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PubMed1975

Decarboxylation of oxalacetate to pyruvate by purified avian liver phosphoenolpyruvate carboxykinase.

Phosphoenolpyruvate carboxykinase, which has been isolated from chicken liver mitochondria in essentially homogenous form, carries out the irreversible decarboxylation of oxalacetate to pyruvate in the presence of catalytic amounts of GDP or IDP, as well as the reversible decarboxylation of oxalacetate to phosphoenolpyruvate in the presence of substrate amounts of GTP or ITP. The pyruvate- and phosphoenolpyruvate-forming reactions are similar in their nucleoside specificity and appear to be carried out by the same protein. However, the two activities vary markedly in their response to added metal ions and sulfhydryl reagents. Phosphoenolpyruvate formation is completely dependent on the presence of a divalent metal ion, with Mn2+ the most effective species. This reaction is also stimulated by sulfhydryl reagents such as 2-mercaptoethanol. In contrast, the pyruvate-forming reaction is strongly inhibited by divalent metal ions, including Mn2+, and also by moderate concentrations of sulfhydryl reagents. These observations and the demonstration that pyruvate kinase-like activity is very low or absent make it unlikely that pyruvate formation proceeds via phosphoenolpyruvate as an intermediate. Although the pyruvate-forming reaction is inhibited by added metal ions, the reaction is also inhibited by metal-chelating agents such as 8-hydroxyquinoline and o-phenanthroline, suggesting that the reaction is dependent on the presence of a metal ion. It has not been possible, however, to demonstrate that the enzyme is a metalloprotein.

2,2'-Dipyridyl↗
PubMed1985

Substrate-dependent inactivation of muscle pyruvate dehydrogenase: identification of the acetyl-substituted enzyme form.

The properties of the pyruvate dehydrogenase component isolated from the pigeon breast muscle pyruvate dehydrogenase complex were studied upon inactivation of the enzyme in an incomplete reaction mixture: in the presence of cofactors and pyruvate, and in the absence of electron acceptors. The substrate-dependent inactivation was shown to result in the modification of two sulfhydryl groups per mole of the enzyme, in the appearance of a maximum at 235 nm in the protein absorption spectrum, and in the involvement of 1.5 moles of the [2-14C]-pyruvate fragment per mole of the pyruvate dehydrogenase. The fragment-protein bond is acid-stable, labile in alkali, and breaks up in the presence of performic acid, neutral hydroxylamine and dithiothreitol. An acetyl-substituted form of pyruvate dehydrogenase appearing with the participation of sulfhydryl enzyme groups is suggested.

Acetylation↗
PubMedDate not supplied

[Effect of Ca2+ ions on the pyruvate kinase isoenzymes from rabbit kidney cortex].

Ca2 ions showed the various effect on isoenzymes of pyruvate kinase from rabbit kidney cortex. Ca2 activated the "L" type of pyruvate kinase at low concentrations of PEP and inhibited -- at high concentrations of the latter. "M2" type of pyruvate kinase was inhibited by Ca2 under all the conditions studied. In presence of Ca2+ the activating effect of PDP on "L" and "M2" types of pyruvate kinase was absent; the inhibitory action of ATP on the "M" type of pyruvate kinase was increased at all the concentrations above 1.3 mM. The effect of Ca2+ on the pyruvate kinase isoenzymes depended on content of Mg2+ in the medium.

Adenosine Triphosphate↗
PubMed1984

In vivo hormonal control of L-type pyruvate kinase gene expression. Effects of glucagon, cyclic AMP, insulin, cortisone, and thyroid hormones on the dietary induction of mRNAs in the liver.

