Showing papers on "Pyruvate dehydrogenase kinase published in 1987"
TL;DR: It is proposed that cells plated at low density in the absence of exogenous pyruvate fail to thrive for two reasons: prolonged depletion of intracellular pyruVate and prolonged vulnerability to oxidant stress.
Abstract: Cells in culture are exposed to marked oxidative stress, H2O2 being one of the predominant agents. Pyruvate and other alpha-ketoacids reacted rapidly, stoichiometrically, and nonenzymatically with H2O2, and they protected cells from its cytolytic effects. All five human and murine cell types studied, both malignant and nonmalignant, released pyruvate at an initial rate of 35-60 microM/h/2.5 X 10(6) cells when placed in 1 ml pyruvate-free medium. After 6-12 h a plateau of 60-150 microM pyruvate was attained, corresponding to concentrations reported for normal human serum and plasma. The rate of pyruvate accumulation was almost doubled in the presence of exogenous catalase, suggesting that released pyruvate functions as an antioxidant. The rate of pyruvate accumulation was dependent on cell number. Succinate, fumarate, citrate, oxaloacetate, alpha-ketoglutarate, and malate were not secreted in significant amounts from P815 cells; export was specific for pyruvate and lactate among the metabolites tested. Extracellular pyruvate was in equilibrium with intracellular stores. Thus, cells conditioned the extracellular medium with pyruvate at the expense of intracellular pyruvate, until homeostatic levels were attained in both compartments. We propose that cells plated at low density in the absence of exogenous pyruvate fail to thrive for two reasons: prolonged depletion of intracellular pyruvate and prolonged vulnerability to oxidant stress.
294 citations
TL;DR: Clinical findings are presented for 30 patients with lactic acidemia in whom activity of the pyruvate dehydrogenase complex in fibroblasts was significantly (P = less than 0.01) below that of control cell lines.
168 citations
TL;DR: Since previous studies demonstrated an increase in steady state glyceraldehyde-3-phosphate dehydrogenase RNA during low Oz exposure it is concluded that the level of this RNA is regulated post transcriptionally whereas the other four glycolytic enzyme RNAs are regulated at least partially at thelevel of transcription by oxygen availability.
Abstract: Cytoplasmic β-actin and five glycolytic enzyme cDNAs were isolated from a rat skeletal muscle cDNA library and together with a genomic clone of rat cytochrome c were used as probes to quantitate the respective RNA transcription rates in isolated nuclei run off transcription assays from stationary cells cultured under normal or 2% oxygen. The transcription rates of lactate dehydrogenase, pyruvate kinase, triosephosphate isomerase and aldolase increased by 2–5 fold during the 72 hr exposure to 2% oxygen. There was a small increase in actin RNA transcription while both cytochrome c and glyceraldehyde-3-phosphate dehydrogenase RNA transcription rates decreased. Since previous studies demonstrated an increase in steady state glyceraldehyde-3-phosphate dehydrogenase RNA during low Oz exposure it is concluded that the level of this RNA is regulated post transcriptionally whereas the other four glycolytic enzyme RNAs are regulated at least partially at the level of transcription by oxygen availability. The relative transcriptional rates of the RNAs in this study are related to their cellular RNA and protein concentrations.
97 citations
TL;DR: Results indicate that pyruvate:NADP+ oxidoreductase in E. gracilis is clearly distinct from either the pyruVate dehydrogenase multienzyme complex or pyruviate:ferredoxin oxidOREductase.
87 citations
TL;DR: An infant with lactic acidosis and developmental delay had neuropathological changes consistent with Leigh's necrotizing encephalomyelopathy, and profound systemic deficiency of PDC was due to lack of both subunit proteins of the first component of P DC.
Abstract: An infant with lactic acidosis and developmental delay had neuropathological changes consistent with Leigh's necrotizing encephalomyelopathy. Total pyruvate dehydrogenase complex (PDC) activity was low relative to controls in lymphocytes (0.2 versus 1.9 ± 0.6 SD nmol/min/mg protein) and cultured skin fibroblasts (0.9 versus 2.7 ± 1.0). Liver, muscle, heart, and kidney mitochondria oxidized several substrates normally, but did not oxidize pyruvate. PDC activity was absent in these mitochondria (0.1 versus 9.8 ± 4.2 in liver and 0.7 versus 75 ± 26 in muscle) and was very low in all tissue homogenates. Activity of the first component was low in liver mitochondria, whereas activities of the second and third components were normal. Western blot analysis of tissue proteins showed normal amounts of second and third component of PDC but undetectable to trace amounts of both a and B subunits of the first component of PDC in liver, brain, kidney, heart, and skin fibroblasts. Thus, profound systemic deficiency of PDC was due to lack of both subunit proteins of the first component of PDC.
