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PubMed2003

[Cloning and expressing the E2 subunit of pyruvate dehydrogenase complex].

OBJECTIVES: To construct the expression vector of the pyruvate dehydrogenase complex E2 subunit gene (PDC-E2). METHODS: The PDC-E2 gene was amplified from human lymphocytes with RT-PCR, and was cloned into pExSecI vector to induce the PDC-E2 expression. The products were identified with western blot and ELISA. RESULTS: The expression vector pExSecI/PDC-E2 was successfully constructed. The products could be identified by the specific self-antibodies in the sera from the primary biliary cirrhosis patients. CONCLUSION: High efficient expression vector of PDC-E2 lays the foundation for serum assay of primary biliary cirrhosis patients with prokaryotic expressing PDC-E2.

Cloning, Molecular↗
PubMed1993

Defect in the lipoyl-bearing protein X subunit of the pyruvate dehydrogenase complex in two patients with encephalomyelopathy.

Among the many metabolic encephalomyelopathies caused by deficiencies in the pyruvate dehydrogenase complex (PDHC), nearly all involve its E1 subunit. We describe two new familial cases of PDHC deficiency with encephalomyelopathy, chronic lactic acidemia, and a normal E1 subunit of PDHC but deficiency in another component. Activity of PDHC was measured in cultured skin fibroblasts and skeletal muscle, and immunoblot studies were performed on mitochondrial extracts from skin fibroblasts. Spectra of muscle tissue, obtained in vivo with phosphorus 31 nuclear magnetic resonance, were recorded both at rest and with exercise. The PDHC activity was markedly reduced to 10% to 20% of normal values in both cultured skin fibroblasts and skeletal muscle. Immunoblotting of skin fibroblast mitochondrial extracts showed a specific deficiency in the protein X component of PDHC but normal E1, E2, and E3 components. Spectra obtained with 31P nuclear magnetic resonance showed alterations compatible with those found in mitochondrial myopathies. This is the second description of an encephalomyelopathy associated with a specific absence of the lipoyl-containing protein X component, which has a structural role in the formation of a functional PDHC.

Child, Preschool↗
PubMed1998

Longitudinal study of tissue- and subunit-specific obesity-induced regulation of the pyruvate dehydrogenase complex.

The tissue-specific expression of the mitochondrial pyruvate dehydrogenase complex (PDHc) has been studied in an animal model of obesity with hyperinsulinemia, the obese (fa/fa) Zucker rat. Liver and heart were obtained from 4 and 8 week-old obese rats and age-matched lean animals, and in each tissue the following parameters were analyzed: (1) total activity of the mitochondrial PDHc; (2) abundance of the mitochondrial PDHc subunits on Western blots; and (3) abundance of the E1alpha and E1beta subunit mRNAs on Northern blots and semi-quantitative RT-PCR. Regardless of age, obese rats showed an increase in liver total PDHc activity and a coordinate increase in liver E1alpha and E1beta PDHc subunit abundance. At 4 weeks, obese rats also showed an increase in liver PDH E1alpha mRNA level, but regardless of age E1beta mRNA level was unchanged. In contrast, neither total PDHc activity nor the concentration of its protein subunits were increased in heart of obese rats. Thus, obese Zucker rats display a liver-specific early increase in PDHc which results from a selective up-regulation of the E1alpha gene expression.

Animals↗
PubMed1983

Regulatory properties of the pyruvate dehydrogenase complex from Escherichia coli. Studies on the thiamin diphosphate-dependent lag phase.

The pyruvate dehydrogenase complex from Escherichia coli shows an appreciable lag phase (tau) of some minutes when its overall reaction rate was tested with very limiting amounts of thiamin diphosphate. tau depends on the concentration of thiamin diphosphate in a nonlinear fashion. Sodium diphosphate, a competitive inhibitor with respect to thiamin diphosphate (Ki = 5.2 . 10(-4) M) prolongs the lag, while the strongly binding transition state analog thiamin thiazolone diphosphate has no effect. tau is independent of the enzyme concentration, thus no dissociation-association step is involved. Incubation of the pyruvate dehydrogenase complex with thiamin diphosphate, Mg2+, and pyruvate leads to a shortening of the lag phase, as well as to a decrease of the intrinsic tryptophan fluorescence in a time-dependent process, which evinces the same characteristics as tau. Dependence of pyruvate, as well as of the substrate analog methylacetylphosphonate, can be established by measurements of fluorescence quenching, thus ruling out an essential role of hydroxyethyl thiamin diphosphate in the process reflected by the lag phase. The results demonstrate that the lag phase is induced after the binding of both thiamin diphosphate . Mg2+ and pyruvate to the catalytic site to form a ternary enzyme complex, which undergoes subsequently a slow conformational change to an active enzyme form. This change is confined to single subunits, and no interactions between neighboring monomers could be observed. A model is proposed to describe the mechanism represented by the lag phase.