Using a cDNA probe complementary to rat L-type pyruvate kinase mRNAs, we studied the respective roles of glucocorticoids, thyroid hormones, glucagon, and insulin in the induction of specific mRNAs in the liver of animals refed either a maltose-rich or a fructose-rich diet. Neither adrenalectomized nor thyroidectomized nor diabetic animals could express L-type pyruvate kinase mRNAs in their liver when refed the carbohydrate-rich diets. When the animals were given the missing hormone, the level of hybridizable mRNAs returned to normal values but administration of the hormone alone failed to induce mRNA synthesis in fasted animals. Both glucagon and cyclic AMP abolished the induction of L-type pyruvate kinase mRNAs in refed animals. Exogenous insulin, whatever the dose, could not reverse the inhibitory action of glucagon. Insulin has usually been regarded as the main regulator of L-type pyruvate kinase gene expression. It appears now that glucagon, beside regulating the enzyme activity by phosphorylation mechanisms, may also modulate L-type pyruvate kinase synthesis at a pre-translational level. Consequently, our results show that three conditions are required for the synthesis of liver L-type pyruvate kinase mRNAs: (i) the presence of dietary carbohydrates, (ii) the cessation of glucagon release, and (iii) the presence of permissive hormones, including insulin.

Adrenalectomy↗
PubMed1982

Studies of the flavin adenine dinucleotide binding region in Escherichia coli pyruvate oxidase.

Experiments have been performed to probe the flavin adenine dinucleotide (FAD) binding region in Escherichia coli pyruvate oxidase. This enzyme functions as a membrane-associated flavoprotein coupled to the aerobic E. coli respiratory chain. The FAD moiety is noncovalently bound to pyruvate oxidase and can be removed reversibly to form apopyruvate oxidase. The addition of free FAD to apoenzyme results in the stoichiometric re-formation of the active flavoprotein. Using this technique, synthetic analogs of FAD were substituted in the flavin binding site and used as structural probes. Spectral analysis indicates that the benzoquinoid forms of 8-mercapto-FAD and 6-hydroxy-FAD are stabilized in the enzyme-binding site. This is consistent with the fact that the native flavoprotein forms a red (anion) radical upon photoreduction. These data suggest that the isoalloxazine ring may be poised for reduction via position N-5 by a carbanionic intermediate. The alpha-carbanion of hydroxyethylthiamin pyrophosphate, formed following the decarboxylation of pyruvate, is a likely candidate. The highly resolved visible spectrum of the native flavoprotein suggests that the flavin is buried in a hydrophobic environment. Reactivity studies using 8-chloro-FAD-pyruvate oxidase and 2-thio-FAD-pyruvate oxidase suggest that the C-8 position and C-2 position of the isoalloxazine ring may not be accessible to the solvent. Spectral perturbations observed with 6-hydroxy-FAD-pyruvate oxidase indicate, however, that the isoalloxazine C-6 position may be located near the binding site for the cofactor thiamin pyrophosphate. Restrictions to the accessibility of the active site of the enzyme are suggested by the fact that sulfite does not form an adduct with the flavin in the native enzyme.

Binding Sites↗
PubMed1982

Effect of dapsone on blood lactic and pyruvic acids in leprosy.

The effect of leprosy and dapsone (DDS) on the basal levels of blood lactic and pyruvic acids has been studied. In untreated tuberculoid and lepromatous leprosy patients both of the acids were found to be significantly raised. The rise in lactic acid was relatively more in tuberculoid patients; whereas pyruvic was relatively more elevated in lepromatous cases. Both the acids showed a tendency to increase with the duration of the disease in lepromatous leprosy. Statistically no significant differences were observed in lactic acid levels between untreated and treated cases of both forms of leprosy, suggesting that DDS was not effective in controlling the conditions responsible for the increased lactic acid. On the other hand, pyruvic acid showed a further increase in cases who were on DDS therapy, particularly in lepromatous cases. This indicated that DDS affects pyruvic acid metabolism. Whether DDS disturbs the normal degradative pathway of pyruvic acid or affects pathways of pyruvic acid production is not clear.

Dapsone↗
PubMedDate not supplied

[Formation of pyruvic and lactic acids in muscles diluted homogenate differing from the generally accepted glycolysis scheme].

