Showing papers on "Pyruvate dehydrogenase kinase published in 1984"

Use of 31P nuclear magnetic resonance spectroscopy and 14C fluorography in studies of glycolysis and regulation of pyruvate kinase in Streptococcus lactis.

Abstract: High-resolution 31P nuclear magnetic resonance spectroscopy and 14C fluorography have been used to identify and quantitate intermediates of the Embden-Meyerhof pathway in intact cells and cell extracts of Streptococcus lactis. Glycolysing cells contained high levels of fructose 1,6-bisphosphate (a positive effector of pyruvate kinase) but comparatively low concentrations of other glycolytic metabolites. By contrast, starved organisms contained only high levels of 3-phosphoglycerate, 2-phosphoglycerate, and phosphoenolpyruvate. The concentration of Pi (a negative effector of pyruvate kinase) in starved cells was fourfold greater than that maintained by glycolysing cells. The following result suggest that retention of the phosphoenolpyruvate pool by starved cells is a consequence of Pi-mediated inhibition of pyruvate kinase: the increase in the phosphoenolpyruvate pool (and Pi) preceded depletion of fructose 1,6-bisphosphate, and reduction in intracellular Pi (by a maltose-plus-arginine phosphate trap) caused the restoration of pyruvate kinase activity in starved cells. Time course studies showed that Pi was conserved by formation of fructose 1,6-bisphosphate during glycolysis. Conversely, during starvation high levels of Pi were generated concomitant with depletion of intracellular fructose 1,6-bisphosphate. The concentrations of Pi and fructose 1,6-bisphosphate present in starved and glycolysing cells of S. lactis varied inversely. The activity of pyruvate kinase in the growing cell may be modulated by the relative concentrations of the two antagonistic effectors.

Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart.

Abstract: Surface fluorometric measurements and indicator metabolite determinations in the isolated perfused rat heart showed that the NADH+NADPH fluorescence of the intact tissue originates largely from the mitochondria. The redox potential of the lactate dehydrogenase system calculated from the endogenous lactate/pyruvate ratios was closely similar to that of the glycerol-3-phosphate dehydrogenase system calculated from the concentrations of glycerol-3-phosphate and dihydroxyacetone phosphate in the tissue. Thus, in contrast to the liver, the cytosolic redox state of the NADH/NAD+ system in isolated perfused heart oxidizing external glucose or fatty acid is not amenable to optical monitoring, but can be assessed from the state of the lactate dehydrogenase or glycerol-3-phosphate, dehydrogenase systems.

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism.

Abstract: A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The Km for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.

Purification and biochemical characterization of pyruvate oxidase from Lactobacillus plantarum.

Abstract: Pyruvate oxidase (EC 1.2.3.3) was isolated and characterized from Lactobacillus plantarum. The enzyme catalyzes the oxidative decarboxylation of pyruvate in the presence of phosphate and oxygen, yielding acetyl phosphate, carbon dioxide, and hydrogen peroxide. This pyruvate oxidase is a flavoprotein, with the relatively tightly bound cofactors flavin adenine dinucleotide, thiamine pyrophosphate, and a divalent metal ion, with Mn2+ being the most effective. The enzyme is only slightly inhibited by EDTA, implying that the enzyme-bound metal ion is poorly accessible to EDTA. Only under relatively drastic conditions, such as acid ammonium sulfate precipitation, could a colorless and entirely inactive apoenzyme be obtained. A partial reactivation of the enzyme was only possible by the combined addition of flavin adenine dinucleotide, thiamine pyrophosphate, and MnSO4. The enzyme has a molecular weight of ca. 260,000 and consists of four subunits with apparently identical molecular weights of 68,000. For catalytic activity the optimum pH is 5.7, and the optimum temperature is 30 degrees C. The Km values for pyruvate, phosphate, and arsenate are 0.4, 2.3, and 1.2 mM, respectively. The substrate specificity revealed that the enzyme reacts also with certain aldehydes and that phosphate can be replaced by arsenate. In addition to oxygen, several artificial compounds can function as electron acceptors.

