Showing papers on "Pyruvate dehydrogenase kinase published in 1999"
TL;DR: It was found that feeding rats WY-14,643, a selective agonist for the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), also induced large increases in pyruvate dehydrogenase kinase activity, PDK4 protein, andPDK4 mRNA in gastrocnemius muscle.
Abstract: Regulation of the activity of the pyruvate dehydrogenase complex in skeletal muscle plays an important role in fuel selection and glucose homeostasis. Activation of the complex promotes disposal of glucose, whereas inactivation conserves substrates for hepatic glucose production. Starvation and diabetes induce a stable increase in pyruvate dehydrogenase kinase activity in skeletal muscle mitochondria that promotes phosphorylation and inactivation of the complex. The present study shows that these metabolic conditions induce a large increase in the expression of PDK4, one of four pyruvate dehydrogenase kinase isoenzymes expressed in mammalian tissues, in the mitochondria of gastrocnemius muscle. Refeeding starved rats and insulin treatment of diabetic rats decreased pyruvate dehydrogenase kinase activity and also reversed the increase in PDK4 protein in gastrocnemius muscle mitochondria. Starvation and diabetes also increased the abundance of PDK4 mRNA in gastrocnemius muscle, and refeeding and insulin treatment again reversed the effects of starvation and diabetes. These findings suggest that an increase in amount of this enzyme contributes to hyperphosphorylation and inactivation of the pyruvate dehydrogenase complex in these metabolic conditions. It was further found that feeding rats WY-14,643, a selective agonist for the peroxisome proliferator-activated receptor-alpha (PPAR-alpha), also induced large increases in pyruvate dehydrogenase kinase activity, PDK4 protein, and PDK4 mRNA in gastrocnemius muscle. Since long-chain fatty acids activate PPAR-alpha endogenously, increased levels of these compounds in starvation and diabetes may signal increased expression of PDK4 in skeletal muscle.
290 citations
TL;DR: Identification and characterization of these glucose‐catabolizing enzymes indicate that chlamydia contains the functional capacity to produce its own ATP and reducing power.
Abstract: Chlamydia trachomatis is an obligate intracellular eubacteria that is dependent on a eukaryotic host cell for a variety of metabolites. For years, it has been speculated that chlamydiae are energy parasites, totally dependent on their host cell for ATP and other high-energy intermediates. To determine whether C. trachomatis contains functional enzymes that produce energy or reducing power, four enzymes involved in glycolysis or the pentose phosphate pathway, specifically pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase, were cloned, sequenced and expressed as recombinant proteins in Escherichia coli. The deduced amino acid sequences obtained show high homology to other pyruvate kinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphate dehydrogenase enzymes. In contrast to numerous other bacterial species, chlamydial glycolytic genes are not arranged in an operon, but are dispersed throughout the genome. Results from reverse transcriptase-polymerase chain reaction (RT-PCR) analysis indicate that all four genes are maximally expressed in the middle of the chlamydial developmental cycle. The chlamydial genes are capable of complementing mutant E. coli strains lacking the respective enzyme activities. In vitro enzyme analysis indicates that recombinant chlamydial enzymes expressed in E. coli are active and, interestingly, recombinant chlamydial pyruvate kinase is not regulated allosterically by fructose 1,6 bisphosphate or AMP, as found with other bacterial pyruvate kinases. In summary, identification and characterization of these glucose-catabolizing enzymes indicate that chlamydia contains the functional capacity to produce its own ATP and reducing power.
100 citations
TL;DR: Modification of GAPDH activity provokes a shift from mixed-acid to homolactic metabolism, confirming the important role of this enzyme in controlling both the flux through glycolysis and the orientation of pyruvate catabolism.
76 citations
TL;DR: The codisruption of PDA1 andALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.
71 citations
TL;DR: In this article, the effects of pyruvate and COHC on sarcoplasmic reticular Ca2+ handling were examined in guinea-pig heart with 45 Ca-loaded hearts.
