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PubMed1983

Age-related changes in activities of hepatic phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase in liver and adipose tissue of the swiss albino mouse.

The activities of phosphofructokinase, pyruvate kinase and pyruvate dehydrogenase were examined in liver as a function of age in Swiss albino mice. The hepatic activity of phosphofructokinase and total pyruvate dehydrogenase peaked in mice between 8 and 12 weeks of age and then decreased to a value that remained stable in mature animals older than 24 weeks of age. Yet, the activity of pyruvate kinase and pyruvate dehydrogenase in the active form in liver remained unchanged in mice up to 12 weeks of age. As mice matured, a progressive increase in the activity of both pyruvate kinase and the active form of pyruvate dehydrogenase in liver was observed while phosphofructokinase was unaltered. The pyruvate dehydrogenase complex, both total activity and the proportion of the enzyme in the active form, in the epididymal fat pad of the mouse showed no consistent age trend. The observed increase in the activity of both pyruvate kinase and the active form of pyruvate dehydrogenase should provide an augmented capacity for the generation of acetyl-CoA units for de novo fatty acid synthesis in livers of mature mice.

Adipose Tissue↗
PubMed1985

Pyruvate and acetate oxidation by leukocytes in vitro. Activation of the pyruvate dehydrogenase complex by uncoupling of oxidative phosphorylation.

Pyruvate oxidation by normal intact leukocytes has been systematically studied to define optimal conditions for detection of enzymatic defects in this process. Leukocytes were isolated by dextran sedimentation and lymphocytes by Ficoll centrifugation. Cells were incubated for 2 h with [1-14C]-pyruvate, [2-14C]-pyruvate or [1-14C]-acetate as substrate. The specific oxidative capacity of lymphocytes was almost three times higher than that of granulocytes from the same blood. Oxidation of both pyruvate and acetate was highly dependent on the substrate concentration in the medium reaching a plateau between 0.5 and 1.0 mmol/l. Addition of succinate (1 mmol/l) stimulated oxidation of [1-14C]-pyruvate by 30%. Uncoupling of phosphorylation by addition of carbonyl cyanide chlorophenylhydrazone (CCCP) (0.1 mumol/l) increased oxidation of [1-14C]-pyruvate by 200% and of [1-14C]-acetate by 70%. Addition of CCCP plus succinate caused further stimulation of pyruvate oxidation (+40%), but not of acetate oxidation. It is therefore concluded that: (1) Lymphocytes are better than mixed leukocytes for oxidative studies. (2) Unlabelled substrate should be added at optimal concentrations. (3) The pyruvate dehydrogenase complex is normally only partially active in lymphocytes. (4) Stimulation of oxidation by CCCP greatly enhances the flux through the PDH step thus facilitating the detection of defects in pyruvate oxidation.

Acetates↗
PubMed1973

Regulation of pyruvate metabolism via pyruvate carboxylase in rat brain mitochondria.

1. The fixation of CO(2) by pyruvate carboxylase in isolated rat brain mitochondria was investigated. 2. In the presence of pyruvate, ATP, inorganic phosphate and magnesium, rat brain mitochondria fixed H(14)CO(3) (-) into tricarboxylic acid-cycle intermediates at a rate of about 250nmol/30min per mg of protein. 3. Citrate and malate were the main radioactive products with citrate containing most of the radioactivity fixed. The observed rates of H(14)CO(3) (-) fixation and citrate formation correlated with the measured activities of pyruvate carboxylase and citrate synthase in the mitochondria. 4. The carboxylation of pyruvate by the mitochondria had an apparent K(m) for pyruvate of about 0.5mm. 5. Pyruvate carboxylation was inhibited by ADP and dinitrophenol. 6. Malate, succinate, fumarate and oxaloacetate inhibited the carboxylation of pyruvate whereas glutamate stimulated it. 7. The results suggest that the metabolism of pyruvate via pyruvate carboxylase in brain mitochondria is regulated, in part, by the intramitochondrial concentrations of pyruvate, oxaloacetate and the ATP:ADP ratio.

