Showing papers on "Pyruvate dehydrogenase kinase published in 1981"
TL;DR: The results demonstrate the inheritance nature of PDC deficiency, and suggest that activation is due to dephosphorylation, and that the present assay is sufficient to detect the heterozygous carriers of the deficiency.
Abstract: Pyruvate dehydrogenase complex (PDC) activity in human skin fibroblasts appears to be regulated by a phosphorylation-dephosphorylation mechanism, as is the case with other animal cells. The enzyme can be activated by pretreating the cells with dichloroacetate (DCA), an inhibitor of pyruvate dehydrogenase kinase, before they are disrupted for measurement of PDC activity. With such treatment, the activity reaches 5-6 nmol/min per mg of protein at 37 degrees C with fibroblasts from infants. Such values represent an activation of about 5-20-fold over those observed with untreated cells. That this assay, based on [1-(14)C]pyruvate decarboxylation, represents a valid measurement of the overall PDC reaction is shown by the dependence of (14)CO(2) production on the presence of thiamin-PP, coenzyme A (CoA), Mg(++), and NAD(+). Also, it has been shown that acetyl-CoA and (14)CO(2) are formed in a 1:1 ratio. A similar degree of activation of PDC can also be achieved by adding purified pyruvate dehydrogenase phosphatase and high concentrations of Mg(++) and Ca(++), or in some cases by adding the metal ions alone to the cell homogenate after disruption. These results strongly suggest that activation is due to dephosphorylation. Addition of NaF, which inhibits dephosphorylation, leads to almost complete loss of PDC activity. Assays of completely activated PDC were performed on two cell lines originating from patients reported to be deficient in this enzyme (Blass, J. P., J. Avigan, and B. W. Ublendorf. 1970. J. Clin. Invest. 49: 423-432; Blass, J. P., J. D. Schuman, D. S. Young, and E. Ham. 1972. J. Clin. Invest. 51: 1545-1551). Even after activation with DCA, fibroblasts from the patients showed values of only 0.1 and 0.3 nmol/min per mg of protein. A familial study of one of these patients showed that both parents exhibited activity in fully activated cells about half that of normal values, whereas cells from a sibling appeared normal. These results demonstrate the inheritance nature of PDC deficiency, and that the present assay is sufficient to detect the heterozygous carriers of the deficiency. Application of the same procedures to fibroblasts obtained from 16 individuals who were believed to have normal PDC activities showed a range from about 2-2.5 nmol/min per mg protein for adults to 5-6 nmol/min per mg protein for cells from infants.
151 citations
148 citations
TL;DR: Dichloroacetate has been tried experimentally in treatment of diabetes, hypercholesterolemia, and hyperlactatemia, but it has neurotoxicity, can cause cataracts, and may be mutagenic.
Abstract: Dichloroacetate activates the pyruvate dehydrogenase complex of many tissues by inhibiting the kinase responsible for phosphorylation and inactivation of the complex. Dichloroacetate also activates the myocardial branched-chain α-keto acid dehydrogenase complex but apparently not by direct inhibition of the analogous kinase. Oxalate and glyoxylate, metabolites of dichloroacetate, are responsible for some in vitro effects of dichloroacetate. Dichloroacetate stimulates leucine oxidation by isolated hepatocytes because glyoxylate transaminates with leucine. Dichloroacetate inhibits lactate gluconeogenesis by hepatocytes incubated in low bicarbonate buffer because oxalate inhibits pyruvate carboxylase under such conditions. In vivo, dichloroacetate decreases blood glucose by limiting the supply of gluconeogenic precursors to the liver. This effect is a consequence of pyruvate dehydrogenase activation in peripheral tissues. Dichloroacetate lowers blood cholesterol in hyperlipidemic patients by uncertain means. Dichloroacetate has been tried experimentally in treatment of diabetes, hypercholesterolemia, and hyperlactatemia, but it has neurotoxicity, can cause cataracts, and may be mutagenic.
116 citations
TL;DR: The investigation of the substrate specificity of the pyruvate dehydrogenase complex from Escherichia coli allows a description of the binding region of pyruve, and alpha-Ketobutyrate is the only alpha-keto acid which is able to substitute for pyruVate as a substrate in the overall reaction of the enzyme complex.
114 citations
96 citations
92 citations
TL;DR: Neither in yeast homogenates nor in the isolated yeast pyruvate dehydrogenase complex, was any evidence found for regulation by interconversion (phosphorylation-dephosphorylated) as occurs in mammals, plants, and Neurospora crassa.