Diphosphates↗
PubMedDate not supplied

[Coenzyme-binding sites of the brain pyruvate dehydrogenase complex].

It is shown that the relative amount of the holoenzyme in the highly purified pyruvate dehydrogenase complex from the bovine brain is higher when the enzyme activity is assayed in the reaction of nonoxidative formation of acetaldehyde as compared to the pyruvate: NAD+ reductase reaction. The S0.5 values for thiamine pyrophosphate are as following: (TPP) (0.314 +/- 0.22) x 10(-7) M with reaction of nonoxidative formation of acetaldehyde, (0.188 +/- 0.08) x 10(-6) M and (1.65 +/- 1.16) x 10(-6) M in case of the pyruvate: NAD+ reductase reaction. TPP in the concentration of (0.5-6.0) x 10(-7) M completely protects the sites of nonoxidative formation of acetaldehyde from modification by the coenzyme analogs, 4'-oxythiamine pyrophosphate and tetrahydrothiamine pyrophosphate. However, the pyruvate: NAD+ reductase activity of the pyruvate dehydrogenase complex is inhibited in this case by 30-34%. The data obtained suggest that in contrast to the pyruvate: NAD+ reductase reaction the conversion of pyruvate to acetaldehyde occurs by the sites which tightly bound TPP.

Animals↗
PubMed1984

In vitro and in situ skeletal muscle pyruvate dehydrogenase activity in adrenalectomized and glucocorticoid treated rats.

Skeletal muscle glucose oxidation is significantly reduced and alanine release enhanced in adrenalectomized rats after short-term glucocorticoid treatment. A possible site of regulation is the pyruvate dehydrogenase complex. Pyruvate dehydrogenase activity was measured in vitro in extracts of the gastrocnemius muscle of normal, adrenalectomized and short-term glucocorticoid treated rats by the p-nitroaniline-arylamine-acetyltransferase method and in situ in non-recirculating perfusions of isolated hindlimbs with physiological pyruvate levels and tracer doses of [1-14C] pyruvate by measuring the off-kinetic of 14CO2 wash out in the effluent. Neither method showed a direct influence of glucocorticoids on skeletal muscle pyruvate dehydrogenase activity.

Adrenal Glands↗
PubMed1990

Molecular cloning of the gene for the E1 alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

The E1 alpha and E1 beta subunits of the pyruvate dehydrogenase complex from the yeast Saccharomyces cerevisiae were purified. Antibodies raised against these subunits were used to clone the corresponding genes from a genomic yeast DNA library in the expression vector lambda gt11. The gene encoding the E1 alpha subunit was unique and localized on a 1.7-kb HindIII fragment from chromosome V. The identify of the gene was confirmed in two ways. (a) Expression of the gene in Escherichia coli produced a protein that reacted with the anti-E1 alpha serum. (b) Gene replacement at the 1.7-kb HindIII fragment abolished both pyruvate dehydrogenase activity and the production of proteins reacting with anti-E1 alpha serum in haploid cells. In addition, the 1.7-kb HindIII fragment hybridized to a set of oligonucleotides derived from amino acid sequences from the N-terminal and central regions of the human E1 alpha peptide. We propose to call the gene encoding the E1 alpha subunit of the yeast pyruvate dehydrogenase complex PDA1. Screening of the lambda gt11 library using the anti-E1 beta serum resulted in the reisolation of the RAP1 gene, which was located on chromosome XIV.

Amino Acid Sequence↗
PubMed2005

Interaction of thiamin diphosphate with phosphorylated and dephosphorylated mammalian pyruvate dehydrogenase complex.

Kinetic and binding studies were carried out on substrate and cofactor interaction with the pyruvate dehydrogenase complex from bovine heart. Fluoropyruvate and pyruvamide, previously described as irreversible and allosteric inhibitors, respectively, are strong competitive inhibitors with respect to pyruvate. Binding of thiamin diphosphate was used to study differences between the active dephosphorylated and inactive phosphorylated enzyme states by spectroscopic methods. The change in both the intrinsic tryptophan fluorescence and the fluorescence of the 6-bromoacetyl-2-dimethylaminonaphthalene-labelled enzyme complex produced on addition of the cofactor showed similar binding behaviour for both enzyme forms, with slightly higher affinity for the phosphorylated form. Changes in the CD spectrum, especially the negative Cotton effect at 330 nm as a function of cofactor concentration, both in the absence and presence of pyruvate, also revealed no drastic differences between the two enzyme forms. Thus, inactivation of the enzyme activity of the pyruvate dehydrogenase complex is not caused by impeding the binding of substrate or cofactor.

