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PubMed1985

The physiological role of oxygen-sensitive pyruvate dehydrogenase in mitochondrial fatty acid synthesis in Euglena gracilis.

In Euglena gracilis a malonyl-CoA-independent fatty acid-synthetic system, in which fatty acids are synthesized directly from acetyl-CoA as both primer and C2 donor, occurs in mitochondria, and the system contributes to the wax ester fermentation. The activity of fatty acid synthesis in the mitochondrial system was enhanced about six times when an artificial acetyl-CoA-regenerating system was present, indicating that the fatty acid-synthetic activity is controlled by the ratio of acetyl-CoA against CoA. When fatty acids were synthesized using pyruvate instead of acetyl-CoA as substrate, a high activity, about 30 times higher than that from acetyl-CoA, was found under anaerobic conditions (below 10(-5) M oxygen), while in aerobiosis fatty acids were not synthesized at all. CoA, NADH, and NADP+ were required as cofactors for fatty acid synthesis from pyruvate. It was indicated that high activity of fatty acid synthesis from pyruvate due to the high ratio of acetyl-CoA against CoA was maintained by the action of the oxygen-sensitive pyruvate dehydrogenase found in Euglena mitochondria. When [2-14C]pyruvate was fed into intact mitochondria under anaerobic conditions, radioactive fatty acids were formed in the presence of malate, which provided reducing power for the matrix.

Acetyl Coenzyme A↗
PubMed1986

Effect on gluconeogenesis of mutants blocking two mitochondrial transport systems in the yeast Saccharomyces cerevisiae.

Two mutants of Saccharomyces cerevisiae, ccr1 and tpy1, have been found to interfere with the transport of small molecules across the inner mitochondrial membrane. Both also have the effect of interfering with the synthesis of a number of cytoplasmically located enzymes involved in gluconeogenesis, even when the cells are released from glucose repression. The ccr1 mutant, defective in the transport of dicarboxylic acids across the inner membrane, represses the synthesis of gluconeogenic enzymes almost totally, but synthesis can be induced on complete medium without a carbon source. This mutant has low levels of intracellular malate under all growth conditions tested. The tpy1 mutant, defective in the transport of pyruvate across the inner membrane, shows repression of gluconeogenesis enzymes under some growth conditions, particularly high levels of ethanol in the medium. These conditions also lead to low levels of malate in the cells. Intracellular levels of malate in these mutants, and in the wild type, are correlated with the levels of gluconeogenic enzymes present. The ability of isolated mutant mitochondria to phosphorylate ADP is shown to be consistent with the interpretation that they are defective in inner membrane transport, although as yet no evidence is available that these defects are the primary lesions in the two mutants. The data are consistent with two general models. In one, the exhaustion of an extramitochondrial corepressor or introduction of a coinducer by mitochondrial activity triggers the induction of gluconeogenic enzyme synthesis. In the second, the mitochondria themselves trigger this induction, but only when the tricarboxylic acid cycle is able to operate at a high level.

Biological Transport↗
PubMed1976

Energy-linked regulation of glucose and pyruvate oxidation in isolated perfused rat heart. Role of pyruvate dehydrogenase.

1. The regulation of glycolysis and pyruvate oxidation under varying conditions of ATP and oxygen consumption was studied in isolated perfused rat hearts. Potassium-induced arrest was employed to inhibit the ATP consumption of the heart. 2. Under the experimental conditions, the beating heart used solely glucose as the oxidisable substrate. The glycolytic flux through the aldolase step decreased in pace with the decreasing oxygen consumption during the potassium-induced arrest of the heart. The decrease in glucose oxidation was larger than the inhibition of the oxygen consumption, suggesting that the arrested heart switches to fatty acid oxidation. The time course and percentage changes of the inhibition of pyruvate oxidation and the decrease in the amount of the active form of pyruvate dehydrogenase suggest that the amount of active pyruvate dehydrogenase is the main regulator of pyruvate oxidation in the perfused heart. 3. To test the relative significance of the possible mechanisms regulating covalent interconversions of pyruvate dehydrogenase, the following parameters were measured in response to the potassium-induced cardiac arrest: concentrations of pyruvate, acetyl-CoA, CoA-SH, citrate, alpha-oxoglutarate, ATP, ADP, AMP, creatine, creatine phosphate and inorganic phosphate and the mitochondrial NADH/NAD+ ratio. In cardiac tissue the adenylate system is not a good indicator of the energy state of the mitochondrion, even when the concentrations of AMP and free cytosolic ADP are calculated from the adenylate kinase and creatine kinase equilibria. Only creatine phosphate and inorganic phosphate undergo significant changes, but evidence of the participation of the latter compounds in the regulation of the pyruvate dehydrogenase interconversions is lacking. The potassium-induced arrest of the heart resulted in a decrease in pyruvate, a slight increase in acetyl-CoA, a large increase in the concentration of citrate and an increase in the mitochondrial NADH/NAD+. The results can be interpreted as showing that in the heart, the pyruvate dehydrogenase interconversions are mainly regulated by the pyruvate concentration and the mitochondrial redox state. Concentrations of all the regulators tested shifted to directions which one would expect to result in a decrease in the amount of active pyruvate dehydrogenase, but the changes were quite small. Therefore, the energy-linked regulation of pyruvate dehydrogenase in intact tissue is possibly mediated by the equilibrium relations between the cellular redox state and the phosphorylation potential recently confirmed in cardiac tissue.