Abstract: Pyruvate augmentation of contractile function and cytosolic free energy of ATP hydrolysis in myocardium could result from pyruvate catabolism in the mitochondria or from increased ratio of the cytosolic NAD+/NADH redox couple via the lactate dehydrogenase equilibrium. Objective: To test the hypothesis that cytosolic oxidation by pyruvate is sufficient to increase cardiac function and energetics. Methods: Isolated working guinea-pig hearts received 0.2 mM octanoate±2.5 mM pyruvate as fuels. α-Cyano-3-hydroxycinnamate (COHC, 0.6 mM) was administered to selectively inhibit mitochondrial pyruvate uptake without inhibiting pyruvate’s cytosolic redox effects or octanoate oxidation. The effects of pyruvate and COHC on sarcoplasmic reticular Ca2+ handling were examined in 45Ca-loaded hearts. Results: Pyruvate increased left ventricular stroke work and power 40%, mechanical efficiency 29%, and cytosolic ATP phosphorylation potential nearly fourfold. 14CO2 formation from [1-14C]pyruvate was inhibited 65% by COHC, and octanoate oxidation, i.e. 14CO2 formation from [1-14C]octanoate, concomitantly increased threefold. COHC prevented pyruvate enhancement of left ventricular function, mechanical efficiency and cytosolic phosphorylation potential, but did not alter respective levels in pyruvate-free control hearts and augmented cytosolic oxidation by pyruvate. Pyruvate increased sarcoplasmic reticular Ca2+ turnover, i.e. Ca2+ uptake and release, as indicated by 62% decrease in caffeine-induced 45Ca release following 40 min 45Ca washout ( P <0.01). In presence of COHC, pyruvate did not lower caffeine-induced 45Ca release; thus, COHC abrogated pyruvate enhancement of Ca2+ turnover ( P <0.001). Conclusion: Pyruvate oxidation of cytosolic redox state is not sufficient to increase cardiac function, cytosolic energetics and sarcoplasmic reticular Ca2+ turnover when mitochondrial pyruvate transport is disabled; thus, mitochondrial metabolism of pyruvate is essential for its metabolic inotropism.
69 citations
TL;DR: Activities of enzymes related to glucose metabolism were measured in canine and feline liver and activities of rate limiting enzymes of gluconeogenesis such as pyruvate carboxylase, fructose-1, 6-bisph phosphatase and glucose-6-phosphatase in feline livers were significantly higher than those in canine livers.
60 citations
TL;DR: Cloning of a pyruvate dehydrogenase kinase cDNA from Arabidopsis thaliana is described and the effects of antisense down-regulation of its expression on plant growth and development are focused on and the potential role for AtPDHK gene manipulation in crop improvement is discussed.
Abstract: Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1α mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed.
58 citations
TL;DR: 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.
55 citations
TL;DR: The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate is suggested to be a general method for the removal of substrate inhibition in L-LDH enzymes.
Abstract: The mutation S163L in human heart lactate dehydrogenase removes substrate inhibition while only modestly reducing the turnover rate for pyruvate. Since this is the third enzyme to show this behaviour, we suggest that the S163L mutation is a general method for the removal of substrate inhibition in L-LDH enzymes. Engineering such enzymatic properties has clear industrial applications in the use of these enzymes to produce enantiomerically pure a-hydroxy acids. The mutation leads to two principal effects. (1) Substrate inhibition is caused by the formation of a covalent adduct between pyruvate and the oxidized form of the cofactor. The inability of S163L mutants to catalyse the formation of this inhibitory adduct is demonstrated. However, NMR experiments show that the orientation of the nicotinamide ring in the mutant NAD F binary complex is not perturbed. (2) The mutation also leads to a large increase in the KM for pyruvate. The kinetic and binding properties of S163L LDH mutants are accounted for by a mechanism which invokes a non-productive, bound form of the cofactor. Molecular modelling suggests a structure for this non-productive enzyme‐NADH complex.
55 citations
54 citations
TL;DR: Results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus of Asn-247Ala, Asp-282Ala and Gly-286Ala.
Abstract: In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent V(max) values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent K(m) value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 microM). In contrast, Gly-284Ala had only a slightly higher K(m) value than the wild-type enzyme (28 versus 16 microM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The K(d) value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 microM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].
TL;DR: The glucose and lactate metabolism of Actinomyces naeslundii and its ecological significance in dental plaque is discussed and it is suggested that this bacterium may modify the dental plaque environment and promote the microbial population shifts inental plaque.