Abstract: Pyruvate dehydrogenase complex, for the first time, was highly purified from commercial baker's yeast (saccharomyces cerevisiae). Proteolytic degradation was prevented by the inclusion of the protease inhibitors pepstatin A, leupeptin, and phenylmethanesulfonyl fluoride during the enzyme purification. The yield from 1 kg of pressed yeast was about 15--20 mg enzyme with a specific activity of 17--30 U/mg. Most of the kinetic and regulatory properties of the yeast enzyme were found similar to those of the mammalian mitochondrial pyruvate dehydrogenase complexes except that Km for pyruvate, when assayed at the pH optimum, was much higher than in the mammalian complexes and resembled the values reported for the complexes of gram-negative bacteria. Furthermore, neither in yeast homogenates nor in the isolated yeast pyruvate dehydrogenase complex, was any evidence found for regulation by interconversion (phosphorylation-dephosphorylation) as occurs in mammals, plants, and Neurospora crassa.
81 citations
TL;DR: In vitro protein synthesis experiments using RNA extracted from rat red cells and liver are described which demonstrate that the difference is reflected in tissue-specific mRNAs and is not due to post-translational processing.
Abstract: Pyruvate kinase subunits of red cells and liver differ in their molecular weights. Peptide mapping of the rat enzymes has shown that this difference is due to a single exon peptide present in the red cell enzyme1‐3. There is strong genetic evidence in man that both enzymes are encoded by the same structural gene (refs 4–8 and G. E. J. Staal et al., personal communication). Here we describe in vitro protein synthesis experiments using RNA extracted from rat red cells and liver which demonstrate that the difference is reflected in tissue-specific mRNAs. Thus the difference is not due to post-translational processing and presumably involves either gene rearrangement or differential processing of a common nuclear RNA precursor.
79 citations
TL;DR: Training alters frontal cortex and reduces the phosphate content of pyruvate dehydrogenase in vivo; this leads to enzyme activation; and an increase in back-titration of sites available for phosphorylation in vitro.
Abstract: The active portion of the alpha subunit of pyruvate dehydrogenase in rat frontal cortex was elevated after a training experience. No change in total pyruvate dehydrogenase activity was observed. The phosphorylation in vitro of pyruvate dehydrogenase (band F-2) was also elevated after training. Since activation of pyruvate dehydrogenase requires its dephosphorylation, the following sequence is proposed. Training alters frontal cortex and reduces the phosphate content of pyruvate dehydrogenase in vivo; this leads to enzyme activation; and an increase in back-titration of sites available for phosphorylation in vitro.
67 citations
TL;DR: Results indicate the presence of a chemical mediator of insulin action in liver similar to that observed in other insulin target tissues.
66 citations
TL;DR: Data provide persuasive evidence that the brain protein is the alpha-subunit of pyruvate dehydrogenase and thereby raise the possibility that even very short periods of synaptic activity influence an enzyme of particular importance to mitochondrial metabolism in brain.
TL;DR: The data suggest that pyruvate transport may control gluconeogenesis from L-lactate and that hormones may stimulate this process through an effect on the respiratory chain and an additional role for pyruVate kinase and pyruviate carboxylase is quite compatible with the data presented.
Abstract: The sensitivity of glucose production from L-lactate by isolated liver cells from starved rats to inhibition by alpha-cyano-4-hydroxycinnamate was studied. A small percentage of the maximal rate of gluconeogenesis was insensitive to inhibition by alpha-cyano-4-hydroxycinnamate, and evidence is presented to show that this is due to pyruvate entry into the mitochondria as alanine. After subtraction of this rate, Dixon plots of the reciprocal of the rate of gluconeogenesis against inhibitor concentration were linear both in the absence and presence of glucagon, phenylephrine or valinomycin, each of which stimulated gluconeogenesis by 30-50%. Pyruvate kinase activity was decreased by glucagon, but not by phenylephrine or valinomycin. Inhibition of gluconeogenesis by quinolinate (inhibitor of phosphoenolpyruvate carboxykinase) or monochloroacetate (probably inhibiting pyruvate carboxylation) caused a significant deviation from linearity of the Dixon plot obtained with alpha-cyano-4-hydroxycinnamate. Amytal, however, inhibited gluconeogenesis without affecting the linearity of this plot. These data, coupled with a computer simulation study, suggest that pyruvate transport may control gluconeogenesis from L-lactate and that hormones may stimulate this process through an effect on the respiratory chain. An additional role for pyruvate kinase and pyruvate carboxylase is quite compatible with the data presented.
