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PubMed1977

Pyruvate metabolism in bovine epididymal spermatozoa.

Treating bovine epididymal spermatozoa with rutamycin or rotenone inhibited both respiration and motility supported by endogenous substrates. When oxidative phosphorylation had been blocked with various inhibitors, pyruvate was metabolized to yield ATP and restored motility. Fructose, which is metabolized via glycolysis to yield ATP, was also able to resuscitate the cells. Other substrates tested (lactate, acetate, alpha-ketoglutarate, or glyoxylate) were unable to restore motility in rutamycin-treated cells. In the presence of pyruvate, the phosphorylation uncoupler, carbonylcyanide-p-trifluoromethyoxphenylhydrazone, reduced motility and ATP to common levels in untreated cells or cells treated with rutamycin or rotenone. Pyruvate is thus metabolized to produce ATP by a pathway independent of oxidative phosphorylation associated with the electron transport chain. 5-Methoxyindole-2-carboxylic acid, an inhibitor of lipoyldehydrogenase, prevented the increase of motility and ATP in rutamycin-treated cells, indicating that alpha-keto acid oxidation is involved in the production of ATP from pyruvate when rutamycin is present. With pyruvate present, bongkrekic acid, antimycin A, and anaerobiosis eliminated motility, reduced ATP to low levels, and also significantly reduced the rate of pyruvate metabolism. Acetate was produced from pyruvate only when cellular ATP concentrations were low. Decreases in free carnitine concentrations showed that pyruvate initially used was converted to acetylcarnitine. The results indicate that the intramitochondrial lactate dehydrogenase X, which is unique to spermatozoa, allows the NADH resulting from pyruvate oxidation to reduce other pyruvate molecules to lactate. Pyruvate thus competes with, and can substitute for, the NADH dehydrogenase of the electron transport chain. Pyruvate rapidly repletes the acetylcarnitine pool under a variety of conditions.

Adenosine Diphosphate↗
PubMed1984

The stimulation of mitochondrial pyruvate carboxylation after dexamethasone treatment of rats.

Treatment of rats for 3 h with dexamethasone was shown to stimulate both pyruvate carboxylation and decarboxylation in the subsequently isolated mitochondria. The effect of hormone treatment on pyruvate carboxylation was also apparent in liver homogenates assayed within minutes of killing the animal and was independent of the temperature at which the assay was performed, suggesting that it was not an artifact of the mitochondrial preparation procedure. The stimulation of both aspects of pyruvate metabolism in the intact organelle was independent of the induction of either pyruvate carboxylase or pyruvate dehydrogenase. Similarly, there was no change in the percentage of pyruvate dehydrogenase in the active form, indicating that the effect of steroid treatment on pyruvate oxidation was not via changes in the degree of phosphorylation of the enzyme. Adrenalectomizing the animals for a period of 14 days before the experiment had no effect on either parameter. Glucocorticoid treatment of the animals increased the rate of pyruvate uptake into the mitochondria, as measured by the titration of pyruvate metabolism with alpha-cyano-4-hydroxycinnamate, a specific inhibitor of the pyruvate translocator. It also increased the intramitochondrial concentrations of acetyl-CoA and ATP and led to an elevated [ATP]/[ADP] ratio within the mitochondria. It is suggested that both enzymes of pyruvate metabolism exist in the mitochondria under considerable restraint and that glucocorticoids act to relieve this restraint by alterations in substrate supply and the intramitochondrial concentrations of effector molecules.

Adrenalectomy↗
PubMed1976

Regulation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria.