When studying formation of pyruvic and lactic acids from fructose diphosphate the effect of dilution is found in the process of muscles homogenate incubation. Pyruvic acid accumulates only in samples with magnesium chloride added. The increase of lactic acid remains constant irrespectively of availability of conditions for pyruvate formation. Phosphoglycerate and phosphoenolpyruvate form pyruvic acid only in samples with magnesium chloride added without forming lactic acid. It is shown that in the muscles diluted homogenate (25 mg per 6 ml of mixture) lactic acid is formed under conditions unsuitable for pyruvic acid formation. Lactic acid is formed omitting the stages of 2-phosphoglycerate, phosphoenolpyruvate and pyruvate i.e. in the pathway different from a classical scheme of glycolysis.

Animals↗
PubMed1980

Dietary alteration of translatable mRNA sequences coding for rat liver pyruvate kinase.

Poly(A+) RNA (RNA containing a polyadenylic acid sequence) was isolated from individual livers of rats fed standard lab chow, fasted, or fasted and refed a high carbohydrate diet. The level of functional mRNA coding for pyruvate kinase was assayed using a rabbit reticulocyte in vitro translation system. The total 35S incorporation into newly synthesized liver pyruvate kinase was measured and compared to 35S incorporation into albumin, total trichloroacetic acid-precipitated proteins, and released polypeptide chains. The relative level of mRNA coding for liver pyruvate kinase decreases almost 60% upon fasting and increases approximately 15-fold upon refeeding with a high carbohydrate diet for 24 h. These observed changes in the amount of mRNA coding for liver pyruvate kinase agree with the previously reported changes in the relative rates of liver pyruvate kinase synthesis measured in vivo during these dietary stresses. Thus, it is suggested that the alterations in the amount of pyruvate kinase in liver in response to these dietary stresses primarily result from alteration in the amount of functional mRNA coding for the enzyme.

Animals↗
PubMed1994

Metabolism of glucose, glutamine and pyruvate in lymphocytes from Walker 256 tumor-bearing rats.

This study examined the effect of Walker 256 tumor growth in vivo on the metabolism of glucose, glutamine and pyruvate in lymphocytes. A comparison between the metabolism of Walker 256 tumor cells obtained in vivo with that of lymphocytes was also carried out. Lymphocytes and tumor cells were isolated and incubated for 1 h for the following measurements: lactate production from glucose (5.6 mM) and pyruvate (3 mM), glutamate and aspartate formation from glutamine (3 mM) and decarboxylation of [U-14C]-glucose, [U-14C]-glutamine, [1-14C]-pyruvate and [3-14C]-pyruvate. The presence of the tumor increased lactate production (2.7-fold from glucose and 2-fold from pyruvate), decarboxylation of [U-14C]-glucose (3.7-fold) and [1-14C]-pyruvate (4.4-fold) and the formation of aspartate (6.3-fold) and glutamate (4.6-fold) from glutamine. The conversion of glucose to lactate and CO2 was higher in tumor cells as compared to lymphocytes. Tumor cells also showed a higher production of glutamate and an 8-fold increased decarboxylation rate of [U-14C]-glutamine in tumor cells, which was more active than that of lymphocytes even from tumor-bearing rats. Tumor growth stimulated glucose and glutamine metabolism in lymphocytes; however, the importance of this fact for the function of these cells remains to be elucidated.

Animals↗
PubMed1976

Use of automated, on-line pyruvate analysis.