TL;DR: The results show that propionyl- CoA inhibits the enzyme competitively with respect to CoASH and that isovaleryl-CoA inhibits competitively and noncompetitively withrespect to NAD+.
TL;DR: The data suggest that the 48000-mol.wt.
Abstract: 1. Incubation of mitochondria from heart, liver and kidney with [32P]phosphate allowed 32P incorporation into two intramitochondrial proteins, the decarboxylase alpha-subunit of the pyruvate dehydrogenase complex (mol.wt 42000) and a protein of mol.wt. 48000. 2. This latter protein incorporated 32P more slowly than did pyruvate dehydrogenase, was not precipitated by antibody to pyruvate dehydrogenase and showed behaviour distinct from that of pyruvate dehydrogenase towards high-speed centrifugation and pyruvate dehydrogenase phosphate phosphatase. 3. 32P incorporation into the protein was greatly diminished by the presence of 0.1 mM-4-methyl-2-oxopentanoate, but enhanced by pyruvate (1 mM), hypo-osmotic treatment of mitochondria and, under some conditions, by uncoupler. 4. The activity of branched-chain 2-oxo acid dehydrogenase was assayed in parallel experiments. Under appropriate conditions the enzyme was inhibited when 32P incorporation was increased and activated when incorporation was decreased. The data suggest that the 48000-mol.wt. phosphorylated protein is identical with the decarboxylase subunit of branched-chain 2-oxo acid dehydrogenase and that this enzyme may be controlled by a phosphorylation-dephosphorylation cycle akin to that for pyruvate dehydrogenase. 5. Strict correlation between activity and 32P incorporation was not observed, and a scheme for the regulation of the enzyme is proposed to account for these discrepancies.
TL;DR: Evidence is presented that anaerobic nitrite reduction with pyruvate involves at least the dehydrogenase subunit of the pyruVate dehydrogenases complex.
Abstract: SUMMARY: A strain of Escherichia coli with a mutation in the ana gene was shown to lack acetaldehyde dehydrogenase and alcohol dehydrogenase. The requirement of this strain for an external oxidant to grow anaerobically on glucose shows that the reduction of acetyl-CoA is the principal means of reoxidation of NADH produced during glycolysis in E. coli. Further mutants derived from the ana strain were shown to be affected in the enzymes involved in the fermentation of pyruvate (pyruvate formate-lyase, phosphotransacetylase, acetate kinase). A gene controlling acetate kinase (ackB) activity has been located at 39 min on the chromosomal map. Evidence is presented that anaerobic nitrite reduction with pyruvate involves at least the dehydrogenase subunit of the pyruvate dehydrogenase complex.
TL;DR: Tumors of chicken and rat contain the M2-type of pyruvate kinase isoenzyme, which is significantly higher in malignant tumors compared with benign tumors.
Abstract: Tumors of chicken and rat contain the M2-type of pyruvate kinase isoenzyme. The amount of this isoenzyme is significantly higher in malignant tumors compared with benign tumors. No alteration of the normal pyruvate kinase isoenzyme pattern was found in hyperplastic tissue.
TL;DR: Evidence is taken as conclusive evidence that the pyruvate dehydrogenase complex in pea leaf mitochondria undergoes interconversion between deactivated and activated states by covalent modification (phosphorylation-dephosphorylated) catalyzed by a kinase and phosphatase.
TL;DR: Methods for the assay of pyruvate dehydrogenase and acetyl-CoA synthetase activities in rat brain subcellular fractions permit the determination of a large number of samples of different turbidity with satisfactory precision.
TL;DR: Leaf slices from Kalanchoe daigremontiana and CAM Mesembryanthemum crystallinum, two species that possess pyruvate, PI dikinase, transfer label from exogenous [3-14C]pyruvates to carbohydrates more rapidly than either Stapelia gigantea, a PEP carboxykinase CAM plant, or C3 Mesembrysum crystall inum, which lack the dkinase.