Reversible phosphorylation of pyruvate dehydrogenase in rat skeletal-muscle mitochondria. Effects of starvation and diabetes.

Abstract: The total activity of pyruvate dehydrogenase (PDH) complex in rat hind-limb muscle mitochondria was 76.4 units/g of mitochondrial protein. The proportion of complex in the active form was 34% (as isolated), 8-14% (incubation with respiratory substrates) and greater than 98% (incubation without respiratory substrates). Complex was also inactivated by ATP in the presence of oligomycin B and carbonyl cyanide m-chlorophenylhydrazone. Ca2+ (which activates PDH phosphatase) and pyruvate or dichloroacetate (which inhibit PDH kinase) each increased the concentration of active PDH complex in a concentration-dependent manner in mitochondria oxidizing 2-oxoglutarate/L-malate. Values giving half-maximal activation were 10 nM-Ca2+, 3 mM-pyruvate and 16 microM-dichloroacetate. Activation by Ca2+ was inhibited by Na+ and Mg2+. Mitochondria incubated with [32P]Pi/2-oxoglutarate/L-malate incorporated 32P into three phosphorylation sites in the alpha-chain of PDH; relative rates of phosphorylation were sites 1 greater than 2 greater than 3, and of dephosphorylation, sites 2 greater than 1 greater than 3. Starvation ( 48h ) or induction of alloxan-diabetes had no effect on the total activity of PDH complex in skeletal-muscle mitochondria, but each decreased the concentration of active complex in mitochondria oxidizing 2-oxoglutarate/L-malate and increased the concentrations of Ca2+, pyruvate or dichloracetate required for half-maximal reactivation. In extracts of mitochondria the activity of PDH kinase was increased 2-3-fold by 48 h starvation or alloxan-diabetes, but the activity of PDH phosphatase was unchanged.

Phosphorylation in vivo of red‐muscle pyruvate kinase from the channelled whelk, Busycotypus canaliculatum, in response to anoxic stress

Abstract: That red muscle pyruvate kinase from anoxic Busycotypus canaliculatum (PK-anoxic) is a phosphoprotein was demonstrated by the anoxia-dependent, in vivo, covalent incorporation of injected [32P]orthophosphate into the enzyme molecule. Specificity in labelling of PK-anoxic was strongly suggested by: (a) coincidental elution of pyruvate kinase activity and radioactivity following chromatography of purified PK-anoxic on Sepharose CL-6B, and (b) comigration of the area containing [32P]phosphate and Coomassie-Blue-staining protein following SDS-polyacrylamide gel electrophoresis of homogenous PK-anoxic. The [32P]phosphate content of the enzyme was calculated to be 7.3 mol phosphate/mol enzyme (233 kDa, 180 units/mg protein). Evidence for the reversibility of this phosphorylation was provided by the consistent kinetic similarities between purified red muscle pyruvate kinase from aerobic animals (PK-aerobic) and homogenous, unlabelled, alkaline phosphatase treated PK-anoxic. Comparison of the electrophoretic mobilities of products derived from acid hydrolysis of purified 32P-labelled PK-anoxic with authentic substances suggest the presence of an O-phospho-L-threonine residue in the protein. That this residue plays a probable role in an interconversion mechanism was suggested by the lack of phosphate exchange of homogenous 32P-labelled PK-anoxic in the presence of all substrates. A possible role of protein phosphorylation as a mechanism for the overall control of molluscan anaerobic metabolism is suggested.

The regulatory properties of isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase from Escherichia coli ML308 and the roles of these activities in the control of isocitrate dehydrogenase.