Animals↗
PubMed1977

Transport of pyruvate and lactate in yeast mitochondria.

Evidence for the existence of mediated transport of pyruvate and lactate in isolated mitochondria of Saccharomyces cerevisiae is presented. 1. The mitochondrial oxidation of pyruvate is specifically inhibited by the monocarboxylic oxoacids alpha-ketoisocaproate and by alpha-cyano-3-hydroxycinnamate, while pyruvate and malate dehydrogenases activities are not inhibited. 2. The stimulation of the mitochondrial oxidations of succinate, alpha-ketoglutarate and citrate by pyruvate are also inhibited by alpha-cyano-3-hydroxycinnamate. 3. The [14C]pyruvate uptake by yeast mitochondria follows saturation kinetics and is completely inhibited by alpha-cyano-3-hydroxycinnamate. 4. Large amplitude passive swellings of mitochondria of the wild type and of cytoplasmic rho- and rho-n mutants are induced by isoosmotic ammonium pyruvate and lactate. These pH-dependent swellings are inhibited by alpha-cyano-3-hydroxycinnamate suggesting that the carrier system is not coded by mitochondrial DNA.

Carboxylic Acids↗
PubMed1980

Persistent changes in the initial rate of pyruvate transport by isolated rat liver mitochondria after preincubation with adenine nucleotides and calcium ions.

1. Preincubation of isolated rat-liver mitochondria in the presence of adenine nucleotides or Ca2+ results in definite and persistent changes in the initial rate of pyruvate transport. 2. These changes in the rate of pyruvate transport are accompanied by equally persistent changes in the opposite direction of the activity of pyruvate dehydrogenase (EC 1.2.4.1). 3. Changes of the transmembrane pH gradient and of the membrane potential, brought about by the pretreatments of the mitochondria, cannot account for the observed changes in the rate of pyruvate transport. 4. It is proposed that the pretreatment of the mitochondria directly modulates the activity of the mitochondrial pyruvate carrier. The possible regulatory role of such a modulation system is discussed.

Adenine Nucleotides↗
PubMed1993

Mitochondrial pyruvate transport in working guinea-pig heart. Work-related vs. carrier-mediated control of pyruvate oxidation.

Myocardial pyruvate oxidation is work- or calcium-load-related, but control of pyruvate dehydrogenase (PDH) by the specific mitochondrial pyruvate transporter has also been proposed. To test the transport hypothesis distribution of pyruvate across the cell membrane as well as rates of mitochondrial pyruvate net transport plus oxidation were examined in isolated perfused but stable and physiologically working guinea-pig hearts. 150 microM-1.2 mM alpha-cyanohydroxycinnamate proved to specifically block mitochondrial pyruvate uptake in these hearts. When perfusate glucose as cytosolic pyruvate precursor was supplied in combination with octanoate (0.2 or 0.5 mM) as diffusible alternative fatty acid substrate, alpha-cyanohydroxycinnamate produced up to 20- and 3-fold increases in pyruvate and lactate efflux, respectively. Cinnamates did not alter myocardial hemodynamics nor sarcolemmal pyruvate and lactate export. In contrast the tested concentrations of cinnamate produced reversible, dose-dependent decreases in 14CO2 production from [1-14C]pyruvate or [U-14C]glucose by inhibiting mitochondrial pyruvate uptake. Linear least-squares estimates of available cinnamate-sensitive total pyruvate transport potential yielded rates close to 110 mumol/min per g dry mass at S0.5 approximately 120 microM, which compared reasonably well with literature values from isolated cardiac mitochondria. This transport potential was severalfold larger than total extractable myocardial PDH activity of approximately 32 mumol/min per g dry mass at 37 degrees C. Even when cytosolic pyruvate levels were in the lower physiologic range of about 90 microM, pyruvate oxidation readily kept pace with mitochondrial respiration over a wide range of workload and inotropism. Furthermore, dichloroacetate, a selective activator of PDH, stimulated pyruvate oxidation without affecting myocardial O2 consumption, regardless of the metabolic or inotropic state of the hearts. Consequently, little or no regulatory function with regard to pyruvate oxidation could be assigned to the native mitochondrial pyruvate carrier of the working heart. Therefore, mitochondrial pyruvate-H+ symport was the normal, highly efficient (rather than controlling) mechanism for pyruvate entry into the mitochondria where PDH regulation controlled pyruvate oxidation.