75 citations
TL;DR: Urine extracts from three cases of succinic semialdehyde dehydrogenase deficiency have shown consistently increased amounts of 2,4-dihydroxybutric acid, and its lactone, and 3-hydroxypropionic acid, metabolites related to the α-oxidation of 4-Hydroxybutyric acid.
Abstract: In addition to the previously reported abnormalities, urine extracts from three cases of succinic semialdehyde dehydrogenase deficiency have shown consistently increased amounts of 2,4-dihydroxybutyric acid, and its lactone, and 3-hydroxypropionic acid, metabolites related to the alpha-oxidation of 4-hydroxybutyric acid. Threo- and erythro-4,5-dihydroxyhexanoic acids have also been identified for the first time and probably arise from the reaction of succinic semialdehyde with an intermediate in the pyruvate dehydrogenase pathway. Adipic acid excretion is also consistently raised, suggesting secondary interference with mitochondrial beta-oxidation. The presence of these metabolites could be a source of diagnostic confusion.
75 citations
TL;DR: The autotroph Methanococcus maripaludis contained high levels of acetate-coenzyme A ligase, pyruvate synthase, phosphoenolpyruVate carboxykinase, citrate synthase and isocitrate dehydrogenase, and the enzymes of the incomplete reductive tricarboxylic acid cycle were not detected.
Abstract: The autotroph Methanococcus maripaludis contained high levels of acetate-coenzyme A ligase, pyruvate synthase, pyruvate, water dikinase, pyruvate carboxylase, and the enzymes of the incomplete reductive tricarboxylic acid cycle. Phosphoenolpyruvate carboxykinase, citrate synthase, and isocitrate dehydrogenase were not detected. In contrast, the heterotroph Methanococcus sp. strain A3 contained acetate kinase, and acetate coenzyme A ligase was virtually absent.
69 citations
TL;DR: Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which are suggested in part to determine specificity toward different hydroxyacids (R-CHOH-COOH).
67 citations
61 citations
TL;DR: The results indicate that insulin and glucagon are involved in the regulation of hepatic gluconeogenesis in trout and that their effects are mediated, at least partly, through the control of pyruvate kinase activity.
Abstract: Gluconeogenesis from lactate in hepatocytes from rainbow trout is activated by glucagon and inhibited by insulin in a dose-dependent fashion. The maximal responses to both hormones occur within their probable physiological concentration ranges. Gluconeogenic activation by glucagon is accompanied by a profound inhibition of pyruvate kinase activity. This is reflected in increases in both the S0.5 and nH for phosphoenolpyruvate and increased sensitivity to inhibition by MgATP compared to the enzyme isolated from cells not treated with hormone (control). Gluconeogenic inhibition by insulin is accompanied by activation of pyruvate kinase activity. This is observed as decreases in both the S0.5 and nH for phosphoenolpyruvate and a decreased sensitivity to inhibition by MgATP compared to enzyme isolated from control cells. These results indicate that insulin and glucagon are involved in the regulation of hepatic gluconeogenesis in trout and that their effects are mediated, at least partly, through the control of pyruvate kinase activity.
59 citations
TL;DR: The antagonistic action of the phospho-oligosaccharide on glucagon effects was dose-dependent and partially antagonized glucagon-stimulated cyclic AMP levels, which support the hypothesis that this phosphorus mediates at least some insulin actions in hepatocytes.
TL;DR: This chapter summarizes some aspects of the structural organization of the mammalian pyruvate dehydrogenase complex and the regulation of its activity by a phosphorylation–dephosphorylation cycle by identifying the two best-studied acute effects of positive inotropic agents.
