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At least 19 recordsLinked to original sources
PubMed1990

Inhibition of pyruvate:ferredoxin oxidoreductase from Trichomonas vaginalis by pyruvate and its analogues. Comparison with the pyruvate decarboxylase component of the pyruvate dehydrogenase complex.

Pyruvate:ferredoxin oxidoreductase and the pyruvate dehydrogenase multi-enzyme complex both catalyse the CoA-dependent oxidative decarboxylation of pyruvate but differ in size, subunit composition and mechanism. Comparison of the pyruvate:ferredoxin oxidoreductase from the protozoon Trichomonas vaginalis and the pyruvate dehydrogenase component of the Escherichia coli pyruvate dehydrogenase complex shows that both are inactivated by incubation with pyruvate under aerobic conditions in the absence of co-substrates. However, only the former is irreversibly inhibited by incubation with hydroxypyruvate, and only the latter by incubation with bromopyruvate. Pyruvate:ferredoxin oxidoreductase activity is potently, but reversibly, inhibited by addition of bromopyruvate in the presence of CoA, and it is suggested that the mechanism involves formation of an adduct between CoA and bromopyruvate in the active site of the enzyme. It is proposed that both enzymes are inactivated by pyruvate through a mechanism involving oxidation of an enzyme-bound thiamin pyrophosphate/substrate adduct to form a tightly bound inhibitory species, possibly thiamin thiazolone pyrophosphate as hypothesized by Sumegi & Alkonyi.

Animals↗
PubMed1983

Specificity of the pyruvate dehydrogenase kinase for pyruvate dehydrogenase component bound to the surface of the kidney pyruvate dehydrogenase complex and evidence for intracore migration of pyruvate dehydrogenase component.

Using the bovine kidney pyruvate dehydrogenase complex we have investigated the mechanism whereby about three pyruvate dehydrogenase (active form) kinase molecules, tightly bound to the dihydrolipoyl transacetylase core, can rapidly phosphorylate and inactivate about 20 pyruvate dehydrogenase (active form) (PDHa) tetramers which are also bound to the 60-subunit core. Evidence is presented that PDHa kinase activity is not serviced by a process of dissociation and reassociation of PDHa. Rapid inactivation of a full complement of PDHa occurs at a rate exceeding the rate of dissociation of PDHa, indicating that a PDHa must move to the fixed kinase subunits without dissociating from the dihydrolipoyl transacetylase core. Consistent with that concept, at low concentrations of complex where a significant portion of PDHa is free, bound PDHa was inactivated at a rate equivalent to that at higher concentrations of complex, and free PDHa was phosphorylated more slowly at a rate closely approximated by the rate of association of free PDHa with the transacetylase core. Thus, with a low number of PDHa molecules bound, PDHa either is preferentially positioned for phosphorylation and inactivation by PDHa kinase or can rapidly become so positioned without dissociating from the transacetylase core.

Animals↗
PubMed1993

Effect of dexamethasone on gluconeogenesis, pyruvate kinase, pyruvate carboxylase and pyruvate dehydrogenase flux in isolated hepatocytes.

Treatment of 18 h-starved rats with dexamethasone and subsequent isolation and incubation of the hepatocytes in the presence of the steroid increased gluconeogenic flux with both 1.0 mM pyruvate and 1.0 mM lactate plus 0.2 mM pyruvate as the substrate. The magnitude of stimulation was comparable with both substrates. The increase in glucose output was accompanied by an increased flux through pyruvate carboxylase, although the absolute flux and magnitude were considerably less in the presence of the more reduced substrate. The effect of the steroid on the flux through pyruvate dehydrogenase was substrate-dependent, an inhibition occurring with the more oxidized substrate. There was no effect of steroid treatment on [1-14C]lactate or pyruvate oxidation or on tricarboxylic-acid-cycle flux as measured by [3-14C]pyruvate oxidation. Dexamethasone treatment resulted in a parallel increase in both pyruvate kinase flux and glucose synthesis with both substrates employed, indicating that the steroid had no effect on the partitioning of phosphoenolpyruvate between pyruvate and lactate formation and gluconeogenesis. Similarly there was no effect of the steroid on either the activity ratio or the total pyruvate kinase activity in the cells. It is suggested that the acute effect of the dexamethasone to increase gluconeogenesis resides at the level of phosphoenolpyruvate formation, i.e. pyruvate carboxylase and possibly phosphoenolpyruvate carboxykinase.