The mechanism by which fatty acid addition leads to the inactivation of pyruvate dehydrogenase in intact rat liver mitochondria was investigated. In all cases the fatty acid octanoate was added to mitochondria oxidizing succinate. Addition of fatty acid caused an inactivation of pyruvate dehydrogenase in mitochondria incubated under State 3 conditions (glucose plus hexokinase), in uncoupled, oligomycin-treated mitochondria, and in rotenone-menadione-treated mitochondria, but not in uncoupled mitochondria or in mitochondria incubated under State 4 conditions. A number of metabolic conditions were found in which pyruvate dehydrogenase was inactivated concomitant with an elevation in the ATP/ADP ratio. This is consistent with the inverse relationship between the ATP/ADP ratio and the pyruvate dehydrogenase activity proposed by various laboratories. However, in several other metabolic conditions pyruvate dehydrogenase was inactivated while the ATP/ADP ratio either was unchanged or even decreased. This observation implies that there are likely other regulatory factors involved in the fatty acid-mediated inactivation of pyruvate dehydrogenase. Incubation conditions in State 3 were found in which the ATP/ADP and the acetyl-CoA/CoASH ratios remained constant and the pyruvate dehydrogenase activity was correlated inversely with the NADH/NAD+ ratio. Other State 3 conditions were found in which the ATP/ADP and the NADH/NAD+ ratios remained constant while the pyruvate dehydrogenase activity was correlated inversely with the acetyl-CoA/CoASH ratio. Further evidence supporting these experiments with intact mitochondria was the observation that the pyruvate dehydrogenase kinase activity of a mitochondrial extract was stimulated strongly by acetyl-CoA and was inhibited by NAD+ and CoASH. In contrast to acetyl-CoA, octanoyl-CoA inhibited the kinase activity. These results indicate that the inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria may be mediated through effects of the NADH/NAD+ ratio and the acetyl-CoA/CoASH ratio on the interconversion of the active and inactive forms of the enzyme complex catalyzed by pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.

Acetyl Coenzyme A↗
PubMed1986

Effect of insulin on the metabolic distribution of carbons 1, 2, and 3 of pyruvate.

It has long been known that the carbons of pyruvate are converted to CO2 at different points in the metabolic process. This report deals with the observation that insulin affects the oxidation of carbons 2 and 3 primarily and has little effect on the oxidation of the carboxyl carbon. Oxidation of different carbons of pyruvate and their incorporation into various metabolic components was studied in isolated rat hepatocytes. Insulin stimulated the 14CO2 production from [2-14C]- and [3-14C]pyruvate and from [U-14C]alanine. However, it had little or no effect on the activity of the pyruvate dehydrogenase complex as measured by the evolution of 14CO2 from [1-14C]pyruvate or [1-14C] alanine. Insulin also stimulated the incorporation of carbons 2 and 3 of pyruvate into protein but had no effect on the incorporation of carbon 1. Incorporation of [1-14C]- and [U-14C]alanine into protein was differentially enhanced by insulin in a manner similar to that of the pyruvate carbons. The fact that insulin stimulates the incorporation of [1-14C]alanine into protein but not [1-14C]pyruvate suggests the possibility of a compartmentation of pyruvate metabolism in the isolated hepatocytes. These studies show that the stimulation of [2-14C]- and [3-14C]pyruvate incorporation into protein involves the stimulatory effect of insulin on the activity of the Krebs cycle which is evident from the fact that insulin did not stimulate the pyruvate carbons to enter protein via alanine but the incorporation via glutamate was increased by about 40%.

Alanine↗
PubMed1990

The protective action of pyruvate on recovery of ischemic rat heart: comparison with other oxidizable substrates.

The recovery of both contractile performance and metabolic response of rat heart following 1 h of ischemia after equilibration with glucose + insulin (glucose-ischemia) or with pyruvate (pyruvate-ischemia), was tested in normoxic reperfusion in the presence of glucose + insulin, pyruvate, lactate or acetate. In glucose-ischemia only the reperfusion with pyruvate results in a complete recovery of the contractile force (left ventricular pressure, LVP) (170%) and good recovery of high energy phosphate compounds. Lower LVP and tissue energy charge were found in glucose reperfusion and even less in lactate and acetate reperfusion. Disappearance of the IMP accumulated during ischemia is evident only in the pyruvate reperfusion indicating a higher metabolic recovery. On the contrary in pyruvate-ischemia all types of reperfusion tested were effective in reactivating the contractile force (although acetate to a lesser extent); the contractile activity was accompanied by a good recovery of phosphocreatine, ATP, energy charge and by the decrease of IMP. Large decreases of adenine nucleotides and NADP and lower decreases of NAD are observed during ischemia/reperfusion in both systems. Pyruvate-ischemia is quite similar to, if not worse than glucose-ischemia, for all the metabolic parameters considered, but not worse for the possibility of recovery. Some specific effect of pyruvate should be exerted during the ischemic phase. The mechanism of pyruvate protection is discussed in relationship to: (i) the possible activation of pyruvate dehydrogenase, (ii) the activation of NADPH-dependent peroxide scavenging systems, (iii) the direct scavenging action of pyruvate on H2O2.

