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Showing papers on "Pyruvate dehydrogenase kinase published in 1973"


Journal ArticleDOI
TL;DR: Homogenates of Tritrichomonas foetus have pyruvate synthase and hydrogenase, but lack pyruVate-formate lyase and formate dehydrogenase, and these activities are in a subcellular particle identified previously as the site of malate and α-glycerophosphate dehydrogensases.

403 citations


Journal ArticleDOI
TL;DR: Evidence is given that dichloroacetate may facilitate the conversion of pyruvate dehydrogenase from an inactive (phosphorylated) form into an active (dephosphorylation) form.
Abstract: The activity of pyruvate dehydrogenase was assayed in extracts of rat hearts perfused in vitro with media containing glucose and insulin±acetate±dichloroacetate. Dichloroacetate (100μm, 1mm or 10mm) increased the activity of pyruvate dehydrogenase in perfusions with glucose or glucose+acetate. Evidence is given that dichloroacetate may facilitate the conversion of pyruvate dehydrogenase from an inactive (phosphorylated) form into an active (dephosphorylated) form.

156 citations


Journal ArticleDOI
TL;DR: Evidence is presented which suggests that these anomalous product inhibition patterns are due to physical association of the flavoprotein with the transacetylase so that combination of acetyl-CoA with thetransacetyl enzyme hinders combination of NAD with the Flavoprotein and combination of NadH with the flavo-NAD, which is consistent with the patterns predicted from rate equations derived by Cleland for three-site ping-pong mechanisms.

152 citations


Journal ArticleDOI
TL;DR: Several other agents known to inhibit lipolysis and to decrease cyclic AMP levels in fat cells, including niacin, 5-methylpyrazole-3-carboxylic acid, and prostaglandin E1 were also effective in activating pyruvate dehydrogenase.

151 citations


Journal ArticleDOI
TL;DR: It is suggested that metabolism of free fatty acids by the heart in vivo, as in vitro, may impair utilization of these substrates as well as possible mechanisms for the changes in cell citrate concentration and for inhibitory effects of dichloroacetate on the oxidation of acetate, 3-hydroxybutyrate and palmitate are discussed.
Abstract: 1. The extractions of glucose, lactate, pyruvate and free fatty acids by dog heart in vivo were calculated from measurements of their arterial and coronary sinus blood concentration. Elevation of plasma free fatty acid concentrations by infusion of intralipid and heparin resulted in increased extraction of free fatty acids and diminished extractions of glucose, lactate and pyruvate by the heart. It is suggested that metabolism of free fatty acids by the heart in vivo, as in vitro, may impair utilization of these substrates. These effects of elevated plasma free fatty acid concentrations on extractions by the heart in vivo were reversed by injection of dichloroacetate, which also improved extraction of lactate and pyruvate by the heart in vivo in alloxan diabetes. 2. Sodium dichloroacetate increased glucose oxidation and pyruvate oxidation in hearts from fed normal or alloxan-diabetic rats perfused with glucose and insulin. Dichloroacetate inhibited oxidation of acetate and 3-hydroxybutyrate and partially reversed inhibitory effects of these substrates on the oxidation of glucose. In rat diaphragm muscle dichloroacetate inhibited oxidation of acetate, 3-hydroxybutyrate and palmitate and increased glucose oxidation and pyruvate oxidation in diaphragms from alloxan-diabetic rats. Dichloroacetate increased the rate of glycolysis in hearts perfused with glucose, insulin and acetate and evidence is given that this results from a lowering of the citrate concentration within the cell, with a consequent activation of phosphofructokinase. 3. In hearts from normal rats perfused with glucose and insulin, dichloroacetate increased cell concentrations of acetyl-CoA, acetylcarnitine and glutamate and lowered those of aspartate and malate. In perfusions with glucose, insulin and acetate, dichloroacetate lowered the cell citrate concentration without lowering the acetyl-CoA or acetylcarnitine concentrations. Measurements of specific radioactivities of acetyl-CoA, acetylcarnitine and citrate in perfusions with [1-14C]acetate indicated that dichloroacetate lowered the specific radio-activity of these substrates in the perfused heart. Evidence is given that dichloroacetate may not be metabolized by the heart to dichloroacetyl-CoA or dichloroacetylcarnitine or citrate or CO2. 4. We suggest that dichloroacetate may activate pyruvate dehydrogenase, thus increasing the oxidation of pyruvate to acetyl-CoA and acetylcarnitine and the conversion of acetyl-CoA into glutamate, with consumption of aspartate and malate. Possible mechanisms for the changes in cell citrate concentration and for inhibitory effects of dichloroacetate on the oxidation of acetate, 3-hydroxybutyrate and palmitate are discussed.