Abstract: Publisher Summary This chapter summarizes some aspects of the structural organization of the mammalian pyruvate dehydrogenase complex and the regulation of its activity by a phosphorylation–dephosphorylation cycle. Phosphorylation and concomitant inactivation of pyruvate dehydrogenase (E1) occurs on three serine residues in the α subunit. The phosphorylation of pyruvate dehydrogenase inhibits the decarboxylation of pyruvate and may also inhibit reductive acetylation of the lipoyl moiety. Pyruvate dehydrogenase kinase appears to be specific for pyruvate dehydrogenase. Pyruvate dehydrogenase phosphatase activity is inhibited by NADH and this inhibition is reversed by nicotinamide adenine dinucleotide (NAD + ). The phosphatase is inactive toward p -nitrophenyl phosphate. The pyruvate dehydrogenase system is well designed for fine regulation of its activity. The interconversion of the active and inactive phosphorylated forms of pyruvate dehydrogenase is a dynamic process that leads rapidly to the establishment of steady states in which the fraction of phosphorylated E1 can be varied progressively over a wide range by changing the concentration or molar ratios of effectors that regulate the activities of the kinase and phosphatase. The acute regulation of pyruvate dehydrogenase in response to hormonal and other influences is mediated by two regulatory mechanisms—namely, end-product inhibition by acetyl-CoA and NADH and reversible covalent phosphorylation. The two best-studied acute effects of hormones on the activity of pyruvate dehydrogenase are those of positive inotropic agents, such as adrenaline on the enzyme in the heart and the effect of insulin on the enzyme in several tissues, but particularly in adipose tissue.
TL;DR: The pyruvate dehydrogenase complex was isolated, partially purified, and characterized from green pea leaf mitochondria and found to be a better substrate than 2-oxo-butyrate; other 2- oxo-acids were not substrates.
Abstract: The pyruvate dehydrogenase complex was isolated, partially purified, and characterized from green pea (Pisum sativum L., cv Little Marvel) leaf mitochondria. The pH optimum for the overall reaction was 7.6. The divalent cation requirement was best satisfied by Mg2+. Reaction velocity was maximal at 40°C. Pyruvate was a better substrate than 2-oxo-butyrate; other 2-oxo-acids were not substrates. Michaelis constants for substrates were; pyruvate, 57 micromolar; NAD, 122 micromolar; Coenzyme-A, 5 micromolar; Mg2+, 0.36 millimolar; Mg-thiamine pyrophosphate, 80 nanomolar. The products, NADH and acetyl-Coenzyme-A, were linear competitive inhibitors with respect to NAD and Coenzyme A. Inhibition constants were 18 and 10 micromolar, respectively. Glyoxylate inhibited complex activity only in the absence of thiol reagents. Glyoxylate inhibition was competitive with respect to pyruvate with an inhibition constant of 51 micromolar. Among mitochondrial metabolites examined as potential effectors, only ADP with an inhibition constant of 0.57 millimolar could be of physiological significance.
TL;DR: The results indicate that Kupffer cells contain a receptor that is involved in the clearance of lactate dehydrogenase M4 and a number of other tissue-derived enzymes from plasma, which is mainly due to endocytosis by macrophages in liver, spleen and bone marrow.
TL;DR: This chapter describes the best methods available for regeneration ofAdenosine triphosphate (ATP) from adenosine diphosphates (ADP) and Adenosine monophosphate and details several applications of these methods in enzyme-catalyzed syntheses.
Abstract: Publisher Summary This chapter describes the best methods available for regeneration of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and adenosine monophosphate (AMP) and details several applications of these methods in enzyme-catalyzed syntheses In particular, it give procedures for synthesis of three phosphate donors—acetyl phosphate, phosphoenolpyruvate, and methoxycarbonyl phosphates—and apply these reagents to syntheses of sn -glycerol 3-phosphate, dihydroxyacetone phosphate, glucose 6-phosphate, 5-phospho-α-D-ribosyl pyrophosphate, and uridine-5'-diphosphoglucose Three procedures for the enzymatic regeneration of ATP are presently available, which are useful in practical-scale organic synthesis One is based on acetyl phosphate (AcP) as the phosphorylating agent and acetate kinase as the catalyst; the second uses phosphoenolpyruvate (PEP) and pyruvate kinase; and the third uses methoxycarbonyl phosphate [CH3OC(O)OPO3 2-, MCP] and acetate kinase The advantages and disadvantages of each method are summarized in the chapter Both pyruvate kinase and acetate kinase have high specific activity and show excellent stability in immobilized form Pyruvate kinase is currently the less expensive enzyme Further, it is effective for regeneration of ATP from ADP at lower concentrations of ADP than is acetate kinase, because the Michaelis constant for pyruvate kinase is lower than that for acetate kinase In practice, for most synthetic applications, either acetyl phosphate/acetate kinase or phosphoenolpyruvate/pyruvate kinase is used for the regeneration of ATP
TL;DR: The results indicate that uptake of pyruvate in E. coli is via a specific active transport system that requires an artificial electron donor system, phenazine methosulfate and sodium ascorbate.