Adenosine Diphosphate↗
PubMed1982

Role of pyruvate transporter in the regulation of the pyruvate dehydrogenase multienzyme complex in perfused rat liver.

Metabolic substrates such as octanoate, beta-hydroxybutyrate, and alpha-ketoisocaproate which produce acetoacetate stimulate the rate of pyruvate decarboxylation in perfused livers from fed rats at perfusate pyruvate concentrations in the physiological range (below 0.2 mM). A quantitative relationship between pyruvate oxidation (14CO2 production from [1-14C]pyruvate) and ketogenesis (production of acetoacetate or total ketone bodies) was observed with all ketogenic substrates when studied over a wide range of concentrations. The ratio of extra pyruvate decarboxylated to extra acetoacetate produced was greater than 1 with octanoate and alpha-ketoisocaproate, but it was less than 1 with beta-hydroxybutyrate. The stimulatory effect of beta-hydroxybutyrate on pyruvate decarboxylation was abolished completely in the presence of 0.1 mM alpha-cyanocinnamate, an inhibitor of the pyruvate transporting system in the mitochondrial membrane. The data suggest that the mechanism by which the flux through the pyruvate dehydrogenase reaction is stimulated in liver under ketogenic conditions involves an acceleration of the net rate of pyruvate transport into the mitochondria compartment due to an exchange with acetoacetate and/or acetoacetate plus beta-hydroxybutyrate.

Acetoacetates↗
PubMed1992

Bicarbonate-dependent ATP cleavage catalysed by pyruvate carboxylase in the absence of pyruvate.

Preparations of pyruvate carboxylase catalyse the cleavage of MgATP in the absence of pyruvate and acetyl-CoA. The rate of this cleavage is higher in the presence of HCO3- than in its absence. Incubation of the enzyme preparations with an excess of the pyruvate carboxylase inhibitor, avidin, completely abolishes the pyruvate carboxylating activity of the enzyme preparations but only abolishes the HCO3(-)-dependent MgATP cleaving activity, with no effect on the HCO3(-)-independent ATPase activity. The HCO3(-)-dependent MgATP cleavage is also sensitive to inhibition by a pyruvate carboxylase inhibitor, oxamate, and the dependence of the reaction on the free Mg2+ concentration is similar to that of the pyruvate-carboxylation reaction, whereas the HCO3(-)-independent MgATP cleavage is not dependent on the concentration of free Mg2+ in the range tested. This indicates that MgATP cleavage by pyruvate carboxylase is entirely dependent on the presence of HCO3- and that there may be a low level of ATPase contamination in the enzyme preparations. In addition, inhibition of the HCO3(-)-dependent MgATP cleavage by both avidin and oxamate indicate that although biotin does not directly participate in the reaction, its presence is required in that part of the active site of the enzyme. The rate of HCO3(-)-dependent MgATP cleavage is about 0.07% of that of the full pyruvate carboxylation reaction under similar conditions with saturating substrates. The reaction mechanism is sequential with respect to MgATP and HCO3- addition and Mg2+ adds at equilibrium before MgATP. Acetyl-CoA stimulates the HCO3(-)-dependent MgATP cleavage at low MgATP concentrations, with the stimulation being greater at low Mg2+ concentrations. At high levels of MgATP in the presence of acetyl-CoA, substrate inhibition is evident and is more pronounced at increasing concentrations of Mg2+. This inhibition appears to be, at least in part, caused by inhibition of decarboxylation of the enzyme-carboxybiotin complex by the binding to this complex of Mg2+ and MgATP, which probably act to reduce the rate of movement of carboxybiotin from the site of the MgATP cleavage reaction to that of the pyruvate carboxylation reaction where it is unstable and decarboxylates.

Acetyl Coenzyme A↗
PubMed1992

Pyruvate catabolism during transient state conditions in chemostat cultures of Enterococcus faecalis NCTC 775: importance of internal pyruvate concentrations and NADH/NAD+ ratios.