Normothermic blood perfusions of isolated canine liver were undertaken to determine the most sensitive and reliable indicators of viability. Perfusate flow rates and arterial and portal venous pressures were monitored. Arterial and venous concentrations of potassium, pyruvate, serum glutamic oxaloacetic transaminase, and oxygen were monitored and the values stored on magnetic tape for computerized calculation of outputs. Continuous evaluation of taurocholate concentration in the perfusate allowed adjustment of infusion rate to provide a constant flow of bile across the hapatocytes. Pyruvate output, portal venous resistance, serum glutamic oxaloacetic transaminase output and potassium output provided the earliest indices of deterioration. Bile flow and oxygen consumption remained constant until irreversible outflow block had occured. The earliest and most constant parameter signaling deteoration of the ex vivo liver was pyruvate output. The deteriorating liver released pyruvate into the perfusate, while the functioning organ consumed or maintained the available substrate. Since pyruvate output occurs before the onset of irreversible damage, measures can be enacted to reverse the deterioration. Restoration of the preparation can be evaluated through the cessation of pyruvate output.

Animals↗
PubMed1994

Enzyme inhibitory autoantibodies to pyruvate dehydrogenase complex in primary biliary cirrhosis: applications of a semiautomated assay.

Sera from patients with primary biliary cirrhosis inhibit the activity of the mitochondrial pyruvate dehydrogenase complex. We utilized this effect to develop a simple, miniaturized, semiautomated spectrophotometric assay as a diagnostic aid. The sera studied were from 71 patients with primary biliary cirrhosis and 62 other subjects. The assays included enzyme inhibition, immunofluorescence on HEp-2 cells, enzyme-linked immunosorbent assay using recombinant pyruvate dehydrogenase complex-E2 and immunoblotting on bovine heart mitochondria. With the 71 primary biliary cirrhosis sera, on which M2 antibody was detected by immunofluorescence in 64 (90%), antibodies against pyruvate dehydrogenase complex were detected in 53 (83%) by means of enzyme inhibition, in 57 (89%) by means of enzyme-linked immunosorbent assay and in 60 (94%) by means of immunoblotting. Of the 64 sera positive by immunofluorescence, 60 reacted with pyruvate dehydrogenase complex-E2 on immunoblotting, and the miniaturized enzyme inhibition assay was positive in 53 of these. The enzyme inhibition assay and enzyme-linked immunosorbent assay were calibrated to give a specificity of 100%. At this level, the sensitivities for detection of pyruvate dehydrogenase complex antibody were 83% and 87%, respectively. We found no significant changes in levels of reactivity with the enzyme inhibition assay or enzyme-linked immunosorbent assay according to disease stage. Treatment with cyclosporine was accompanied by a significant decrease in levels of antibody to pyruvate dehydrogenase complex-E2 that matched improved indexes of biochemical liver function.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies↗
PubMed1994

Molecular cloning of the p45 subunit of pyruvate dehydrogenase kinase.

Purified preparations of rat heart pyruvate dehydrogenase kinase have two polypeptides with molecular weights of 48,000 (p48) and 45,000 (p45). Recently, we reported the primary structure of p48 (Popov, K. M., Kedishvili, N. Y., Zhao, Y., Shimomura, Y., Crabb, D. W., and Harris, R. A. (1993) J. Biol. Chem. 268, 26602-26606) and presented evidence that (i) it exhibits kinase activity for pyruvate dehydrogenase and (ii) it belongs to a family of mitochondrial protein kinases unique from other eukaryotic protein kinases. Here, we report the molecular cloning and deduced amino acid sequence of p45. The protein sequence of p45 has 70% identity to the protein sequence of p48. Minor differences exist throughout the protein sequences with the greatest difference occurring at the amino termini. Recombinant p45 protein, expressed in Escherichia coli and purified to homogeneity, catalyzed the phosphorylation and inactivation of kinase-depleted pyruvate dehydrogenase complex, indicating that p45 and p48 correspond to different isoforms of pyruvate dehydrogenase kinase. Northern blot analysis revealed a single hybridizing species of 2.5 kilobases. The highest level of p45 message expression was found in heart and skeletal muscle and the lowest in spleen and lung. Liver, kidney, brain, and testis express intermediate amounts of p45 mRNA. In contrast, p48 mRNA is predominantly expressed in heart, with other tissues expressing only a modest amount of this message. Tissue-specific expression of isoforms of pyruvate dehydrogenase kinase may indicate the existence of tissue-specific mechanisms for the regulation of pyruvate dehydrogenase activity.