Animals↗
PubMed1996

Measurement of metabolic fluxes through pyruvate kinase, phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase, and pyruvate carboxylate in hepatocytes of different acinar origin.

Isolated hepatocytes were prepared from the periportal and perivenous regions of the liver of 18-h-starved rats. These showed characteristics enzyme patterns and an enhanced rate of ureagenesis in the periportal cells; however, total cellular ATP content was unchanged in the two cell types. Measurements of pyruvate kinase flux showed no significant difference in the overall rate in the two cell types; however, the flux through phosphoenolpyruvate (PEP) carboxykinase was significantly higher in the periportal cells, such that the percentage of PEP being metabolized by pyruvate kinase was enhanced in the perivenous cells. The increase in partitioning of PEP through pyruvate kinase could account for only a small percentage of the difference in gluconeogenic flux in the two cell types, suggesting that the rate of provision of PEP was the principal limiting factor for glucose synthesis. The flux through pyruvate dehydrogenase showed no significant metabolic zonation, whereas pyruvate carboxylase flux was enhanced in the periportal zone. The partitioning of pyruvate between pyruvate carboxylase and pyruvate dehydrogenase was increase 2.8-fold in the periportal cells compared to that in the perivenous cells and it is suggested that this, together with possible alterations in phosphoenolpyruvate carboxykinase, is primarily responsible for the different gluconeogenic rates in the two zones of the liver.

Animals↗
PubMed1995

Use of pyruvate oxidase to overcome pyruvate inhibition during the lactate to pyruvate reaction for assaying lactate dehydrogenase in serum.

Automated assays of lactate dehydrogenase (LD) in serum are based on measuring the rate of NADH produced in a reverse LD reaction using lactate and NAD. The observed nonlinearity of LD reaction used in earlier assays performed in phosphate buffers has generally been attributed to the formation of a ternary complex of NAD, pyruvate, and phosphate. this is not satisfactory to explain the course of assay reaction carried out in organic buffers. Investigation of the possible causes of nonlinearity during the course of the reverse LD reaction during LD assays performed in Tris or other organic buffers indicated that inhibition of LD activity by pyruvate may be chiefly responsible for the observed effects, especially in serum exhibiting abnormally high LD enzyme activity. Most of the LD activity in serum was inhibited by 5 mMoles/L pyruvate. By contrast, the LD isoenzyme activities were inhibited partially at 0.5 mMole/L pyruvate, LD1 being the most and LD4 the least susceptible. In assays of serum samples with abnormally high LD and PYR concentration using LD reagent containing Tris buffer, pH 9.3, the inclusion of a bacterial pyruvate oxidase (PO) enabled the removal of pyruvate accumulating in situ, making it possible to assay LD activity in the absence of inhibitory concentration of pyruvate. The inclusion of 10 U/L of PO in our routine LD reagent was sufficient to overcome pyruvate inhibition, thus permitting the assay of serum exhibiting high LD activity, hence the extension of the upper limits of linearity of LD assay without compromising assay performance.

Coloring Agents↗
PubMed2002

X-ray structure of pyruvate formate-lyase in complex with pyruvate and CoA. How the enzyme uses the Cys-418 thiyl radical for pyruvate cleavage.

The glycyl radical enzyme pyruvate formate-lyase (PFL) synthesizes acetyl-CoA and formate from pyruvate and CoA. With the crystal structure of the non-radical form of PFL in complex with its two substrates, we have trapped the moment prior to pyruvate cleavage. The structure reveals how the active site aligns the scissile bond of pyruvate for radical attack, prevents non-radical side reactions of the pyruvate, and confines radical migration. The structure shows CoA in a syn conformation awaiting pyruvate cleavage. By changing to an anti conformation, without affecting the adenine binding mode of CoA, the thiol of CoA could pick up the acetyl group resulting from pyruvate cleavage.

Acetyltransferases↗
PubMed2003

Chronic high glucose lowers pyruvate dehydrogenase activity in islets through enhanced production of long chain acyl-CoA: prevention of impaired glucose oxidation by enhanced pyruvate recycling through the malate-pyruvate shuttle.