Abstract: Actinomyces are among the predominant bacteria in the oral microflora This review discusses the glucose and lactate metabolism of Actinomyces naeslundii and its ecological significance in dental plaque This bacterium has the Embden-Meyerhof-Parnas (EMP) pathway as the main route to degrade glucose The EMP pathway-derived metabolic intermediates, phosphoenolpyruvate (PEP) and pyruvate, are further converted into different end-products, depending on the environment Under anaerobic conditions in the absence of bicarbonate, the pyruvate is converted into lactate by a lactate dehydrogenase In the presence of bicarbonate, the PEP is combined with bicarbonate and then converted into succinate through the succinate pathway, while the pyruvate is converted into formate and acetate through the pyruvate formate-lyase pathway Under aerobic conditions, the pyruvate liberates acetate and CO2 through a pathway initiated by a pyruvate dehydrogenase A naeslundii strains also degrade lactate, aerobically, to acetate and CO2 through the conversion of lactate into pyruvate by a NAD-independent lactate dehydrogenase These strains also synthesize glycogen from a glycolytic intermediate, glucose 6-phosphate Besides atmospheric conditions and bicarbonate, the intracellular reduction-oxidation potential, carbohydrate concentration, and environmental pH also modulate the metabolism of A naeslundii Some of the phosphorylating enzymes involved in A naeslundii metabolism--eg, GTP/polyphosphate (PPn)-dependent glucokinase, pyrophosphate (PPi)-dependent phosphofructokinase, UDP-glucose pyrophosphorylase, and GDP/IDP-dependent PEP carboxykinase--are unique to A naeslundii and have not been found in other oral bacteria The utilization of PPn and PPi as phosphoryl donors, together with glycogen synthesis and lactate utilization, could contribute to the efficient energy metabolism found in A naeslundii Through this flexible and efficient metabolic capacity, A naeslundii can adapt to fluctuating environments and compete with other bacteria in dental plaque Further, this bacterium may modify the dental plaque environment and promote the microbial population shifts in dental plaque
TL;DR: It is indicated that sepsis induces stable changes in PDHK in skeletal muscle through modification of the activity of the PDHK intrinsic to the PDH and free PDHK.
Abstract: Chronic sepsis promotes a stable increase in pyruvate dehydrogenase kinase (PDHK) activity in skeletal muscle PDHK is found tightly bound to the pyruvate dehydrogenase (PDH) complex and as free kinase We investigated the ability of sepsis to modify the activity of the PDHK intrinsic to the PDH and free PDHK Sepsis was induced by the intraabdominal introduction of a fecal-agar pellet infected with E coli and B fragilis Five days later, mitochondria were isolated from skeletal muscle and PDHK measured in mitochondrial extracts Sepsis caused an approximate 2-fold stimulation of PDHK The mitochondrial extracts from control and septic rats were fractionated by gel chromatography on Sephacryl S-300 to separate PDHK intrinsic to PDH complex and free PDHK PDH complex eluted at void volume and was assayed for PDHK intrinsic to the complex The activity of PDHK intrinsic to PDH complex was a significantly increased 3 fold during sepsis Free PDHK activity eluted after the PDH complex and its activity was enhanced by 70% during sepsis Incubation of PDHK intrinsic to PDH with dichloroactate, an uncompetitive inhibitor of PDHK, showed the PDHK from septic rats relatively less sensitive to inhibition than controls These results indicate that sepsis induces stable changes in PDHK in skeletal muscle
TL;DR: The stimulation of metabolic flux through the reaction catalyzed by the pyruvate dehydrogenase complex appears to result from an increase in intramitochondrial [Ca2+] ions in astrocytes.
Abstract: Noradrenaline effects on the rate of metabolism of pyruvate to acetyl coenzyme A, catalyzed by the pyruvate dehydrogenase complex, was measured in primary cultures of mouse astrocytes as rate of production of labeled CO(2) from 1-[(14) C]pyruvate in the absence of competing glucose in the medium. The subtype specificity of a noradrenaline-stimulated increase in rate of CO(2) formation was identical to that for noradrenaline-induced increase in free intracellular calcium ([Ca(2+)](i)), suggesting a causal relationship between these two phenomena. The noradrenaline-induced stimulation of pyruvate decarboxylation was abolished in the presence of 10 mM magnesium chloride in the medium, combined with the omission of calcium, a procedure known to prevent an increased [Ca(2+)] in the cytosol from raising intramitochondrial [Ca(2+)]. Thus, the stimulation of metabolic flux through the reaction catalyzed by the pyruvate dehydrogenase complex appears to result from an increase in intramitochondrial [Ca(2+)] ions in astrocytes. Such a mechanism for stimulation of the same enzyme has been convincingly demonstrated in other cell types, primarily heart muscle and hepatic cells, but it has not previously been demonstrated in any cell type from the central nervous system.
TL;DR: The oxime 12 and dehydroabietyl amine 2 exhibit a blood glucose lowering effect in the diabetic ob/ob mouse after a single oral dose of 100 micromol/kg, however, the mechanism of the blood glucose lowered effect is likely unrelated to PDK inhibition.
TL;DR: Findings indicate that purified PDC and its catalytic components are affected by lipoic compounds based on their stereoselectivity; and the oxidation of pyruvate by intact HepG2 cells is not inhibited by R-LA, in support of therapeutic role of R- LA as an antioxidant.
