of insulin-stimulated glucose transport in cell culture from subjects were compared with their corresponding in vivo glucose disposal determined by hyperinsulinemic glucose clamp. In summary, differentiated human skeletal muscle cultures exhibit biochemical and molecular features of insulin-stimulated glucose transport and intracellular enzyme activity comparable with the in vivo situation. Defective insulin-stimulated glucose transport persists in muscle cultures from NIDDM subjects and resembles the reduced insulin-mediated glucose uptake present in vivo. We conclude that this technique provides a relevant cellular model to study insulin action and glucose metabolism in normal subjects and determine the mechanisms of insulin resistance in NIDDM.

Adult↗
PubMed1992

Isolation, characterization, and physiological role of the pyruvate dehydrogenase complex and alpha-acetolactate synthase of Lactococcus lactis subsp. lactis bv. diacetylactis.

The pyruvate dehydrogenase complex of Lactococcus lactis subsp. lactis bv. diacetylactis has a specific activity of 6.6 U/mg and a Km of 1 mM for pyruvate. The specific activities of E2 and E3 in the complex are 30 and 0.36 U/mg, respectively. The complex is very sensitive to NADH inhibition and consists of four subunits: E1 alpha (44 kDa), E1 beta (35 kDa), E2 (73 kDa), and E3 (60 kDa). The L. lactis alpha-acetolactate synthase has a specific activity of 103 U/mg and a Km of 50 mM for pyruvate. Thiamine pyrophosphate (Km = 3.2 microM) and divalent cations are essential for activity. The native enzyme measures 172 kDa and consists of 62-kDa monomers. The role of both enzymes in product formation is discussed in view of NADH inhibition and competition for pyruvate.

Acetolactate Synthase↗