Amino Acid Sequence↗
PubMed1994

Pyruvate carboxylase activity in the heart and skeletal muscles of the rat. Evidence for a stimulating effect of exercise.

The mitochondrial pyruvate carboxylase catalyses the ATP-dependent carboxylation of pyruvate to oxaloacetate. Since pyruvate carboxylase generates oxaloacetate for Krebs cycle function, it is proposed that the enzyme activity may be enhanced by exercise. To investigate this proposition, pyruvate carboxylase activity was determined in the heart, soleus and gastrocnemius (white portion) muscles of sedentary and swimming-trained adult rats (1 hour per day, 5 days a week, during 5 weeks) under the following conditions: rest, one hour of exercise and exhaustion. The results show that the pyruvate carboxylase activity is increased during exercise in both the sedentary and trained groups of rats. The stimulatory mechanism is unknown but it is possibly related to the generation of pyruvate from the breakdown of glycogen and acetyl CoA during fatty acid oxidation.

Analysis of Variance↗
PubMed1994

Enzyme inhibitory autoantibodies to pyruvate dehydrogenase complex in primary biliary cirrhosis differ for mammalian, yeast and bacterial enzymes: implications for molecular mimicry.

Primary biliary cirrhosis is a chronic autoimmune disease in which serum autoantibodies against the mitochondrial 2-oxo acid dehydrogenase enzyme complexes (M2 antibodies) are regularly present. Molecular mimicry of host proteins by bacterial counterparts is a suggested explanation for the origin of these autoantibodies. We tested this hypothesis by measuring the functional reactivity of serum autoantibodies by means of an enzyme inhibition assay against pyruvate dehydrogenase complex from different sources: mammalian, Saccharomyces cerevisiae and Escherichia coli. The 10 primary biliary cirrhosis sera all reacted on immunofluorescence study for M2 antibodies and on immunoblotting with the pyruvate dehydrogenase complex E2 subunit from each of the three enzymes, but there were strikingly different inhibitory capacities. The primary biliary cirrhosis sera were highly inhibitory for mammalian pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level of inhibition, 99%), moderately inhibitory for yeast pyruvate dehydrogenase complex (10 of 10 inhibitory; mean level, 70%) and weakly inhibitory for Escherichia coli pyruvate dehydrogenase complex (4 of 10 inhibitory; mean level, 26%). Thus, with a functional assay that depends on epitope recognition of primary biliary cirrhosis sera, cross-reactivity between mammalian and bacterial pyruvate dehydrogenase complex enzymes is low and molecular mimicry, at least at the B-lymphocyte level, is not supported.

Animals↗
PubMedDate not supplied

[Paracatalytic inactivation of pyruvate decarboxylase in the presence of quinones].

Pyruvate promotes the yeast pyruvate decarboxylase inactivation under the influence of substituted p-benzoquinones. Pyruvate decarboxylase activity is not renewed after the removal of low-molecular impurities by gel filtration and subsequent addition of dithiothreitol, thiamine diphosphate, magnesium chloride. The inactivation rate under joint action of 2-methyl-5-isopropyl-p-benzoquinone and pyruvate is regulated by the pseudo-first-order equation. The relationship between pseudo-first-order rate constant and pyruvate concentration takes the shape of hyperbola. The inactivation order with respect to quinone is determined by oxidant concentration and pH value. Maximum pseudo-first-order rate constant values in the presence of the excess substrate and 2-methyl-5-isopropyl-p-benzoquinone are observed at pH 5.9-6.0. The data obtained evidence for the fact that during inactivation quinone interacts with "active acetaldehyde" being the intermediate in the process of catalysis with pyruvate decarboxylase.