NADH/NAD+ ratios and internal pyruvate concentrations were determined during switches between aerobic and anaerobic steady-state conditions of glucose-limited chemostat cultures of Enterococcus faecalis. During the switch experiments, changes in catabolic fluxes were observed: transition from anaerobic to aerobic conditions resulted in a complete and instantaneous conversion of glucose into acetate and CO2 via the pyruvate dehydrogenase complex, while during a switch from aerobic to anaerobic conditions the culture became homolactic. A similar switch to a homolactic fermentation was observed upon release of the limitation by addition of a glucose pulse to the culture. In sharp contrast to this, a pyruvate pulse resulted in an increase of both pyruvate formate-lyase and pyruvate dehydrogenase complex activity. Furthermore, acetoin was formed during a pyruvate pulse, probably due to a dramatic increase in internal pyruvate concentration. Regulation of the catabolic fluxes over the various pyruvate-catabolizing enzymes is discussed in view of the observed changes in internal pyruvate concentrations and NADH/NAD+ ratios.

Acetates↗
PubMed1977

Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants.

Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase. Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars. This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP. In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars. No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.

Escherichia coli↗
PubMed1982

Studies on pyruvate carboxylase, pyruvate decarboxylase and lipoamide dehydrogenase in subacute necrotizing encephalomyelopathy.

In two autopsy-proven cases of subacute necrotizing encephalomyelopathy (SNE, Leigh's Disease) the activities of pyruvate carboxylase, pyruvate decarboxylase and lipoamide dehydrogenase were investigated in cultured fibroblasts. Normal activities of pyruvate carboxylase and lipoamide dehydrogenase were found in both cases. The activity of pyruvate decarboxylase was low in one of the cases (p less than 0.05), while the activity in the other was within normal limits. The concentrations of alanine, lactate and pyruvate were normal or only slightly increased. The relationship between SNE and a defect in pyruvate metabolism is under discussion, and it is concluded that the general assumption that pyruvate carboxylase deficiency is the cause of SNE is not in agreement with our results or the present literature. However, pyruvate decarboxylase deficiency may in some cases contribute to the development of SNE.

Alanine↗
PubMed1978

The genetic system of the L-type pyruvate kinase forms in man. Subunit structure, interrelation and kinetic characteristics of the pyruvate kinase enzymes from erythrocytes and liver.

Pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from human liver and red cells has been purified to homogeneity; its subunit structure and some of its kinetic characteristics have been studied. The influence of a partial proteolysis by trypsin on the subunit structure, the isozymic pattern and the kinetic characteristics of red cell and liver enzyme have been investigated. From the results of this study we may conclude that: 1. Liver (L-type) pyruvate kinase is composed of 4 identical L subunits while the major form of erythrocyte enzyme (PK-R2) is a heterotetramer designated as L2L2', the molecular weight of L' being slightly higher than that of L subunits (63 000 and 58 000 respectively). Pyruvate kinase PK-R1, predominant in the erythroblasts and the young red cells, is composed of four identical L' subunits. 2. A mild tryptic attack is able to transform PK-R1 into PK-R2, then PK-R2 into pyruvate kinase L (PK-L). The same proteolytic treatment transforms the L' subunits into L ones. 3. Consequently L-type pyruvate kinase seems to be initially synthesized in the erythroid precursors as an L4' enzyme secondarily partially proteolysed into L2L2'. In liver a very active proteolytic system would be responsible for the total transformation into L4 pyruvate kinase. 4. L4' enzyme exhibits Michaelis-Menten kinetic behaviour with an apparent Michaelis constant of 3.8 mM whereas L4 enzyme shows both positive and negative homotropic interactions towards phosphoenolpyruvate and has [S] 0.5 of 1.2 mM. The characteristics of L2L2' are roughly intermediate between those of L4' and of L4. Fructose 1,6-biphosphate decreases [S]0.5 for these three pyruvate kinase forms without suppressing the differences in the apparent affinity for phosphoenolpyruvate of these enzymes. 5. L4 pyruvate kinase is more inhibited by Mg-ATP than L4', with L2L2' in the intermediate range. 6. Tryptic treatment of each enzyme form studied transforms its kinetic behaviour into that observed for L4.