Acetates↗
PubMed1980

The regulation of pyruvate oxidation during membrane depolarization of rat brain synaptosomes.

Studies were performed to elucidate factors involved in the regulation of pyruvate dehydrogenase activity in rat brain synaptosomes during membrane depolarization. Addition of 24 mM-KCl to synaptosomes resulted in increases in rates of O2 consumption (90%) and [1-(14)C]pyruvate decarboxylation (85%) and in the active/total ratio of extractable pyruvate dehydrogenase (90--100%) within 10 s. Neither pyruvate (10 mM) nor dichloroacetate (10 mM) affected the activation state of the enzyme complex. Also, the activation state of pyruvate dehydrogenase was unaffected by addition of 1 mM-octanoate, L-(--)-carnitine, 3-hydroxybutyrate, glutamate, citrate, lactate, L-malate, acetate, acetaldehyde or ethanol. Removal of Ca2+ by using EGTA lowered the active/total ratio to about 70%, although the rate of O2 consumption and pyruvate decarboxylation was unaffected. Rates of pyruvate decarboxylation in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone in the presence and absence of NaF and EGTA demonstrated a linear correlation with changes in the activity of the enzyme complex. This observation indicated that a change in the activation state of pyruvate dehydrogenase from 90 to 100% active could result in a 27% increase in the rate of pyruvate decarboxylation. It is suggested that the pyruvate dehydrogenase complex is an important site for the regulation of substrate utilization in rat brain synaptosomes. Further, the phosphorylation/dephosphorylation system and direct feedback-inhibitory effects on the enzyme complex both play a significant role in rapidly adapting pyruvate decarboxylation to changes in the requirements for mitochondrial energy production.

Animals↗
PubMed1982

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.

Acetates↗
PubMed1984

Influence of oleate oxidation on pyruvate production and utilization in hepatocytes isolated from fed rats. Effect of 2-[5-(4-chlorophenyl)pentyl]oxiran-2-carboxylate.

Oleate (0.35 and 1.5 mM) decreases, in a concentration-dependent manner, lactate and pyruvate concentrations in hepatocytes, isolated from fed rats, incubated without exogenous substrate. The glycolytic flux, estimated at 18 mM-glucose by [6-3H]-glucose detritiation and apparent production of lactate and pyruvate, is decreased by oleate. The measurement of glycolytic intermediates shows a cross-over at the phosphofructokinase level, which might result from an increased citrate concentration. All those effects are dependent on oleate oxidation in mitochondria, since they are suppressed by 1 microM-2-[5-(4-chlorophenyl)pentyl]oxiran-2-carboxylate (POCA), an inhibitor of the mitochondrial entry of oleate, but not of its uptake by hepatocytes. The decrease of lactate and pyruvate also results from an oleate-induced enhancement of pyruvate utilization by hepatocytes, as shown by the increase of 14CO2 formation from [1-14C]- and [3-14C]-pyruvate, especially at low (0.4 mM) pyruvate concentration. Those oleate effects are also suppressed by POCA. They might be due to an enhanced flux through pyruvate carboxylase and pyruvate dehydrogenase, as a result of an oleate-induced increase in the mitochondrial concentrations of pyruvate and acetyl-CoA. Thus oleate oxidation inhibits production of lactate and pyruvate in fed-rat hepatocytes, as it does in other tissues. But, in the liver, it also enhances the mitochondrial utilization of pyruvate. The physiological implications of those findings are discussed.

Animals↗
PubMed2004

Engineering Escherichia coli for efficient conversion of glucose to pyruvate.