Abstract: A mutant of Escherichia coli K-12 lacking pyruvate dehydrogenase and phosphoenolpyruvate synthase was used to study the transport of pyruvate by whole cells. Uptake of pyruvate was maximal in mid-log phase cells, with a Michaelis constant for transport of 20 microM. Pretreatment of the cells with respiratory chain poisons or uncouplers, except for arsenate, inhibited transport up to 95%. Lactate and alanine were competitive inhibitors, but at nonphysiological concentrations. The synthetic analogs 3-bromopyruvate and pyruvic acid methyl ester inhibited competitively. The uptake of pyruvate was also characterized in membrane vesicles from wild-type E. coli K-12. Transport required an artificial electron donor system, phenazine methosulfate and sodium ascorbate. Pyruvate was concentrated in vesicles 7- to 10-fold over the external concentration, with a Michaelis constant of 15 microM. Energy poisons, except arsenate, inhibited the transport of pyruvate. Synthetic analogs such as 3-bromopyruvate were competitive inhibitors of transport. Lactate initially appeared to be a competitive inhibitor of pyruvate transport in vesicles, but this was a result of oxidation of lactate to pyruvate. The results indicate that uptake of pyruvate in E. coli is via a specific active transport system.
TL;DR: It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/,[NAD+] ratios.
Abstract: 1. Increasing concentrations of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a mild respiratory-chain inhibitor [Halestrap (1987) Biochim. Biophys. Acta 927, 280-290], caused progressive inhibition of glucose production from lactate + pyruvate by hepatocytes from starved rats incubated in the presence or absence of oleate and gluconeogenic hormones. 2. No significant changes in tissue ATP content were observed, but there were concomitant decreases in ketone-body output and cytochrome c reduction and increases in NADH fluorescence and the ratios of [lactate]/[pyruvate] and [beta-hydroxybutyrate]/[acetoacetate]. 3. The inhibition by DCMU of palmitoylcarnitine oxidation by isolated liver mitochondria was used to calculate a flux control coefficient of the respiratory chain towards gluconeogenesis. In the presence of 1 mM-oleate, the calculated values were 0.61, 0.39 and 0.25 in the absence of hormone and in the presence of glucagon or phenylephrine respectively, consistent with activation of the respiratory chain in situ as previously suggested [Quinlan & Halestrap (1986) Biochem. J. 236, 789-800]. 4. Cytoplasmic oxaloacetate concentrations were shown to decrease under these conditions, implying inhibition of pyruvate carboxylase. 5. Inhibition of gluconeogenesis from fructose and dihydroxyacetone was also observed with DCMU and was accompanied by an increased output of lactate + pyruvate, suggesting that activation of pyruvate kinase was occurring. With the latter substrate, measurements of tissue ADP and ATP contents showed that DCMU caused a small fall in [ATP]/[ADP] ratio. 6. Two inhibitors of fatty acid oxidation, pent-4-enoate and 2-tetradecylglycidate, were shown to abolish and to decrease respectively the effects of hormones, but not valinomycin, on gluconeogenesis from lactate + pyruvate, without changing tissue ATP content. 7. It is concluded that the hormonal increase in mitochondrial matrix volume stimulates fatty acid oxidation and respiratory-chain activity, allowing stimulation of pyruvate carboxylation and thus gluconeogenesis to occur without major changes in [ATP]/[ADP] or [NADH]/[NAD+] ratios. 8. The high flux control coefficient of the respiratory chain towards gluconeogenesis may account for the hypoglycaemic effect of mild respiratory-chain inhibitors.
TL;DR: The subcellular distribution of pyruvate-degrading enzymes has been determined in Chlamydomonas reinhardtii by protoplast induction with autolysine, dig-itonin lysis and further fractionation by differential centrifugation using a Percoll cushion.
Abstract: The subcellular distribution of pyruvate-degrading enzymes has been determined in Chlamydomonas reinhardtii (Dangeard) by protoplast induction with autolysine, dig-itonin lysis and further fractionation by differential centrifugation using a Percoll cushion. Mitochondrial and plastidic fractions contained intact and physiologically competent organelles - RC 1.7, ADP/O 2.7 and rate of malate oxidation 76 nmol O, (mg protein)-1min-1 for mitochondria, CO2; fixation 46.8 μmol (mg Chi)-1 h-1 for chloroplasts.