TL;DR: Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetelatable protein.
Abstract: The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 M NaCl and 0.1 M glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78-kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2-14C]pyruvate. Affinity-purified antibodies against the 55-kDa protein do not cross-react with the 78-kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55-kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2-14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro-eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetylatable protein.
TL;DR: This study suggests that arginine-349 is critical for E1's activity, methionine-181 is involved in the binding of TPP, and proline-188 is necessary for structural integrity of E1.
TL;DR: Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met, and steady-state fluorescence titration data suggest that the substrate PEP binds to K240M with the same affinity as it does to wild-type YPK.
Abstract: Site-directed mutagenesis was used to change Lys 240 of yeast pyruvate kinase (Lys 269 in muscle PK) to Met. K240M has an absolute requirement for FBP for catalysis. K240M is 100- and 1000-fold les...
TL;DR: A simple photometric assay that uses a cyclic detection system which, due to the sequential action of pyruvate kinase and hexokinase, results in an exponential increase of ADP and glucose 6-phosphate.
TL;DR: The failure to reverse completely the effects of prolonged starvation in suppressing PDHC activity by acute inhibition of FA oxidation suggests additional regulatory mechanisms that dampen the PDHC response to acute changes in substrate supply.
Abstract: The present study evaluated the substrate competition between fatty acids (FA) and glucose in the kidney in vivo in relation to the operation of the "glucose-FA" and "reverse glucose-FA" cycles. In fed rats, neither inhibition of adipocyte lipolysis by 5-methylpyrazole-3-carboxylic acid (MPCA) nor inhibition of mitochondrial long-chain FA oxidation by 2-tetradecylglycidate (TDG) influenced the renal ratio of free/acylated carnitine or the percentage of total renal pyruvate dehydrogenase complex (PDHC) in the active (dephosphorylated) form (PDHa). The additional provision of glucose, a precursor for the synthesis of malonyl-coenzyme A (coA), did not influence renal PDHa activity or the renal ratio of free to acylated carnitine, implying that FA oxidation is maximally suppressed in the fed state. A reverse glucose-FA cycle may therefore be important in suppressing renal FA oxidation in the fed state. After 48 hours of starvation, MPCA and TDG decreased short- and long-chain acylcarnitine concentrations (40% to 50%, P < .01) and elevated the renal ratio of free/acylated carnitine (2.5-fold, P < .001, and 3.3-fold, P < .001, respectively), indicating that FA oxidation is increased after starvation. Despite suppression of renal FA oxidation, renal PDHa activity in 48-hour starved rats was only partially restored by treatment with MPCA or TDG. The additional administration of glucose did not remedy this. The failure to reverse completely the effects of prolonged starvation in suppressing PDHC activity by acute inhibition of FA oxidation suggests additional regulatory mechanisms that dampen the PDHC response to acute changes in substrate supply. Estimations of PDH kinase (PDK) activity in renal mitochondria showed a significant 1.7-fold stable increase (P < .01) after 48 hours of starvation. Analysis of PDK pyruvate sensitivity in renal mitochondria incubated with respiratory substrate (5 mmol/L 2-oxoglutarate/0.5 mmol/L L-malate) showed that the pyruvate concentration required for 50% activation was substantially decreased by starvation. Enzyme-linked immunosorbent assay (ELISA) analysis over a range of PDHC activities demonstrated that increased PDK activity was concomitant with a significant (at least P < .01) 1.8-fold increase in the protein expression of the ubiquitously expressed PDK isoform, PDK2. We hypothesize that changes in protein expression and activity of individual PDK isoforms may dictate the renal response to incoming FA lesterification v oxidation) through modulation of the relationship between glycolytic flux and PDHC activity, and thus the provision of precursor for malonyl-coA production.
TL;DR: Results indicate that alphaC62 and betaW135 facilitate coenzyme binding, andalphaC62 could be near the substrate-binding site in E1 catalysis, and pyruvate induced inactivation of human E1 could be restored by thiol reagents.
TL;DR: Functional studies of the promoter indicate the presence of both enhancer and repressor elements that are common to other genes that are only expressed in mature sperm.
TL;DR: A mouse homologue cDNA of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) with differential mRNA display as an up‐regulated gene in the hypertrophied ventricles of juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency was isolated.