Escherichia coli TC44, a derivative of W3110, was engineered for the production of pyruvate from glucose by combining mutations to minimize ATP yield, cell growth, and CO2 production (DeltaatpFH DeltaadhE DeltasucA) with mutations that eliminate acetate production [poxB::FRT (FLP recognition target) DeltaackA] and fermentation products (DeltafocA-pflB DeltafrdBC DeltaldhA DeltaadhE). In mineral salts medium containing glucose as the sole carbon source, strain TC44(DeltafocA-pflB DeltafrdBC DeltaldhA DeltaatpFH DeltaadhE DeltasucA poxB::FRT DeltaackA) converted glucose to pyruvate with a yield of 0.75 g of pyruvate per g of glucose (77.9% of theoretical yield; 1.2 g of pyruvate liters(-1).h(-1)). A maximum of 749 mM pyruvate was produced with excess glucose. Glycolytic flux was >50% faster for TC44 producing pyruvate than for the wild-type W3110 during fully aerobic metabolism. The tolerance of E. coli to such drastic changes in metabolic flow and energy production implies considerable elasticity in permitted pool sizes for key metabolic intermediates such as pyruvate and acetyl-CoA. In strain TC44, pyruvate yield, pyruvate titer, and the rate of pyruvate production in mineral salts medium were equivalent or better than previously reported for other biocatalysts (yeast and bacteria) requiring complex vitamin feeding strategies and complex nutrients. TC44 offers the potential to improve the economics of pyruvate production by reducing the costs of materials, product purification, and waste disposal.

Escherichia coli↗
PubMed2006

Ligand-induced effects on pyruvate dehydrogenase kinase isoform 2.

Tryptophan fluorescence was used to analyze binding of ligands to human pyruvate dehydrogenase isoform 2 (PDHK2) and to demonstrate effects of ligand binding on distal structure of PDHK2 that is required for binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase. Ligand-altered binding of PDHK2 to L2 and effects of specific ligands on PDHK2 oligomeric state were characterized by analytical ultracentrifugation. ATP, ADP, and pyruvate markedly quenched the tryptophan fluorescence of PDHK2 and gave maximum quenching/L0.5 estimates: approximately 53%/3 microM for ATP; approximately 49%/15 microM for ADP; and approximately 71%/approximately 590 microM for pyruvate. The conversion of Trp-383 to phenylalanine completely removed ATP- and ADP-induced quenching and > or = 80% of the absolute decrease in fluorescence due to pyruvate. The W383F-PDHK2 mutant retained high catalytic activity. Pyruvate, added after ADP, quenched Trp fluorescence with an L0.5 of 3.4 microM pyruvate, > or = 150-fold lower concentration than needed with pyruvate alone. ADP-enhanced binding of pyruvate was maintained with W383F-PDHK2. Binding of PDHK2 dimer to L2 is enhanced when L2 are housed in oligomeric structures, including the glutathione S-transferase (GST)-L2 dimer, and further strengthened by reduction of the lipoyl groups (GST-L2(red)) (Hiromasa and Roche (2003) J. Biol. Chem. 278, 33681-33693). Binding of PDHK2 to GST-L2(red) was modestly hindered by 200 microM level of ATP or ADP or 5.0 mM pyruvate; a marked change to nearly complete prevention of binding was observed with ATP or ADP plus pyruvate at only 100 microM levels, and these conditions caused PDHK2 dimer to associate to a tetramer. These changes should make major contributions to synergistic inhibition of PDHK2 activity by ADP and pyruvate. Ligand-induced changes that interfere with PDHK2 binding to GST-L2(red) may involve release of an interdomain cross arm between PDHK2 subunits in which Trp-383 plays a critical anchoring role.

Adenosine Diphosphate↗
PubMed1997

Metabolic responses of pyruvate decarboxylase-negative Saccharomyces cerevisiae to glucose excess.