Abstract: Pyruvate, PI dikinase (EC 2.7.9.1) was present in crassulacean acid metabolism (CAM) plants that lack phosphoenolpyruvate (PEP) carboxykinase (EC 4.1.1.32) but was not detected in plants that contain PEP carboxykinase or in C3 plants. It is suggested that, during deacidification in CAM plants that contain NAD and NADP malic enzymes (EC 1.1.1.38 and EC 1.1.1.40) but not PEP carboxykinase, pyruvate, P*i dikinase reverses the glycolytic reaction catalysed by pyruvate kinase (EC 2.7.1.40) and converts pyruvate to PEP as the first step in the gluconeogenic conservation of pyruvate as storage carbohydrate. The enzyme is not required by CAM plants that contain PEP carboxykinase and produce mainly PEP during decarboxylation. Leaf slices from Kalanchoe daigremontiana and CAM Mesembryanthemum crystallinum, two species that possess pyruvate, PI dikinase, transfer label from exogenous [3-14C]pyruvate to carbohydrates more rapidly than either Stapelia gigantea, a PEP carboxykinase CAM plant, or C3 Mesembryanthemum crystallinum, which lack the dikinase. Label from [2-14C]- and [3-14C]pyruvate is converted to carbohydrate at the same rate in K. daigremontiana while in S. gigantea label from [2-14C]pyruvate accumulates in carbohydrates twice as rapidly as label from [3-14C]pyruvate. The patterns observed for K. daigremontiana and for CAM M. crystallinum are consistent with the gluconeogenic anabolism of pyruvate whereas the patterns observed for S. gigantea and for C.3 M. crystallinum suggest pyruvate is oxidized possibly via the tricarboxylic acid cycle in these species. Deacidification in Aloe arborescens, a PEP carboxykinase CAM plant that also possesses NAD and NADP malic enzyme activity, was inhibited 80% by 0.1 mM 3-mercaptopicolinic acid (3-MPA), an inhibitor of PEP carboxykinase. It is thus likely that, in this species and probably also in other CAM plants with high PEP carboxykinase activities, a small proportion of the malic acid may be decarboxylated by malic enzymes. However, as 0.5 mM 3-MPA inhibited deacidification in K. daigremontiana by 40%, the inhibitor is probably only specific at low concentrations. 14CO2 fixation in the light by mesophyll cells isolated from K. daigremontiana was stimulated by 20-50% in the presence of 10 mM pyruvate, but there was no increase in 14CO2 fixation by mesophyll cells isolated from S. gigantea.
TL;DR: N-Phenyl maleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvates and alpha-Cyanocinnamate protects the pyruVate transporter from attack by this thiol-blocking reagent.
Abstract: 1. N-Phenylmaleimide irreversibly inhibits pyruvate transport into rat heart and liver mitochondria to a much greater extent than does N-ethylmaleimide, iodoacetate or bromopyruvate. alpha-Cyanocinnamate protects the pyruvate transporter from attack by this thiol-blocking reagent. 2. In both heart and liver mitochondria alpha-cyanocinnamate diminishes labelling by [3H]N-phenylmaleimide of a membrane protein of subunit mol.wt. 15000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. Exposure of mitochondrial to unlabelled N-phenylmaleimide in the presence of alpha-cyanocinnamate, followed by removal of alpha-cyanocinnamate and exposure to [3H]N-phenylmaleimide, produced specific labelling of the same protein. 4. Both labelling and kinetic experiments with inhibitors gave values for the approximate amount of carrier present in liver and heart mitochondria of 100 and 450 pmol/mg of mitochondrial protein respectively. 5. The turnover numbers for net pyruvate transport and pyruvate exchange at 0 degrees C were 6 and 200 min-1 respectively.
TL;DR: Researchers studied a child with a rapidly progressive neurologic disorder, with psychomotor retardation, hypotonia, seizures, and respiratory disturbances, and found an isolated deficiency of pyruvate dehydrogenase complex in brain tissue with the morphologic picture of progressive polio-dystrophy with hypomyelination.
Abstract: We studied a child with a rapidly progressive neurologic disorder, with psychomotor retardation, hypotonia, seizures, and respiratory disturbances. Laboratory studies showed elevated levels of lactate and pyruvate in cerebrospiral fluid (CSF), without notable elevated levels in serum. In liver, muscle, leukocytes, and cultured fibroblasts we found no abnormality in pyruvate oxidation; biochemical studies of a brain biopsy showed an isolated deficiency of pyruvate dehydrogenase complex in brain tissue with the morphologic picture of progressive polio-dystrophy with hypomyelination.
TL;DR: It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide, which might best account for the biphasic inactivation kinetics.