Benzoquinones↗
PubMed1993

Comparison of the kinetic properties of the pyruvate dehydrogenase complex from pig kidney cortex and medulla.

The activity of the pyruvate dehydrogenase complex (PDC) purified from pig kidney medulla was affected by K+, Na+, Cl-, HCO3-, HPO4(2-) and changes in ionic strength. Increased ionic strength influenced the activity of PDC from medulla by decreasing the Vmax and S0.5 for pyruvate and increasing the Hill coefficient. The magnitude of these changes was smaller than the corresponding changes for PDC purified from the cortex. In the presence of K+ (80 mM), Na+ (20 mM), Cl- (20 mM), HCO3- (20 mM), HPO4(2-) (10 mM) and at ionic strength of 0.15 M the S0.5 for pyruvate of PDC from medulla was 117 microM and the enzyme complex was saturated by 1.1 mM pyruvate. Under these conditions the S0.5 for pyruvate of PDC derived from cortex was 159 microM and the enzyme was saturated at 4.5 mM pyruvate. Based on the results presented in this report it is suggested that PDC in kidney medulla may be regulated not only by a phosphorylation/dephosphorylation system and end-product inhibition but also via changes in ionic strength.

Animals↗
PubMed1993

[The effect of phosphorylation on catalytic function of muscle pyruvate dehydrogenase complex].

It has been shown that phosphorylation of the pyruvate dehydrogenase complex from pigeon breast muscle by endogenous ATP-dependent protein kinase suppresses the substrate conversion in the pyruvate: acceptor oxidoreductase reactions and nonoxidative reactions monitored by pyruvate decline in the absence of CoA and NAD. To identify the catalytic step blocked by phosphorylation, CD spectroscopy was used which revealed the appearance and decay of the charge transfer complex between component E1 and thiamine pyrophosphate during the enzymatic reaction. Phosphorylation of the pyruvate dehydrogenase complex while lowering the affinity for thiamine pyrophosphate does not preclude the formation of holo-E1 but inhibits its interaction with pyruvate. Phosphorylated pyruvate dehydrogenase, like the dephosphorylated enzyme, reacts with 2-hydroxyethyl thiamine pyrophosphate in half of the active sites. In the presence of deacylating agents (CoA or dithiothreitol) all the sites are reactive. A conclusion is drawn that the alternating functioning of the active centers is preserved in reductive acetylation of the acceptor substrates by phospho-E1.

Animals↗
PubMed1995

Trypanosoma evansi: measurement of pyruvate production as an indicator of the drug sensitivity of isolates in vitro.

In a previous study, three in vitro methods for the assessment of drug sensitivity among Trypanosoma evansi isolates were compared--a direct counting method, pyruvate production method and uptake of radiolabelled hypoxanthine. The pyruvate assay system, which measures the amount of pyruvate in the supernatant of growing populations of trypanosomes by a spectrophotometric method, was selected for further investigation with regard to its suitability for field studies. The effect of initial seeding density and incubation time on the growth of three stocks of T. evansi--TREU 1840 and TREU 1981 (suramin sensitive) and TREU 2136 (suramin resistant)--and drug sensitivities revealed by the pyruvate assay and direct counting were examined to optimise assay conditions. Maximum densities and pyruvate production achieved were not affected by varying the initial seeding densities in the range of 5 x 10(4)-5 x 10(5)/ml and had been reached after 48 hours incubation with one exception: Pyruvate levels continued to increase up to 72 hours in the suramin resistant stock. However, inhibition curves were affected by initial seeding density and incubation period. Results suggested that an initial seeding density of 1 x 10(5)/ml and an incubation time of 48 hours are optimal for the assay. Using these assay conditions, the isolates were screened against suramin, quinapyramine sulphate and Cymelarsan, the trypanocides used most commonly against T. evansi. This assay proved to be a relatively simple and cheap technique applicable to screening large numbers of isolates of differing sensitivities to trypanocidal drugs.

Animals↗