In Saccharomyces cerevisiae, oxidation of pyruvate to acetyl coenzyme A can occur via two routes. In pyruvate decarboxylase-negative (Pdc-) mutants, the pyruvate dehydrogenase complex is the sole functional link between glycolysis and the tricarboxylic acid (TCA) cycle. Such mutants therefore provide a useful experimental system with which to study regulation of the pyruvate dehydrogenase complex. In this study, a possible in vivo inactivation of the pyruvate dehydrogenase complex was investigated. When respiring, carbon-limited chemostat cultures of wild-type S. cerevisiae were pulsed with excess glucose, an immediate onset of respiro-fermentative metabolism occurred, accompanied by a strong increase of the glycolytic flux. When the same experiment was performed with an isogenic Pdc- mutant, only a small increase of the glycolytic flux was observed and pyruvate was the only major metabolite excreted. This finding supports the hypothesis that reoxidation of cytosolic NADH via pyruvate decarboxylase and alcohol dehydrogenase is a prerequisite for high glycolytic fluxes in S. cerevisiae. In Pdc- cultures, the specific rate of oxygen consumption increased by ca. 40% after a glucose pulse. Calculations showed that pyruvate excretion by the mutant was not due to a decrease of the pyruvate flux into the TCA cycle. We therefore conclude that rapid inactivation of the pyruvate dehydrogenase complex (e.g., by phosphorylation of its E1 alpha subunit, a mechanism demonstrated in many higher organisms) is not a relevant mechanism in the response of respiring S. cerevisiae cells to excess glucose. Consistently, pyruvate dehydrogenase activities in cell extracts did not exhibit a strong decrease after a glucose pulse.

Acetyl Coenzyme A↗
PubMed1982

Effects of glucose, pyruvate, lactate, and amino acids on muscle protein synthesis.

Protein synthesis was measured in rat diaphragms incubated with serum amino acids + 0.35 mM L-[2,6-3H]tyrosine and different energy-yielding substrates. Muscles incubated with 5.5 mM glucose (with or without actinomycin D) synthesized more protein than those incubated with 11 mM pyruvate or 11 mM lactate. Tissue ATP decreased during incubation with lactate, but pyruvate maintained ATP, ADP, and creatine phosphate as well as glucose. Glucose 6-phosphate decreased in muscles incubated in glucose-free media. 14CO2 production from substrates was [1-14C]pyruvate greater than [1-14C]lactate greater than [3,4-14C]glucose. Intracellular lactate/pyruvate was measured to assess cytoplasmic free NADH/NAD+; the effect of different media on these ratios was lactate greater than glucose = lactate + pyruvate greater than pyruvate + glucose greater than pyruvate. Lactate + pyruvate (8.8 + 2.2 mM) supported protein synthesis better than pyruvate and as well as glucose. Adding glucose to pyruvate accelerated protein synthesis and increased NADH/NAD+. Iodoacetate (0.1 mM) inhibited glycolytic NAD reduction and abolished the stimulatory effect of glucose on protein synthesis in the presence of pyruvate. Supplementation of pyruvate media with 1 mM leucine or isoleucine stimulated protein synthesis, but beta-hydroxybutyrate, malate, alpha-ketoisocaproate, and all other amino acids were ineffective. The cytoplasmic redox potential may act as a translational modulator of protein synthesis in skeletal muscle.

Adenosine Triphosphate↗
PubMed1981

Relationships between pyruvate decarboxylation and branched-chain volatile acid synthesis in Ascaris mitochondria.

The rate of 14CO2 evolution from 1-[14C]pyruvate by intact Ascaris mitochondria was very slow, but increased with increasing concentrations of pyruvate. At all concentrations of pyruvate, in an aerobic environment, pyruvate decarboxylation was stimulated greatly by the addition of fumarate, malate, or succinate. However, under anaerobic conditions, only malate and fumarate stimulated pyruvate decarboxylation; succinate had no effect. This implies that the aerobic metabolism of succinate, presumably to other dicarboxylic acids, may be required for the stimulation. Incubation of sonicated mitochondria with pyruvate plus fumarate, under rate-limiting concentrations of NAD+, resulted in approximately equal quantities of pyruvate utilized and succinate formed, suggesting that pyruvate oxidation and fumarate reduction may be linked. Branched-chain, volatile fatty acids were not formed during incubations with either malate or succinate, or succinate plus acetate. However, incubations of intact Ascaris mitochondria with pyruvate plus succinate yielded 2-methylbutyrate and 2-methylvalerate, whereas incubations with pyruvate plus propionate yielded almost exclusively 2-methylvalerate. Oxygen dramatically inhibited the synthesis of the branched-chain acids from succinate plus pyruvate, attesting to the apparent anaerobic nature of Ascaris mitochondrial metabolism. Significantly, the addition of glucose plus ADP stimulated the formation of all volatile fatty acids. Therefore, the synthesis of branched-chain acids may be related directly to increased energy generation. Alternatively, they may function in the regulatory role of maintaining the mitochondrial redox balance.