Abstract: Isocitrate dehydrogenase kinase can use ATP but not other nucleoside triphosphates as a phosphate donor. It responds hyperbolically to both ATP and isocitrate dehydrogenase. The kinase is inhibited sigmoidally by low concentrations of DL-isocitrate and hyperbolically by ADP, AMP, NADPH, phosphoenolpyruvate and several other effectors. Isocitrate dehydrogenase phosphatase requires a nucleotide for activity; ADP and ATP are the best activators. The phosphatase responds hyperbolically to ADP or ATP, to Mg2+ ions and to phosphorylated isocitrate dehydrogenase. The phosphatase is activated twofold to threefold by AMP, oxaloacetate, pyruvate, phosphoenolpyruvate, 2-oxoglutarate and DL-isocitrate. It is inhibited hyperbolically by NADPH. The pH optima and the Km values for substrates of the kinase and the phosphatase are reported. We propose that the role of the phosphorylation of isocitrate dehydrogenase during growth of Escherichia coli on acetate is to render this enzyme rate-limiting in the citric acid cycle; this should cause an increase in the level of isocitrate and divert the flux of carbon through the glyoxylate bypass. We suggest that the phosphorylation state of isocitrate dehydrogenase in intact cells is controlled by the levels of isocitrate, phosphoenolpyruvate, NADPH and the adenine nucleotides. This theory can explain many recent observations on the control of the activity of isocitrate dehydrogenase.

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell

Abstract: The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.

Role of Ca2+ ions in the regulation of intramitochondrial metabolism in rat epididymal adipose tissue. Evidence against a role for Ca2+ in the activation of pyruvate dehydrogenase by insulin.

Abstract: The sensitivity of rat epididymal-adipose-tissue pyruvate dehydrogenase phosphate phosphatase, NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase to Ca2+ ions was studied both in mitochondrial extracts and within intact coupled mitochondria. It is concluded that all three enzymes may be activated by increases in the intramitochondrial concentration of Ca2+ and that the distribution of Ca2+ across the mitochondrial inner membrane is determined, as in rat heart mitochondria, by the relative activities of a uniporter (which transports Ca2+ into mitochondria and is inhibited by Mg2+ and Ruthenium Red) and an antiporter (which allows Ca2+ to leave mitochondria in exchange for Na+ and is inhibited by diltiazem). Previous studies with incubated fat-cell mitochondria have indicated that the increases in the amount of active non-phosphorylated pyruvate dehydrogenase in rat epididymal tissue exposed to insulin are the result of activation of pyruvate dehydrogenase phosphate phosphatase. In the present studies, no changes in the activity of the phosphatase were found in extracts of mitochondria, and thus it seemed likely that insulin altered the intramitochondrial concentration of some effector of the phosphatase. Incubation of rat epididymal adipose tissue with medium containing a high concentration of CaCl2 (5mM) was found to increase the active form of pyruvate dehydrogenase to much the same extent as insulin. However, the increases caused by high [Ca2+] in the medium were blocked by Ruthenium Red, whereas those caused by insulin were not. Moreover, whereas the increases resulting from both treatments persisted during the preparation of mitochondria and their subsequent incubation in the absence of Na+, only the increases caused by treatment of the tissue with insulin persisted when the mitochondria were incubated in the presence of Na+ under conditions where the mitochondria are largely depleted of Ca2+. It is concluded that insulin does not act by increasing the intramitochondrial concentration of Ca2+. This conclusion was supported by finding no increases in the activities of the other two Ca2+-responsive intramitochondrial enzymes (NAD+-isocitrate dehydrogenase and 2-oxoglutarate dehydrogenase) in mitochondria prepared from insulin-treated tissue compared with controls.

Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12.

Abstract: We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli. Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS). We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues). In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively. This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate. Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel.

Effects of dichloroacetate on brain pyruvate dehydrogenase.

Abstract: The action of dichloroacetate (DCA) on pyruvate dehydrogenase (PDH) activity of rat brain has been studied in vitro and in vivo. In a crude brain mitochondrial fraction, DCA inhibits PDH kinase and in rat brain slices this compound increases PDH activity and stimulates glucose oxidation. In the whole animal, intraperitoneal injection of DCA causes activation of brain PDH, indicating that this inhibitor crosses the blood-brain barrier. The same treatment with DCA also produced a large increase in heart PDH activity. Further studies of the effects of DCA on the CNS should lead to results of considerable importance.

Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308

Abstract: Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000-fold from Escherichia coli ML308 by procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE-cellulose, blue-dextran-Sepharose and Sephadex G150. The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D.C. and Koshland, D.E. (1982) Nature (Lond.) 300, 458-460] that a single protein bears both activities. Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation. Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine. Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P-phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions. The active and inactive forms of isocitrate dehydrogenase can be resolved by non-denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.