123 citations



Journal ArticleDOI
TL;DR: Supporting evidence for hormonal control of pyruvate dehydrogenase in isolated liver cells is provided by the fact that glucagon decreases and insulin increases decarboxylation of [1-(14)C]pyruVate.
Abstract: A simplified procedure was developed for isolation of intact, hormone-sensitive liver cells in a high and reproducible yield. These cells produce glucose from various precursors at rates comparable to those achieved in isolated perfused liver. Glucagon enhanced glucose synthesis from pyruvate, dihydroxyacetone, fructose, or xylitol more effectively at low than at high substrate concentration. At high pyruvate concentrations (>2 mM), glucagon or adenosine 3′:5′-cyclic monophosphate (0.1 mM) exerts a curious inhibition of gluconeogenesis that can be reverted to stimulation on addition of ethanol. It is suggested that glucagon and cyclic AMP inhibit pyruvate dehydrogenase and thus limit the supply of reducing equivalents needed for glucose formation. Supporting evidence for hormonal control of pyruvate dehydrogenase in isolated liver cells is provided by the fact that glucagon decreases and insulin increases decarboxylation of [1-14C]pyruvate. Calcium salts (1.3 mM) enhance glucose formation from pyruvate but greatly enhance the inhibition exerted by the divalent cationophore, A23187. Inhibition by glucagon of glucose synthesis from pyruvate is additive with the effects of A23187 + Ca++. However, with dihydroxyacetone as substrate, glucagon partially reverses the inhibition exerted by A23187 + Ca++. The results are consistent with glucagon effecting an inhibition of pyruvate dehydrogenase and a stimulation of hexosediphosphatase activities.

92 citations


Journal ArticleDOI
TL;DR: P-enolpyruvate carboxykinase mutants have established that the enzyme is needed for gluconeogenesis and, under normal growth conditions, for sporulation, and can be restored by the continuous feeding of gluconate.

84 citations


Journal ArticleDOI
TL;DR: The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruVate and MgATP, and that dead-end complexes, between pyruvates,ADP and M g(2+) form randomly and exist in equilibrium with themselves and other substrate complexes.
Abstract: The paper reports a study of the kinetics of the reaction between phosphoenolpyruvate, ADP and Mg(2+) catalysed by rabbit muscle pyruvate kinase. The experimental results indicate that the reaction mechanism is equilibrium random-order in type, that the substrates and products are phosphoenolpyruvate, ADP, Mg(2+), pyruvate and MgATP, and that dead-end complexes, between pyruvate, ADP and Mg(2+), form randomly and exist in equilibrium with themselves and other substrate complexes. Values were determined for the Michaelis, dissociation and inhibition constants of the reaction and are compared with values ascertained by previous workers.