Adenosine Diphosphate↗
PubMed1992

The role of the matrix calcium level in the enhancement of mitochondrial pyruvate carboxylation by glucagon pretreatment.

The effect of Ca2+ on the rate of pyruvate carboxylation was studied in liver mitochondria from control and glucagon-treated rats, prepared under conditions that maintain low Ca2+ levels (1-3 nmol/mg of protein). When the matrix-free [Ca2+] was low (less than 100 nM), the rate of pyruvate carboxylation was not significantly different in mitochondria from control and glucagon-treated rats. Accumulation of 5-8 nmol of Ca2+/mg, which increased the matrix [Ca2+] to 2-5 microM in both preparations, significantly enhanced pyruvate carboxylase flux by 20-30% in the mitochondria from glucagon-treated rats, but had little effect in control preparations. Higher levels of Ca2+ (up to 75 nmol/mg) inhibited pyruvate carboxylation in both preparations, but the difference between the mitochondria from control and glucagon-treated animals was maintained. The enhancement of pyruvate dehydrogenase flux by mitochondrial Ca2+ uptake was also significantly greater in mitochondria from glucagon-treated rats. These differential effects of Ca2+ uptake on enzyme fluxes did not correlate with changes in the mitochondrial ATP/ADP ratio, the pyrophosphate level, or the matrix volume. Arsenite completely prevented 14CO2 incorporation when pyruvate was the only substrate, but caused only partial inhibition when succinate and acetyl carnitine were present as alternative sources of energy and acetyl-CoA. Under these conditions, mitochondria from glucagon-treated rats were less sensitive to arsenite than the control preparations, even at low Ca2+ levels. We conclude that the Ca(2+)-dependent enhancement of pyruvate carboxylation in mitochondria from glucagon-treated rats is a secondary consequence of pyruvate dehydrogenase activation; glucagon treatment is suggested to affect the conditions in the mitochondria that change the sensitivity of the pyruvate dehydrogenase complex to dephosphorylation by the Ca(2+)-sensitive pyruvate dehydrogenase phosphatase.

Adenosine Diphosphate↗
PubMed2001

Intracellular compartmentation of pyruvate in primary cultures of cortical neurons as detected by (13)C NMR spectroscopy with multiple (13)C labels.

The intracellular compartmentation of pyruvate in primary cultures of cortical neurons was investigated by high resolution (13)C NMR using mixtures of different pyruvate precursors conveniently labeled with (13)C or unlabeled. Cells were incubated with 1-5 mM (1-(13)C, 1,2-(13)C(2) or U-(13)C(6)) glucose only or with mixtures containing 1.5 mM (1-(13)C or U-(13)C(6)) glucose, 0.25-2.5 mM (2-(13)C or 3-(13)C) pyruvate and 1 mM malate. Extracts from cells and incubation media were analyzed by (13)C NMR to determine the relative contributions of the different precursors to the intracellular pyruvate pool. When ((13)C) glucose was used as the sole substrate fractional (13)C enrichments and (13)C isotopomer populations in lactate and glutamate carbons were compatible with a unique intracellular pool of pyruvate. When mixtures of ((13)C) glucose, ((13)C) pyruvate and malate were used, however, the fractional (13)C enrichments of the C2 and C3 carbons of lactate were higher than those of the C2 and C3 carbons of alanine and depicted a different (13)C isotopomer distribution. Moreover, neurons incubated with 1 mM (1,2-(13)C(2)) glucose and 0.25-5 mM (3-(13)C) pyruvate produced exclusively (3-(13)C) lactate, revealing that extracellular pyruvate is the unique precursor of lactate under these conditions. These results reveal the presence of two different pools of intracellular pyruvate; one derived from extracellular pyruvate, used mainly for lactate and alanine production and one derived from glucose used primarily for oxidation. A red-ox switch using the cytosolic NAD(+)/NADH ratio is proposed to modulate glycolytic flux, controlling which one of the two pyruvate pools is metabolized in the tricarboxylic acid cycle when substrates more oxidized or reduced than glucose are used.