Results from protoplast fractionation were further confirmed by the determination of enzyme activities within trypsin-treated organelles. Mitochondria (formate fermentation) and chloroplasts (chlorofermentation) were shown to possess the capacity for anaerobic pyruvate degradation. Pyruvate dehydrogenase (NAD+, EC 1.2.4.1), pyruvate formate-lyase (EC 2.3.1.54) and lactate dehydrogenase (NADH, EC 1.1.1.27) showed equal distribution between mitochondria and chloroplasts, whereas activities of phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) were only detectable in the mitochondrial fraction. NADH- and NADPH-dependent activities of both alcohol dehydrogenase (EC 1.1.1.1) and aldehyde dehydrogenase (acylating, EC 1.2.1.10) were localized in the mitochondrial and cytoplasmic or the plastidic and cytoplasmic fractions, respectively, whereas pyruvate decarboxylase (EC 4.1.1.1) was only detected in the cytoplasmic fraction.
TL;DR: The results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects, and a dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.
TL;DR: A cytoplasmic pyruvate oxidase was partially purified from Streptococcus sanguis ATCC 10556 and its activity was not influenced by various low molecular-weight substances known to be present in the cy toplasm.
Abstract: A cytoplasmic pyruvate oxidase was partially purified from Streptococcus sanguis ATCC 10556. The enzyme used pyruvate, inorganic phosphate and oxygen as substrates, and formed acetyl phosphate and hydrogen peroxide. In this reaction carbon dioxide can also be expected, but this product was not looked for. The enzyme was dependent on thiamine pyrophosphate (TPP), flavin adenine dinucleotide (FAD) and magnesium ions for activity. Its relative molecular weight (Afr) was 260,000 as calculated from its migration distance in polyacrylamide gradient gel. The enzyme was hysteretic and its activity was not influenced by various low molecular-weight substances known to be present in the cytoplasm. The pH optimum of the enzyme was 6.7 to 7.5 and its activity was not inhibited by 1 mM hydrogen peroxide or iodoacetamide, or by 10 mM potassium cyanide, sodium azide, iodoacetate, sodium fluoride, zinc chloride, EDTA, or dithiothreitol.
TL;DR: Bovine heart pyruvate dehydrogenase complex was acetylated by using [3-14C]pyruVate in the presence of N-ethylmaleimide, with approx.
Abstract: Bovine heart pyruvate dehydrogenase complex was acetylated by using [3-14C]pyruvate in the presence of N-ethylmaleimide, with approx. 1 mol of acetyl groups being incorporated per mol of E2 polypeptide. After peptic digestion, lipoate-containing peptides were purified by high-voltage electrophoresis and ion-exchange and reverse-phase h.p.l.c. The amino acid sequence around the lipoic acid-attachment site of E2 was determined by automated Edman degradation. Acetylation of a lipoate cofactor bound to a lysine residue was verified by fast-atom-bombardment m.s.
TL;DR: In situ and in vitro studies indicate that the ATP:ADP ratio has little or no effect in regulating the mitochondrial pyruvate dehydrogenase complex from green pea seedlings.
TL;DR: 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate.
Abstract: The alanine analogue 1-aminoethylphosphinate [H3C-CH(NH2)-PO2H2] effectively inhibited anthocyanin synthesis in buckwheat hypocotyls and caused an increase in the concentrations of alanine and alanine-derived metabolites. Aminotransferase inhibitors partially alleviated the effects of the analogue. 1-Aminoethylphosphinate did not affect the growth of Klebsiella pneumoniae under anaerobic conditions, but under aerobic conditions it inhibited growth and caused the massive excretion of pyruvate. The analogue inhibited the pyruvate dehydrogenase complex in vitro in the presence of an aminotransferase activity. The transamination product of 1-aminoethylphosphinate, acetylphosphinate (H3C-CO-PO2H2), was found to inhibit the pyruvate dehydrogenase complex in a time-dependent reaction that followed first-order and saturation kinetics and required the presence of thiamin pyrophosphate.
TL;DR: Inhibition of a process as crucial as mitochondrial respiration should serve to emphasize the care necessary in interpretation of whole-organism calmodulin antagonist studies.
TL;DR: A Brevibacterium flavum mutant lacking pyruvate kinase, No. 70, grew on glucose, fructose and sucrose as well as the original wild strain did, but was unable to grow on ribose or gluconate unless pyruVate was added.