In islet beta-cells, the high expression of pyruvate carboxylase and the functional importance of the downstream anaplerosis pathways result in a unique characteristic whereby high glucose and fatty acids both increase production of a key fatty acid metabolite, long chain acyl-CoA, for signaling and enzyme regulation in beta-cells. We showed previously in islets that pyruvate dehydrogenase (PDH) activity is lowered by excess fatty acids (the so-called Randle effect). We have now investigated PDH activity and pyruvate metabolism in islets after 48-h culture at 16.7 mmol/liter glucose. Active PDH V(max) was lowered 65% by 48 h of high glucose, and this effect was markedly attenuated by co-culture with triacsin C, which inhibits acyl-CoA synthase. Despite the large reduction in PDH activity, glucose oxidation was twice normal. The reason was continued metabolism of pyruvate through pyruvate carboxylase (V(max), 83% of control) and diversion of flux through the pyruvate-malate shuttle. The result was a 3-fold increase of the pyruvate concentration that overcame the lowered PDH activity by mass action as shown by glucose oxidation measured with [6-(14)C]glucose being twice normal. In addition, glucose-induced insulin secretion was 3-fold increased after 48 h of high glucose, and this effect was totally blocked by co-culture with triacsin C. These results show that a unique feature of islet beta-cells is not only fatty acids but also excess glucose that impairs PDH activity. Also, a specialized trait of beta-cells is a long chain acyl-CoA-mediated defense mechanism that prevents a reduction in glucose oxidation and consequently in insulin secretion.

Acyl Coenzyme A↗
PubMed1973

Effect of insulin on pyruvate metabolism in epididymal adipose tissue of the rat. Correlation of intracellular pyruvate contents and pyruvate dehydrogenase activity.

A method is described to measure the intracellular content of pyruvate and lactate in epididymal adipose tissue of the rat. The intracellular pyruvate concentration was approx. 330mum. Intracellular pyruvate contents and the rates of pyruvate output were increased when NNN'N'-tetramethyl-p-phenylenediamine was added, and decreased in the presence of alanine. Insulin addition caused an increase in intracellular pyruvate contents only at the earlier time-period studied (1.5min as against 20min). Pyruvate dehydrogenase activity was increased in adipose tissue incubated in vitro with insulin. This increase occurred subsequent to the rise in the intracellular pyruvate content induced by insulin addition. The possible physiological implications are discussed.

Adipose Tissue↗
PubMed1983

Control of the activation/inactivation of pyruvate, Pi dikinase from the C4 plant maize by adenylate energy charge, pyruvate, and analogs of pyruvate.

Pyruvate, Pi dikinase, which is localized in the mesophyll chloroplasts of C4 plants, requires a high adenylate energy charge for conversion of the enzyme from the inactive to the active form. The inactivation process is favored by a low energy charge, being maximal at values below 0.7. Pyruvate and analogs of pyruvate, oxamate and oxalate, strongly inhibit the inactivation process at millimolar levels. The results suggest that light activation of the enzyme in vivo may be mediated by an increased adenylate energy charge in the chloroplast. Pyruvate may allow a higher steady-state level of activation to be achieved in vivo by inhibiting inactivation.

Adenine Nucleotides↗
PubMed1993

The relationships between transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase of the pyruvate dehydrogenase complex.

The amino acid sequences of four thiamine pyrophosphate-requiring enzymes were aligned with the published amino acid sequence of the transketolase of Hansenula polymorpha. Sequences of the combined alpha and beta subunits of the E1 enzyme of the pyruvate dehydrogenase complexes of Homo sapiens and Bacillus stearothermophilus aligned well with the transketolase while the E1 of the pyruvate dehydrogenase complex of Escherichia coli aligned easily provided a non-aligning segment of 77 amino acids was omitted. The non-acetylating pyruvate decarboxylase of Saccharomyces cerevisiae could only be aligned if the sequence was cut in two with the C-terminus corresponding to the N-terminus of the other TPP-dependent enzymes. Using the published 2.5 A resolution of the X-ray crystal structure of Saccharomyces cerevisiae transketolase as a template we show that a hydrophobic region of the beta-subunit of the PDH E1 alpha beta enzymes likely contains a binding site for the thiazolium ring of TPP and key motifs are retained in common by all the TPP-dependent enzymes considered, which are essential for catalysis.

Amino Acid Sequence↗
PubMed1985

Pyruvate dehydrogenase kinase activity of pig heart pyruvate dehydrogenase (E1 component of pyruvate dehydrogenase complex).