Abstract: We isolated a mouse homologue cDNA of pyruvate dehydrogenase (PDH) kinase 4 (PDK4) with differential mRNA display as an up-regulated gene in the hypertrophied ventricles of juvenile visceral steatosis (JVS) mice with systemic carnitine deficiency. The PDK4 mRNA level was 5 times higher in JVS mice than in control mice under fed conditions. After 24 h starvation, this level increased to 20 times in JVS and 7 times in control, compared with the control fed level. On the other hand, carnitine administration reduced the high level of PDK4 mRNA in JVS mice to the control fed level. In control mice, the change in PDK4 mRNA was inversely correlated with the change in PDH activity. In JVS mice, however, the PDK4 mRNA level was not always correlated with the active-form PDH level.
TL;DR: The results confirm the limited role of renal gluconeogenesis during the perinatal development of rat liver and kidney and indicate an early development of the holocarboxylase synthetase activity in theperinatal period.
Abstract: The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period
TL;DR: The higher the NH4Cl provided, the lower the oxidation rate of state 3 with pyruvate in liver mitochondria, and the actual activity of pyruVate dehydrogenase complex, as expressed as units of produced CO2 per g wet weight of liver, which were accompanied by the lower pH in blood.
TL;DR: The results suggest that the C-terminus of the PDK is not required for subunit association of the homodimer or catalysis, but instead seems to be involved in the binding of thePDKs to the dihydrolipoyl transacetylase core of the complex.
Abstract: Pyruvate dehydrogenase kinases (PDKs) from the anaerobic parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans were functionally expressed with hexahistidine tags at their N-termini and purified to apparent homogeneity. Both recombinant PDKs (rPDKs) were dimers, were not autophosphorylated and exhibited similar specific activities with the A. suum pyruvate dehydrogenase (E1) as substrate. In addition, the activities of both PDKs were activated by incubation with PDK-depleted A. suum muscle pyruvate dehydrogenase complex (PDC) and were stimulated by NADH and acetyl-CoA. However, the recombinant A. suum PDK (rAPDK) required higher NADH/NAD+ ratios for half-maximal stimulation than the recombinant C. elegans PDK (rCPDK) or values reported for mammalian PDKs, as might be predicted by the more reduced microaerobic mitochondrial environment of the APDK. Limited tryptic digestion of both rPDKs yielded stable fragments truncated at the C-termini (trPDKs). The trPDKs retained their dimeric structure and exhibited substantial PDK activity with the A. suum E1 as substrate, but PDK activity was not activated by incubation with PDK-depleted A. suum PDC or stimulated by elevated NADH/NAD+ or acetyl-CoA/CoA ratios. Direct-binding assays demonstrated that increasing amounts of rCPDK bound to the A. suum PDK-depleted PDC. No additional rCPDK binding was observed at ratios greater than 20 mol of rCPDK/mol of PDC. In contrast, the truncated rCPDK (trCPDK) did not exhibit significant binding to the PDC. Similarly, a truncated form of rCPDK, rCPDK1-334, generated by mutagenesis, exhibited properties similar to those observed for trCPDK. These results suggest that the C-terminus of the PDK is not required for subunit association of the homodimer or catalysis, but instead seems to be involved in the binding of the PDKs to the dihydrolipoyl transacetylase core of the complex.
TL;DR: A microplate assay for mevalonate and 5-phosphomevalonates kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase and showed Michaelis-Menten kinetics with respect to ATP and their specific substrates.
TL;DR: Results indicate that acetyl-CoA for the TCA cycle is produced effectively in the tissues of the termite itself, both from pyruvate by the pyruVate dehydrogenase complex and from acetate by acetyl -CoA synthetase.
TL;DR: Evidence suggests that TNF functions as a proximal mediator in sepsis based on the development of hyperlactatemia in the absence of overt signs of hypoperfusion and changes in lactate concentrations may reflect altered regulation of glucose metabolism.
Abstract: Sepsis shifts the dynamic metabolic interactions involved in the regulation of inter-organ substrate fluxes, and ultimately results in derangements in the plasma concentrations of metabolites. One manifestation of the septic process is the development of hyperlactatemia in the absence of overt signs of hypoperfusion. Lactate is the end-product of glycolysis. Consequently, changes in lactate concentrations during sepsis may reflect altered regulation of glucose metabolism. In skeletal muscle, the PDH complex is the link between the glycolytic pathway and oxidative tricarboxylic acid cycle activity in the metabolism of glucose. In sepsis, production of pyruvate via glycolysis is accelerated in skeletal muscle. Because the proportion of the PDH complex in the active, nonphosphorylated form is diminished during sepsis, pyruvate is preferentially converted to lactate, and subsequently released into the circulation. The inhibition of PDH activity resides in a stimulation of the PDH kinase during sepsis. Although the factors responsible for lactate metabolism dyshomeostasis are unknown, evidence suggests that TNF functions as a proximal mediator.