Erythrocytes↗
PubMed1987

13C NMR study of effects of fasting and diabetes on the metabolism of pyruvate in the tricarboxylic acid cycle and the utilization of pyruvate and ethanol in lipogenesis in perfused rat liver.

13C NMR has been used to study the competition of pyruvate dehydrogenase with pyruvate carboxylase for entry of pyruvate into the tricarboxylic acid (TCA) cycle in perfused liver from streptozotocin-diabetic and normal donor rats. The relative proportion of pyruvate entering the TCA cycle by these two routes was estimated from the 13C enrichments at the individual carbons of glutamate when [3-13C]alanine was the only exogenous substrate present. In this way, the proportion of pyruvate entering by the pyruvate dehydrogenase route relative to the pyruvate carboxylase route was determined to be 1:1.2 +/- 0.1 in liver from fed controls, 1:7.7 +/- 2 in liver from 24-fasted controls, and 1:2.6 +/- 0.3 in diabetic liver. Pursuant to this observation that conversion of pyruvate to acetyl coenzyme A (acetyl-CoA) was greatest in perfused liver from fed controls, the incorporation of 13C label into fatty acids was monitored in this liver preparation. Livers were perfused under steady-state conditions with labeled substrates that are converted to either [2-13C]acetyl-CoA or [1-13C]acetyl-CoA, which in the de novo synthesis pathway label alternate carbons in fatty acids. With the exception of the repeating methylene carbons, fatty acyl carbons labeled by [1-13C]acetyl-CoA (from [2-13C]pyruvate) gave rise to resonances distinguishable on the basis of chemical shift from those observed when label was introduced by [3-13C]alanine plus [2-13C]ethanol, which are converted to [2-13C]acetyl-CoA. Thus, measurement of 13C enrichment at several specific sites in the fatty acyl chains in time-resolved spectra of perfused liver offers a novel way of monitoring the kinetics of the biosynthesis of fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1982

Studies on the interactions of Ca2+ and pyruvate in the regulation of rat heart pyruvate dehydrogenase activity. Effects of starvation and diabetes.

1. Previous studies showed that the activation of pyruvate dehydrogenase within intact rat heart mitochondria of pyruvate is much diminished in mitochondria from starved or diabetic animals [see Kerbey, Randle, Cooper, Whitehouse, Pask & Denton (1976) Biochem. J. 154, 327-348]. In the present study, diminished responses to added Ca2+ and ADP were also found in these mitochondria. 2. Starvation or diabetes did not affect the mitochondrial respiratory control ratio of the ATP content. Moreover, starvation and diabetes did not alter the response of the intramitochondrial Ca2+-sensitive enzyme, 2-oxoglutarate dehydrogenase, to changes in the extramitochondrial concentration of Ca2+ and 2-oxoglutarate, thus indicating that there were no appreciable changes in the distribution of Ca2+ and H+ across the mitochondrial inner membrane. 3. Pyruvate, Ca2+ and ADP were found to have synergistic effects on pyruvate dehydrogenase activity, particularly in mitochondria from starved and diabetic rats. 4. The results suggest that the effects of diabetes and starvation on pyruvate dehydrogenase are not brought about by changes in the distribution of these effectors across the mitochondrial inner membrane or by changes in the intrinsic sensitivity of the kinase or phosphatase of the pyruvate dehydrogenase system to pyruvate, Ca2+ or ADP; rather it is probably that there is an increase in the maximum activity of kinase relative to that of the phosphatase. 6. The results also lend further support to the hypothesis that adrenaline may bring about the activation of pyruvate dehydrogenase in the rat heart by an increase in the intramitochondrial concentration of Ca2+.