Abstract: Reaction of rabbit muscle pyruvate kinase with the affinity label 5'-[p-(fluorosulfonyl) benzoyl] guanosine (5'-FSBG), at pH 7.65 and 7.93, leads to a loss in enzyme activity. The inactivation is characterized by a biphasic kinetic profile, with the initial phase accounting for approximately 55% of the reduction in enzymatic activity. For both the rapid and slow phases, at pH 7.93, the inactivation rate constants are linearly proportional to the reagent concentration (from 0.48 to 3.0 mM), yielding second-order rate constants of 195 min-1 M-1 and 19 min-1 m-1, respectively. The effect of ligands was tested on the two phases of inactivation. For both, a decrease in the inactivation rate was produced by Mg2+ alone, but the best protection was provided by Mg2+ plus either ADP or GDP, suggesting that the reaction occurs in the region of the metal-nucleotide binding site. Modified pyruvate kinase is completely reactivated by incubation with 20 mM dithiothreitol, indicating the involvement of cysteine in the inactivation, indicating the involvement of cysteine in the inactivation process. Reaction with [5'=3H]-5'-FSBG leads to the incorporation of up to 1.3 mol of radioactive reagent per mol of enzyme subunit; however, identical radiolabel incorporation is observed before or after dithiothreitol reactivation of modified enzyme. This result implies that the labeled amino acid residue, measured by means of incorporation, is not directly involved in the inactivation process. In contrast, inactivation was found to correlate well with the loss of two free sulfhydryl groups per enzyme subunit and the restoration of activity to correlate with the regeneration of two free sulfhydryls after treatment of modified enzyme with dithiothreitol. It is proposed that inactivation of pyruvate kinase by 5'-FSBG proceeds by formation of thiol sulfonate followed by a rapid displacement of the sulfinic acid moiety by a second cysteine to yield a disulfide. A negative cooperatively in the interaction of pyruvate kinase subunits with 5'-[p-(fluorosulfonyl)-benzoyl] guanosine might best account for the biphasic inactivation kinetics.
TL;DR: A factor derived from the supernatant of adipocytes plasma membranes which mimics the effects of insulin on mitochondrial pyruvate dehydrogenase was shown to activate a high affinity (Ca2+ + Mg2+)-ATPase and ATP-dependent Ca2+ transport of adipocyte plasma membranes.
TL;DR: Developmental and senescent patterns of a number of heart enzyme activities linked to energy metabolism have been studied in rats aged between 4 days and 21 months, and these patterns could point towards a partial impairment of Krebs cycle function, and a reduced energy-producing capacity in the aged rat heart.
TL;DR: The hypothesis that substrates of pp 60 src - kinase, essential for cell proliferation, are among the enzymes that control glycolysis is supported.
TL;DR: The organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of Gram-negative bacteria.
Abstract: 1. Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (s20,w 77 S) and flavin content (1.3--1.4 nmol/mg) to the complexes from mammalian mitochondria. 2. The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58 000; lipoamide dehydrogenase (E3), 56 000; pyruvate dehydrogenase (E1), alpha-subunit, 45 000, and beta-subunit, 35 000. Gel chromatography in the presence of 6 M guanidine . HCl gave a value of 52 000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes. Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria. 3. The complex was resolved into its component enzymes by the following methods. E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution. E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea. The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.
TL;DR: The high activity of carnitine transacetylase in mitochondria from various tissues is highly correlated with the necessity of carn itine esters for fatty acid oxidation.
TL;DR: A function of leaf L-lactate dehydrogenase is proposed as a component of the systems regulating the cellular pH and/or controlling the concentration of reducing equivalents in the cytoplasm of leaf cells.
Abstract: 1. L-Lactate dehydrogenase from lettuce (Lactuca sativa) leaves was purified to electrophoretic homogeneity by affinity chromatography. 2. In addition to its NAD(H)-dependent activity with L-lactate and pyruvate, the enzyme also catalyses the reduction of hydroxypyruvate and glyoxylate. The latter activities are not due to a contamination of the enzyme preparations with hydroxypyruvate reductase. 3. The enzyme shows allosteric properties that are markedly by the pH. 4. ATP is a potent inhibitor of the enzyme. The kinetic data suggest that the inhibition by ATP is competitive with respect to NADH at pH 7.0 and 6.2. The existence of regulatory binding sites for ATP and NADH is discussed. 5. Bivalent metal cations and fructose 6-phosphate relieve the ATP inhibition of the enzyme. 6. A function of leaf L-lactate dehydrogenase is proposed as a component of the systems regulating the cellular pH and/or controlling the concentration of reducing equivalents in the cytoplasm of leaf cells.