79 citations


Journal ArticleDOI
TL;DR: It is shown that fatty acid oxidation is required for the inactivation of pyruvate dehydrogenase by interconversion of the active to the inactive form by fatty acids in the regulation of pyrivate dehydration by means of fatty acids and fatty acids.
Abstract: Active form and total activity of pyruvate dehydrogenase were measured in homogenates prepared from tissue samples obtained from the isolated rat liver during perfusion. In the absence of substrate, the active portion accounted for about 20% of total activity in livers from fed rats, whereas only about 10% were found to be active in livers from starved rats. These activities remained rather constant during a perfusion period of 120 min. Addition of 10 mM pyruvate to the perfusion medium resulted in an immediate, three-fold increase of the active pyruvate dehydrogenase. A similar effect was exerted by 20 mM fructose, which is known to be rapidly converted to pyruvate in liver. 10 mM lactate led to a considerably smaller increase of pyruvate dehydrogenase activity as compared to the effects obtained with pyruvate or fructose. Total activities of the enzyme did not change significantly during perfusion. If the activities of active pyruvate dehydrogenase from livers perfused with and without substrate were plotted against medium pyruvate concentrations an exponential correlation could be observed. Addition of 2 mM oleate abolished the effects of fructose or pyruvate on the formation of active pyruvate dehydrogenase. The simultaneous addition of d-(+)-decanoylcarnitine inhibited the action of oleate, indicating that fatty acid oxidation is required for the inactivation of pyruvate dehydrogenase by interconversion of the active to the inactive form. The possible physiological significance of pyruvate and fatty acids in the regulation of pyruvate dehydrogenase interconversion is discussed.

77 citations



Journal ArticleDOI
TL;DR: Ten independent lipoamide dehydrogenase mutants (lpd) of Escherichia coli were isolated by selecting strains which required supplements of acetate plus succinate for best growth on glucose and genetic studies indicated that the wild-type phenotype could be restored by single reversion or transduction events.
Abstract: SUMMARY: Ten independent lipoamide dehydrogenase mutants (lpd) of Escherichia coli were isolated by selecting strains which required supplements of acetate plus succinate for best growth on glucose. They would not grow on unsupplemented medium (except anaerobically) nor would they grow with single supplements of acetate or lipoate, but they responded slowly to lysine plus methionine or succinate. Bacteria-free extracts of the mutants had between 1 and 10% of parental lipoamide dehydrogenase activity and no activity for the pyruvate and α-ketoglutarate dehydrogenase complexes could be detected. Evidence that the mutants contained the dehydrogenase (E1) and transacylase (E2) components of the complexes and were deficient only in the lipoamide dehydrogenase (E3) components was obtained from studies with mixtures containing lpd mutant extracts and either extracts of other mutants having defined lesions or purified lipoamide dehydrogenases, e.g. overall pyruvate dehydrogenase complex could be reconstituted with extracts of aceE and F mutants and the α-ketoglutarate complex was similarly reconstituted with sucA and B extracts. Furthermore, both complexes could be restored by adding extract of an aceE, sucA double-amber mutant (which lacks both types of E1 and E2 component but has 30% of parental lipoamide dehydrogenase activity) or with purified bacterial and mammalian lipoamide dehydrogenases. The bacterial enzymes were several times more efficient than the mammalian enzyme for restoring pyruvate dehydrogenase complex activity. Genetic studies indicated that the wild-type phenotype could be restored by single reversion or transduction events and they confirmed that the mutants are deficient only in lipoamide dehydrogenase. The mutant phenotype was introduced into a recipient strain by cotransduction with leu +. This indicates that there is a lipoamide dehydrogenase gene in the leu region of the Escherichia coli linkage map and strongly supports the view that the E3 components of both α-ketoacid dehydrogenase complexes are specified by a single lipoamide dehydrogenase gene (lpd).

Journal ArticleDOI
TL;DR: It is concluded that citrate can prevent the activation of pyruvate dehydrogenase by a mechanism independent of Mg++ and Ca++ chelation.

Journal ArticleDOI
TL;DR: Two additional methods for the detection of abnormal pyruvate kinase in hemolysates are described: heat stability test and activation studies with the allosteric effector fructose 1,6-diphosphate.