Adenosine Diphosphate↗
PubMed1998

Pyruvate augments mechanical function via activation of the pyruvate dehydrogenase complex in reperfused ischemic immature rabbit hearts.

BACKGROUND: Reperfusion of ischemic adult hearts is associated with increased fatty acid oxidation, reduced pyruvate oxidation, and reduced pyruvate dehydrogenase (PDH) activity, leading to a decrease in cardiac efficiency. These effects may be amplified in newborn hearts because of the immaturity of their PDH pathway. We hypothesize that pyruvate can augment mechanical function in the immature heart by activating the PDH complex (PDC) during reperfusion in severely ischemic hearts. MATERIALS AND METHODS: Seven-day old isolated working rabbit hearts (n = 12) were perfused with modified Krebs solution containing 0.4 mM palmitate. Pyruvate (5 mM) was added for a 10-min period either before or after a 30-min period of normothermic global ischemia. Cardiac functional indices before global ischemia and during reperfusion were correlated with active and total PDC activity measured in 28 additional hearts frozen at the various time points throughout the perfusion protocol. RESULTS: Addition of pyruvate before ischemia increased the proportion of active PDC but did not affect any measured functional indices. During early reperfusion, aortic flow, cardiac output, and cardiac work were all significantly depressed compared to preischemic values. Addition of pyruvate significantly increased the proportion of active PDC and was also associated with a significant increase in aortic flow, cardiac work, and developed pressure. Removal of pyruvate from the perfusate resulted in a subsequent significant decrease in PDC activity and these functional parameters. CONCLUSION: During reperfusion of neonatal rabbit hearts, addition of pyruvate improves cardiac performance in association with activation of PDC.

Animals↗
PubMed1981

Pyruvate dehydrogenase component of the pyruvate dehydrogenase complex from Escherichia coli K12. Purification and characterization.

Free pyruvate dehydrogenase component of the Escherichia coli K12 pyruvate dehydrogenase complex was isolated from a mutant lacking the dihydrolipoamide transacetylase component. The procedure, employing three chromatographic steps, yields a product that is electrophoretically pure. The purified enzyme reassociates with the residual complex lacking this component to a fully active enzyme complex. The kinetic characteristics of the free component were compared to that of the enzyme integrated into the native complex molecule. No essential differences could be detected regarding the behaviour of the catalytic reaction with variations in temperature, pH and substrate concentration. An inhibition, competitive to pyruvate, of the pyruvate dehydrogenase component (Ki = 18 microM) by fluoropyruvate was observed with both enzyme forms. Pyruvate exerts a cooperative effect both upon the partial enzyme reaction of the pyruvate dehydrogenase component as well as upon the overall reaction of the native enzyme complex. However, there is a clear difference in the shape of the saturation curves of both types of reaction. Experiments with the free enzyme, with the component integrated into the native complex molecule and with enzyme complexes which are partially deficient in the pyruvate dehydrogenase component demonstrate that the type of saturation curves obtained were characteristic for the reaction observed rather than for the interaction of a high number of subunits of the pyruvate dehydrogenase complex.

Escherichia coli↗
PubMed1991

Pyruvate dehydrogenase and pyruvate carboxylase. Sites of pretranslational regulation by glucose of glucose-induced insulin release in pancreatic islets.