The pyruvate dehydrogenase (E1) and acetyltransferase (E2) components of pig heart and ox kidney pyruvate dehydrogenase (PDH) complex were separated and purified. The E1 component was phosphorylated (alpha-chain) and inactivated by MgATP. Phosphorylation was mainly confined to site 1. Addition of E2 accelerated phosphorylation of all three sites in E1 alpha and inactivation of E1. On the basis of histone H1 phosphorylation, E2 is presumed to contain PDH kinase, which was removed (greater than 98%) by treatment with p-hydroxymercuriphenylsulphonate. Stimulation of ATP-dependent inactivation of E1 by E2 was independent of histone H1 kinase activity of E2. The effect of E2 is attributed to conformational change(s) induced in E1 and/or E1-associated PDH kinase. PDH kinase activity associated with E1 could not be separated from it be gel filtration or DEAE-cellulose chromatography. Subunits of PDH kinase were not detected on sodium dodecyl sulphate/polyacrylamide gels of E1 or E2, presumably because of low concentration. The activity of pig heart PDH complex was increased by E2, but not by E1, indicating that E2 is rate-limiting in the holocomplex reaction. ATP-dependent inactivation of PDH complex was accelerated by E1 or by phosphorylated E1 plus associated PDH kinase, but not by E2 plus presumed PDH kinase. It is suggested that a substantial proportion of PDH kinase may accompany E1 when PDH complex is dissociated into its component enzymes. The possibility that E1 may possess intrinsic PDH kinase activity is considered unlikely, but may not have been fully excluded.

Acetyltransferases↗
PubMed1977

The phosphoenol-pyruvate branchpoint in adult Hymenolepis diminuta (Cestoda): a study of pyruvate kinase and phosphoenol-pyruvate carboxykinase.

The properties of pyruvate kinase (PK) and phosphoenol pyruvate carboxykinase (PEP CK), two enzymes that determine the preferrential accumulation of either succinate or lactate as endproducts of carbohydrate metabolism, are described in adult Hymenolepis diminuta. PK activity at Vmax and Km levels of PEP was unaffected by ATP, alanine, FDP4, OR H+ ions, but was inhibited by 50% at 6.3 mM L-lactate and 30 mM HCO3. The addition of 30 mM HCO3 increased the Km(PEP) by 6-fold but did not alter the Vmax. The inhibition of PK by HCO3 cannot be explained entirely by an effect of ionic strength, but probably represents a specific modulator-enzyme interaction. Under similar conditions PEP CK was maximally activated. Although L-lactate inhibited PEP CK (Ki(lac) = 1.8 mM), this effector may play a minor role in regulation of PEP flux. These results implicate the poise of the HCO3-:CO2 system as a major determiner of endproduct accumulation in H. diminuta.

Animals↗
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↗
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↗
PubMed1972

Effects of intrabrachial arterial infusion of pyruvate on forearm tissue metabolism. Interrelationships between pyruvate, lactate, and alanine.

Postabsorptive release of alanine from forearm skeletal muscle is large relative to other amino acids, suggesting new synthesis by transamination of pyruvate. This hypothesis was tested and the pathway quantified in six subjects, each given two 30 min intrabrachial arterial pyruvate infusions. The first (12 mumoles/min) supplied approximately that amount of pyruvate produced endogenously by glycolysis in resting muscle. The second (36 mumoles/min) approximated endogenous pyruvate production by glycolysis during moderate exercise. Changes in balance across forearm tissues of pyruvate, glucose, lactate, and amino acids were measured. The time-course of pyruvate equilibration across fore-arm muscles was detailed in three additional subjects. The two infusions increased arterial pyruvate from 64 to 674 and 1776 mumoles/liter respectively. Muscle consumed 72% of the exogenous pyruvate during both infusions. Outputs of lactate and alanine increased, accounting respectively for 30.3 and 6.7% of the pyruvate at the low infusion rate, and 17.1 and 3.8% at the high rate. The remaining pyruvate probably was oxidized. Muscle release of valine, isoleucine, and leucine decreased during the high dose infusion. Additionally, adipose tissue plus skin released more alanine and lactate during the high dose infusion. Other metabolies were unchanged.Thus, both muscle and adipose tissue plus skin synthesize alanine from pyruvate. Lactate production considerably exceeds that of alanine. In muscle, increased availability of intracellular pyruvate serving as a nitrogen acceptor may facilitate branched chain amino acid oxidation. Muscle consumption of infused pyruvate is rapid, and detailed studies of its equilibration suggest that passage across the muscle cell membrane is rate limiting.

Adipose Tissue↗
PubMed1986

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex.

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.

Alanine↗