Journal ArticleDOI
TL;DR: It is suggested that cotton seed pyruvate kinase is a regulatory enzyme involved in the conversion of fat into carbohydrate in fatstoring seeds.
Abstract: A number of plant species were examined for the presence of pyruvate kinase (pyruvate-ATP phosphotransferase, EC 2.7.1.40), and of a phosphatase activity which hydrolyzes phosphoenolpyruvate. Of those examined, only cotton (Gossypium sp. L.) seeds were found to be sufficiently free of the phosphatase to permit a kinetic study of pyruvate kinase.During germination of cotton seeds, pyruvate kinase activity rises for the first 3 days, after which it falls back to its original level. This developmental pattern is characteristic of enzymes involved in the conversion of fat into carbohydrate in fatstoring seeds. The phosphatase also rose rapidly during germination, which precluded the use of extracts from seedlings in the study of pyruvate kinase. No evidence was found for the presence of more than one pyruvate kinase in cotton seedlings.In crude extracts from ungerminated seeds, the enzyme shows slight deviations from normal kinetics with respect to phosphoenolpyruvate, magnesium, and to a lesser extent, ADP. After partial purification of the enzyme by ion exchange chromatography, the enzyme shows normal kinetics. The enzyme is activated by AMP, and inhibited by both ATP and citrate, in both crude and partially purified preparations. It is suggested that cotton seed pyruvate kinase is a regulatory enzyme.

Journal ArticleDOI
TL;DR: Reduced L-type pyruvate kinase from rat liver can be converted into an oxidized form by incubation with oxidized mercaptoethanol and oxidized glutathione and this interconversion can be completely reversed by incubating with reduced mercapteethanol.


Journal ArticleDOI
TL;DR: The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney, and similar dependence on ionic strength and independence of the nature of the buffer anions or cations characterized preparations.
Abstract: The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney. A broad pH optimum around 7.8, similar dependence on ionic strength, and independence of the nature of the buffer anions or cations characterized preparations from both tissues. Michaelis constants for the binding of pyruvate, thiamin pyrophosphate, NAD+ and CoA were also similar. Enzyme from both tissues was inhibited by NADH, by copper and other heavy metals, by high concentrations of tricarboxylic acid-cycle intermediates, and by preincubation with ATP. Acetyl-CoA itself did not appear to inhibit these preparations, although some commercial preparations of acetyl-CoA did contain an inhibitor. Although oxaloacetate and α-oxobutyrate were weak inhibitors, a number of other α-oxo acids including phenylpyruvate did not inhibit. The properties of the pyruvate dehydrogenase complex from brain and kidney appeared similar.

Journal ArticleDOI
TL;DR: This is the first instance in which regulatory properties have been reported for a pyruvate kinase from a higher plant and the kinetics of the activations by AMP and by fumarate suggest the existence of separate activation sites for the two compounds.

Journal ArticleDOI
TL;DR: Pyruvate kinase from pea seeds and carrots and partially purified by fractionation with (NH4)2SO4 showed hyperbolic kinetics for the substrates phosphoenolpyruvates and ADP, and the possible role of pyruvATE kinase in the regulation of carbohydrate metabolism in plants is discussed.

Journal ArticleDOI
TL;DR: It is suggested that the resistance to substrate inhibition of bound lactate dehydrogenase may possibly be due to the prevention of dissociation of the enzyme into monomeric or other subunits because of attachment to the particulate structures.

Journal ArticleDOI
TL;DR: Temperature-dependent kinetic properties may give the LDH isozymes of G. mirabilis a major role in determining the metabolic fate of pyruvate, which can be channneled preferentially towards the Krebs cycle at low temperatures and efficiently converted to lactate at higher temperatures.
Abstract: 1. 1. The lactate dehydrogenase (LDH) isozymes present in white skeletal muscle of Gillichthys mirabilis exhibit substrate (pyruvate) inhibition only at temperature below 25°C. The extent of pyruvate inhibition is inversely proportional to temperature. 2. 2. The apparent K m of pyruvate is essentially temperature-independent between 10 and 25°C. At higher temperature in the organism's physiolohgical range the apparent K m of pyruvate increases as temperature is raised. 3. 3. These temperature-dependent kinetic properties may give the LDH isozymes of G. mirabilis a major role in determining the metabolic fate of pyruvate. 4. 4. At higher temperatures, when oxygen availability is reduced and the orgnism's activity level is apt to be relatively high, pyruvate may be efficiently converted to lactate, i.e. the fish can rely on anaerobic glycolysis. 5. 5. At low temperatures, when the opposite conditions of oxygen avalability and organismal activity pertain, pyruvate can be channneled preferentially towards the Krebs cycle.