It has been shown previously that glucose-induced insulin release is completely absent in rat pancreatic islets that had been cultured for 1 day at low glucose (1 mM) and that it is restored by culturing islets for a 2nd day at high (20 mM) glucose (MacDonald, M. J., Fahien, L. A., McKenzie, D. I., and Moran, S. M. (1991) Am. J. Physiol. 259, E548-E554). It has been suggested that the incapacitation of glucose's insulinotropism is due to down-regulation of the synthesis of enzymes that process glucose's metabolic signal for insulin release. In the current study, results of metabolic, enzymic, and molecular biologic experiments were each consistent with (an) intramitochondrial site(s) of down-regulation in islets cultured at low glucose. Glucose metabolism was inhibited 80% in islets cultured at 1 mM glucose. The suppression of release of 14CO2 from [6-14C]glucose greater than from [U-14C]glucose greater than [3,4-14C]glucose greater than from [1-14C]glucose in islets cultured at low glucose indicated a mitochondrial site of down-regulation because C-6 of glucose can only be converted to CO2 in the citric acid cycle, whereas C-1 can be released as CO2 in the 6-phosphogluconate dehydrogenase [corrected] reaction, and C-6 of glucose dwells in the citric acid cycle longer than carbons 2-5 of glucose. Since carbons 3 and 4 of glucose can be decarboxylated in the pyruvate dehydrogenase reaction, incomplete suppression of CO2 formation from these carbons is consistent with suppression of pyruvate carboxylation as well as decarboxylation. Formation of 3HOH from [5-3H]glucose was equal in the two groups of islets, indicating that glycolysis as far as phosphoenolpyruvate was intact. This idea was supported by assays which showed that activities of enzymes of the glycolytic pathway between glucokinase/hexokinase and pyruvate kinase were equal in both types of islets. Additional studies indicated that regulation by glucose was at transcription of genes coding for some mitochondrial enzymes. Glucokinase, malic enzyme, and fumarase mRNAs were not affected by glucose, whereas the pyruvate dehydrogenase E1 alpha subunit and pyruvate carboxylase mRNAs were decreased 85-90% in islets cultured at 1 mM glucose. Pyruvate dehydrogenase enzyme activity was decreased to a similar extent in these islets. About 24 h was required for maximal (de)induction of pyruvate dehydrogenase E1 alpha and pyruvate carboxylase mRNAs, and the amounts of transcripts were proportional to the concentrations of glucose between 1 and 20 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1979

Effect of thyroid hormone on the turnover of rat liver pyruvate carboxylase and pyruvate dehydrogenase.

Immunochemical techniques have been utilized to study the effect of thyroid status on the content and rates of synthesis and degradation of pyruvate carboxylase and pyruvate dehydrogenase in rat liver. Liver from hyperthyroid rats had twice the pyruvate carboxylase activity of normal rats while thyroidectomized rats had about two-thirds of normal activity. Pyruvate dehydrogenase complex activity was unchanged in the hyperthyroid state but was significantly reduced (by a third) in hypothyroid rats. Changes in catalytic activity during altered thyroid status were by immunochemical means to be closely related to the amount of the hepatic enzymes present. Isotopic studies showed that the changes in the content of pyruvate carboxylase and pyruvate dehydrogenase reflected alterations in the rate of the synthesis of the enzymes with the degradation rates little affected by thyroid status. The half-life for pyruvate carboxylase was 4.6 days, and that for pyruvate dehydrogenase, 8.1 days. In both cases, the turnover time was slower than that of the average mitochondrial protein (t1/2 = 3.8 days) for the control animals.

Animals↗
PubMed1977

Evidence for separate enzymes of pyruvate decarboxylation and pyruvate synthesis in soluble extracts of Clostridium pasteurianum.

Additional evidence to that already presented (Sauer, F. D., Bush, R. S., and Stevenson, I. L. (1976) Biochim. Biophys. Acta 445, 518-520) suggests that pyruvate-ferredoxin oxidoreductase isolated from Clostridium pasteurianum consists of two separate enzymes: (a) pyruvate lyase, which catalyzes the CoA and electron acceptor-dependent decarboxylation of pyruvate, and (b) pyruvate synthase, which catalyzes the reduced ferredoxin-dependent carboxylation of acetyl-CoA to pyruvate. The enzymes separated on Sephadex G-200 and with acrylamide gel electrophoresis but complete separation of one enzyme free of the other was not achieved. Extensive purification procedures were not used because both enzymes are unstable. The results confirm published reports that pyruvate lyase contains thiamin and a chromophore which participates in electron transfer. Pyruvate synthase, however, did not appear to be a thiamin enzyme and there was no evidence to indicate participation of an enzyme chromophore in the pyruvate synthase reaction.

Carboxy-Lyases↗