The concentration of the mitochondrial pyruvate carrier in rat liver and heart mitochondria determined with alpha-cyano-beta-(1-phenylindol-3-yl)acrylate

Abstract: alpha-Cyano-beta-(1-phenylindol-3-yl)acrylate inhibited pyruvate transport into both liver and heart mitochondria approximately linearly with respect to its concentration until 65% inhibition was achieved. The extent of inhibition was dependent on the mitochondrial protein concentration. By extrapolation of plots of inhibition versus inhibitor concentration to total inhibition, or by mathematical analysis of the plots, the concentration of pyruvate transporter molecules per mg of protein was calculated to be approximately 100 pmol/mg for both heart and liver mitochondria, and the Ki about 7 nM. The data also suggest that pyruvate transport is rate-limiting for pyruvate oxidation by heart mitochondria in State 3, but not by liver mitochondria.

A re-evaluation of the role of mitochondrial pyruvate transport in the hormonal control of rat liver mitochondrial pyruvate metabolism.

Abstract: The inhibitor of mitochondrial pyruvate transport alpha-cyano-beta-(1-phenylindol-3-yl)-acrylate was used to inhibit progressively pyruvate carboxylation by liver mitochondria from control and glucagon-treated rats. The data showed that, contrary to our previous conclusions [Halestrap (1978) Biochem. J. 172, 389-398], pyruvate transport could not regulate metabolism under these conditions. This was confirmed by measuring the intramitochondrial pyruvate concentration, which almost equilibrated with the extramitochondrial pyruvate concentration in control mitochondria, but was significantly decreased in mitochondria from glucagon-treated rats, where rates of pyruvate metabolism were elevated. Computer-simulation studies explain how this is compatible with linear Dixon plots of the inhibition of pyruvate metabolism by alpha-cyano-4-hydroxycinnamate. Parallel measurements of the mitochondrial membrane potential by using [3H]triphenylmethylphosphonium ions showed that it was elevated by about 3 mV after pretreatment of rats with both glucagon and phenylephrine. There was no significant change in the transmembrane pH gradient. It is shown that the increase in pyruvate metabolism can be explained by a stimulation of the respiratory chain, producing an elevation in the protonmotive force and a consequent rise in the intramitochondrial ATP/ADP ratio, which in turn increases pyruvate carboxylase activity. Mild inhibition of the respiratory chain with Amytal reversed the effects of hormone treatment on mitochondrial pyruvate metabolism and ATP concentrations, but not on citrulline synthesis. The significance of these observations for the hormonal regulation of gluconeogenesis from L-lactate in vivo is discussed.

Bromopyruvate as an active-site-directed inhibitor of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Abstract: Bromopyruvate behaves as an active-site-directed inhibitor of the pyruvate decarboxylase (E1) component of the pyruvate dehydrogenase complex of Escherichia coli. It requires the cofactor thiamin pyrophosphate (TPP) and acts initially as an inhibitor competitive with pyruvate (Ki ca. 90 microM) but then proceeds to react irreversibly with the enzyme, probably with the thiol group of a cysteine residue. E1 catalyzes the decomposition of bromopyruvate, the enzyme becoming inactivated once every 40-60 turnovers. Bromopyruvate also inactivates the intact pyruvate dehydrogenase complex in a TPP-dependent process, but the inhibition is more rapid and is mechanistically different. Under these conditions, bromopyruvate is decarboxylated, and the lipoic acid residues in the lipoate acetyltransferase (E2) component become reductively bromoacetylated. Further bromopyruvate then reacts with the new thiol groups thus generated in the lipoic acid residues, inactivating the complex. If reaction with the lipoic acid residues is prevented by prior treatment of the complex with N-ethylmaleimide in the presence of pyruvate, the mode of inhibition reverts to irreversible reaction with the E1 component. In both types of inhibition of E1, reaction of 1 mol of bromopyruvate/mol of E1 chain is required for complete inactivation, and all the evidence is consistent with reaction taking place at or near the pyruvate binding site.