Adrenergic Agonists↗
PubMed1975

Separation of lipoamide dehydrogenase isoenzymes by affinity chromatography.

1. Lipoamide dehydrogenase NADH: lipoamide oxidoreductase, (EC 1.6.4.3) from pig heart has been separated into two sets of isoenzymes by chromatography on lipoyl- and NAD+-derivatized Sepharose-4B matrices. The first fraction is eluted at 30 mM sodium phosphate buffer (pH 7.2), the other requires a higher ionic strength. The two groups originate from the alpha-ketoglutarate and the pyruvate dehydrogenase complex respectively. 2, Hydrophobic chromatography on a homologous series of alkyl-Sepharoses lead to similar results. The first fraction is eluted with 30 mM phosphate buffer in the case of propyl- and butyl-Sepharose but a high ionic strength is required in the case of an increased chain length (C5--C6). The second fraction is reversibly bound on Sepharose-NC3 and -NC4 but binding becomes irreversible at higher chain lengths. 3. Aminoalkyl-Sepharose behave qualitatively as the alkyl derivatives although elution, particularly in the case of the second fraction, can be realized at lower ionic strength. 4. Matrices which are negatively charged (Sepharose-NCnCOOH, n equal 3--7) have no affinity at pH 7.2. 5. The influence of a neutral polar substituent has been studied by comparing the following matrices: Sepharose-NC6OH, Sepharose-NC6NH2 and Sepharose NC6. Binding of the various isoenzymes is completely reversible in the case of a Sepharose-NC6OH matrix and the elution behaviour is identical to that on propyl- and butyl matrices.

Alkanes↗
PubMed1980

Phosphonate analogues of pyruvate. Probes of substrate binding to pyruvate oxidase and other thiamin pyrophosphate-dependent decarboxylases.

A number of enzymes catalyze the removal of carbon dioxide from pyruvate through covalent participation of the coenzyme thiamin pyrophosphate. The conversions of the decarboxylated adduct, hydroxyethyl thiamin pyrophosphate, to subsequent products distinguishes the function of these enzymes. Acetaldehyde is produced by pyruvate decarboxylase, acetic acid by pyruvate oxidase and acetyl coenzyme A by pyruvate dehydrogenase. Differences and details of steps prior to decomposition of hydroxyethyl thiamin pyrophosphate can be evaluated through the use of two substrate analogues, methyl acetylphosphonate and acetylphosphonate. Methyl acetylphosphonate and acetylphosphonate are competitive inhibitors toward pyruvate with Escherichia coli pyruvate oxidase and E. coli pyruvate dehydrogenase but the value of the Ki for the oxidase is more than three orders of magnitude higher than for the dehydrogenase. Yeast pyruvate decarboxylase is not inhibited at all under the same conditions. The binding of methyl acetylphosphonate results in ligand-induced changes in the near ultraviolet circular dichorism spectrum of the oxidase. This spectral perturbation is only seen in the presence of the cofactor, thiamin pyrophosphate, strongly suggesting that the inhibitor is binding at the same site as the substrate, pyruvate, on the enzyme. Kinetic data suggest that lipid activators of pyruvate oxidase increase the affinity of the enzyme for pyruvate and its analogues.

Acetaldehyde↗
PubMed1990

Importance of spontaneous alpha-ketoacid decarboxylation in experiments involving peroxide.

The potential role of spontaneous alpha-ketoacid decarboxylation as a source of interference in experiments involving peroxide was investigated. The assay of pyruvate dehydrogenase activity in isolated renal mitochondria was employed as an example. Spontaneous peroxide-induced pyruvate decarboxylation competed significantly with enzymatic decarboxylation at peroxide concentrations greater than 50 microM. Corrected values for enzymatic decarboxylation could be obtained by subtracting spontaneous decarboxylation rates from rates obtained in the presence of mitochondria. At higher peroxide concentrations (greater than 200 microM), reaction product accumulates (acetoacetate) to levels which may have regulatory effects on mitochondrial metabolism. The divalent cations, Ca2+ and Mg2+, both accelerate spontaneous peroxide-induced pyruvate decarboxylation while other components of the assay medium had an inhibitory effect on the reaction. The results are discussed in relation to the currently accepted reaction mechanism. Investigators who perform experiments involving reactive oxygen species should be familiar with this often overlooked reaction.

Animals↗