Animals↗
PubMed1996

Insulinotropic action of methyl pyruvate: enzymatic and metabolic aspects.

The metabolism of methyl pyruvate was compared to that of pyruvate in isolated rat pancreatic islets. Methyl pyruvate was found to be more efficient than pyruvate in supporting the intramitochondrial conversion of pyruvate metabolites to amino acids, inhibiting D-[5-3H]glucose utilization, maintaining a high ratio between D-[3,4-14C]glucose or D-[6-14C]glucose oxidation and D-[5-3H]glucose utilization, inhibiting the intramitochondrial conversion of glucose-derived 2-keto acids to their corresponding amino acids, and augmenting 14CO2 output from islets prelabeled with L-[U-14C]glutamine. Methyl pyruvate also apparently caused a more marked mitochondrial alkalinization than pyruvate, as judged from comparisons of pH measurements based on the use of either a fluorescein probe or 14C-labeled 5,5-dimethyl-oxazolidine-2,4-dione. Inversely, pyruvate was more efficient than methyl pyruvate in increasing lactate output and generating L-alanine. These converging findings indicate that, by comparison with exogenous pyruvate, its methyl ester is preferentially metabolized in the mitochondrial, rather than cytosolic, domain of islet cells. It is proposed that both the positive and the negative components of methyl pyruvate insulinotropic action are linked to changes in the net generation of reducing equivalents, ATP and H+.

Alanine Transaminase↗
PubMed1997

The monovalent cation requirement of rabbit muscle pyruvate kinase is eliminated by substitution of lysine for glutamate 117.

The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+ [T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994) Biochemistry 33, 6301-6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases from Corynebacterium glutamicum and Escherichia coli, which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+ binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+ site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+ in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated epsilon-amino group of Lys 117 in these bacterial enzymes may provide an "internal monovalent cation." Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is > 200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated epsilon-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.

Amino Acid Sequence↗
PubMed2002

Intracellular pyruvate flux in the methane-producing archaeon Methanococcus maripaludis.

During growth of the methanogenic archaeon Methanococcus maripaludis on alanine as the sole nitrogen source under H(2)/CO(2), alanine was incorporated into amino acids derived from pyruvate including leucine, isoleucine, and valine. Thus, growth with alanine was an efficient means of labeling intracellular pools of pyruvate in this lithotroph. Cells were grown with 18% [U-(13)C]alanine, and the distribution of the isotope in the branched-chain amino acids was determined by (13)C-NMR. Carbons derived from pyruvate contained 14.5% (13)C, indicating that most of the cellular pyruvate was obtained from alanine. In contrast, carbons derived from acetyl-CoA contained only 3-5% (13)C, indicating that only small amounts of acetyl-CoA were formed from pyruvate. Thus, autotrophic acetyl-CoA biosynthesis continued even in the presence of an organic carbon source. Moreover, the labeling of acetyl-CoA was lower than would be predicted if pyruvate was a C-1 donor for acetyl-CoA biosynthesis. Carbon derived from the C-1 of acetyl-CoA contained less (13)C than carbon derived from the C-2 of acetyl-CoA, and this difference was attributed to the acetyl-CoA:CO(2) exchange activity of acetyl-CoA synthase. No enrichment was detected for the C-1 of valine, which was derived from the C-1 of pyruvate. This result was attributed to the pyruvate:CO(2) exchange activity of pyruvate oxidoreductase and may have important implications for isotope tracer studies utilizing pyruvate. Lastly, these results demonstrate that the breakdown of pyruvate by methanococci is very limited even under conditions where it is the sole nitrogen and major carbon source.

Alanine↗