Journal ArticleDOI
02 Mar 1973-Science
TL;DR: Changes in brain glycolytic intermediates were consistent with inhibition of pyruvate kinase in vivo, and no decrease in adenosine triphosphate or creatine phosphate was found.
Abstract: The hypothesis that brain damage in phenylketonuria is related to inhibition of pyruvate kinase by phenylalanine was examined in rat brain in vivo. One hour after a single injection of phenylalanine into the rat, the brains were removed and completely frozen in less than a second. The concentration of phenylalanine in the brain was comparable to that found in phenylketonuric patients. Changes in brain glycolytic intermediates were consistent with inhibition of pyruvate kinase in vivo. The inhibition of pyruvate kinase was apparently compensated for by an increase in phosphoenolpyruvate; no decrease in adenosine triphosphate or creatine phosphate was found.

Journal ArticleDOI
TL;DR: It is concluded that erythrocyte pyruvate kinase deficiency can be a consequence of an increased oxidized glutathione concentration in the red blood cell.

Journal ArticleDOI
TL;DR: About 80% of the aminoacid sequence of dogfish (Squalus acanthius) M(4) lactate dehydrogenase (EC 1.1.27) has been elucidated and several sequence homologies with peptides from pig H(4).
Abstract: About 80% of the aminoacid sequence of dogfish (Squalus acanthius) M4 lactate dehydrogenase (EC 1.1.1.27) has been elucidated. Several sequence homologies with peptides from pig H4 and pig M4 lactate dehydrogenase are identified. Histidine 195 is homologous to the essential histidine residue in pig H4 lactate dehydrogenase. Similarities in the sequence around the “essential” cysteine residue of lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and yeast and liver alcohol dehydrogenase are delineated.

Journal ArticleDOI
TL;DR: The results from both tubular and mitochondrial studies were consistent with a model in which an increase in H+ concentration leads to an activation of isocitric dehydrogenase, succinic dehydrogenases, and possibly glutamic dehydrogensase, but an inhibition of pyruvate metabolism by an inhibited inhibition of either citrate synthetase or pyruVate dehydrogen enzyme.

Journal ArticleDOI
TL;DR: It is suggested that adaptive changes in pyruvate kinase and cytochrome oxidase activity resulting from alterations in either serum concentration or ambient oxygen tension are regulated by two independent mechanisms.
Abstract: Adaptive changes in energy metabolism, as reflected by pyruvate kinase and cytochrome oxidase activities, were examined during in vitro differentiation of the cultivated macrophage. Serum concentrations of tissue culture media, which directly influence endocytic activity, and ambient oxygen tension were both shown to influence pyruvate kinase and cytochrome oxidase activities. Cells maintained in high serum concentrations (30% newborn calf serum [NBCS]) exhibited a 300–400% increase in pyruvate kinase activity and a 40% increase in cytochrome oxidase activity, whereas cells maintained in low serum concentrations (2% NBCS) exhibited a lesser increase (65%) in pyruvate kinase activity and no change in cytochrome oxidase activity. Anaerobiosis resulted in additional alterations in pyruvate kinase and cytochrome oxidase activities. Cells maintained for 48–72 h under anaerobic conditions exhibited a 500–600% increase in pyruvate kinase activity and a 40% decrease in cytochrome oxidase activity. Increased pyruvate kinase activity was dependent on continued protein synthesis. Enzyme increases occurred in anaerobically cultured cells despite an overall reduction in cell protein synthesis. It is suggested that adaptive changes in pyruvate kinase and cytochrome oxidase activity resulting from alterations in either serum concentration or ambient oxygen tension are regulated by two independent mechanisms. One mechanism is aimed at providing energy for endocytic activity and the other in compensating for impaired oxidative metabolism during anaerobiosis.


Journal ArticleDOI
TL;DR: The influence of pH and amino acids on the activity of pyruvate kinase from leucocytes was studied and it is proposed that this model is also valid for the other various types of pyRuvate Kinase.