Abstract: A Brevibacterium flavum mutant lacking pyruvate kinase, No. 70, grew on glucose, fructose and sucrose as well as the original wild strain did, but was unable to grow on ribose or gluconate unless pyruvate was added. Mutants that required pyruvate for growth on ribose were derived directly from the wild strain. Many of them were completely or partially defective in pyruvate kinase activity. These pyruvate kinase mutants were also unable to grow on gluconate. A phosphoenolpy- ruvate (PEP): sugar phosphotransferase system (PTS) was found in B. flavum, which catalyzed the formation of pyruvate and sugar phosphate from PEP and sugar. The system required Mg2 +, acted on glucose, fructose, mannose, glucosamine and 2-deoxyglucose, and existed in the cells grown on any of the carbon sources tested. Cells grown on fructose, mannitol and sucrose, however, exhibited higher PTS activities on fructose than those grown on others. Glucose PTS activity was about 20-fold stronger than that of glucokinase. Other sugar metab...
TL;DR: The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented and this complex appears to be able to synthesize acetoin from acetaldehyde plus pyruVate and is competitively inhibited by acetoin.
TL;DR: Cells of Clostridium acetobutylicum contained an NADH-dependent L(+)-lactate dehydrogenase which was activated specifically by fructose-1,6-bisphosphate (F-1-6-P2), with calcium or magnesium ions as positive effectors.
Abstract: Cells of Clostridium acetobutylicum contained an NADH-dependent L(+)-lactate dehydrogenase which was activated specifically by fructose-1,6-bisphosphate (F-1,6-P2), with calcium or magnesium ions as positive effectors. During the purification steps the enzyme was very unstable. The purified enzyme existed in a tetrameric structure (apparent Mr of about 159 kDa) and had its pH optimum at pH 5.8. Little activity was left at pH values below 5.0. The enzyme was unidirectional, catalysing only the reduction of pyruvate. The half maximal activation of the reaction velocity with F-1,6-P2 depended on the pyruvate concentration.
TL;DR: Evidence is presented here that the level of pyruvate carboxylase activity as well as the native and subunit molecular weights of this enzyme are identical in the mutant and the wild type yeast.
TL;DR: It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylations of site 1.
Abstract: Mitochondria from rat epididymal white adipose tissue were made permeable to small molecules by toluene treatment and were used to investigate the effects of Mg2+ and Ca2+ on the re-activation of pyruvate dehydrogenase phosphate by endogenous phosphatase. Re-activation of fully phosphorylated enzyme after addition of 0.18 mM-Mg2+ showed a marked lag of 5-10 min before a maximum rate of reactivation was achieved. Increasing the Mg2+ concentration to 1.8 mM (near saturating) or the addition of 100 microM-Ca2+ resulted in loss of the lag phase, which was also greatly diminished if pyruvate dehydrogenase was not fully phosphorylated. It is concluded that, within intact mitochondria, phosphatase activity is highly sensitive to the degree of phosphorylation of pyruvate dehydrogenase and that the major effect of Ca2+ may be to overcome the inhibitory effects of sites 2 and 3 on the dephosphorylation of site 1. Apparent K0.5 values for Mg2+ and Ca2+ were determined from the increases in pyruvate dehydrogenase activity observed after 5 min. The K0.5 for Mg2+ was diminished from 0.60 mM at less than 1 nM-Ca2+ to 0.32 mM at 100 microM-Ca2+; at 0.18 mM-Mg2+, the K0.5 for Ca2+ was 0.40 microM. Ca2+ had little or no effect at saturating Mg2+ concentrations. Since effects of Ca2+ are readily observed in intact coupled mitochondria, it follows that Mg2+ concentrations within mitochondria are sub-saturating for pyruvate dehydrogenase phosphate phosphatase and hence less than 0.5 mM.
01 Jan 1987
TL;DR: Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+, then this results in an increase in the intramitochondrial concentration ofCa2+.
Abstract: Three key dehydrogenases in the mitochondria of higher animals have been found to be activated by Ca2+ ions; these are pyruvate dehydrogenase and two enzymes in the citric acid cycle, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase Activation can also be demonstrated within permeabilized and intact mitochondria Evidence is summarized that when hormones and other extracellular stimuli increase the cytoplasmic concentration of Ca2+, then this results in an increase in the intramitochondrial concentration of Ca2+ In this way, rates of pyruvate oxidation and citric acid cycle flux are increased, and hence there is an increase in NADH supply for the respiratory chain under conditions where there is an enhanced demand for ATP In contrast, the activation of pyruvate dehydrogenase which is observed in adipose and other tissues exposed to insulin is brought about by a Ca2+-independent mechanism