Animals↗
PubMed1987

The effect of 2-oxoglutarate or 3-hydroxybutyrate on pyruvate dehydrogenase complex in isolated cerebrocortical mitochondria.

The oxidation of pyruvate is mediated by the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12 and EC 1.6.4.3) whose catalytic activity is influenced by phosphorylation and by product inhibition. 2-Oxoglutarate and 3-hydroxybutyrate are readily utilized by brain mitochondria and inhibit pyruvate oxidation. To further elucidate the regulatory behavior of brain PDHC, the effects of 2-oxoglutarate and 3-hydroxybutyrate on the flux of PDHC (as determined by [1-14C]pyruvate decarboxylation) and the activation (phosphorylation) state of PDHC were determined in isolated, non-synaptic cerebro-cortical mitochondria in the presence or absence of added adenine nucleotides (ADP or ATP). [1-14C]Pyruvate decarboxylation by these mitochondria is consistently depressed by either 3-hydroxybutyrate or 2-oxoglutarate in the presence of ADP when mitochondrial respiration is stimulated. In the presence of exogenous ADP, 3-hydroxybutyrate inhibits pyruvate oxidation mainly through the phosphorylation of PDHC, since the reduction of the PDHC flux parallels the depression of PDHC activation state under these conditions. On the other hand, in addition to the phosphorylation of PDHC, 2-oxoglutarate may also regulate pyruvate oxidation by product inhibition of PDHC in the presence of 0.5 mM pyruvate plus ADP or 5 mM pyruvate alone. This conclusion is based upon the observation that 2-oxoglutarate inhibits [1-14C]pyruvate decarboxylation to a much greater extent than that predicted from the PDHC activation state (i.e. catalytic capacity) alone. In conjunction with the results from our previous study (Lai, J. C. K. and Sheu, K.-F. R. (1985) J. Neurochem. 45, 1861-1868), the data of the present study are consistent with the notion that the relative importance of the various mechanisms that regulate brain and peripheral tissue PDHCs shows interesting differences.

3-Hydroxybutyric Acid↗
PubMed1993

The regulatory properties of kidney pyruvate dehydrogenase complex components.

The activities of the enzyme components of the pyruvate dehydrogenase complex are affected to different extents by changes in ionic strength and pH. At pH 7.4 the optimum activity of pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3) occur in the ranges of ionic strengths of 0.06-0.08, 0.01-0.02, and 0.10-0.15 M, respectively. The activity of dihydrolipoamide dehydrogenase is least sensitive to changes in the ionic strength of the assay medium. At constant ionic strength (0.15 M) the optimum activity of E1, E2, and E3 occur at pH 7.4, 7.0, and 8.0, respectively. Changes in pH mostly affect the dihydrolipoamide acetyltransferase activity. Cl- and HCO3- anions inhibit the activity of pyruvate dehydrogenase. In the presence of 80 mM Cl- or HCO3- ions the activity of E1 is inhibited by 25 and 10% respectively. K+, Na+, and HPO4(2-) ions affect the activity of dihydrolipoamide acetyltransferase. The activity of this enzyme component is stimulated by 28 and 25% in the presence of 80 mM K+ and Na+ cations, respectively. HPO4(2-) stimulates the dihydrolipoamide acetyltransferase in a calcium-dependent manner. In the presence of 20 mM HPO4(2-) the activity of the dihydrolipoamide acetyltransferase increases 20 and 40% in the absence and presence of 0.1 mM Ca2+, respectively. The activity of dihydrolipoamide dehydrogenase is not affected by K+, Na+, HPO4(2-), Cl-, or HCO3-.

Acetyltransferases↗
PubMed1978

Demonstration of a lag period in the time-course of the reaction catalyzed by pyruvate dehydrogenase complex.

At low thiamine pyrophosphate concentrations the time-course of the reaction catalyzed by mammalian pyruvate dehydrogenase complex shows a lag period of some minutes when the reaction is started by either enzyme, pyruvate or thiamine pyrophosphate. However, started by CoASH or NAD+, the lag period disappears. An increase in enzyme concentration to 25 mU/ml causes a concomitant shortening of the duration of the lag period (tau), while above this value tau is independent of the enzyme concentration. An increase in thiamine pyrophosphate concentration decreases the value of tau and the lag period vanishes at infinite thiamine pyrophosphate concentration. It is suggested that both isomerization and aggregation-dissociation reactions may play an improtant role in the development of the lag period of pyruvate dehydrogenase complex.

Animals↗
PubMedDate not supplied

[Effect of thiamine phosphates on the activity of regulatory enzymes of the pyruvate dehydrogenase complex].

The effect of thiamine triphosphate (ThTP) and thiamine diphosphate (ThDP) on the activity of rat liver pyruvate dehydrogenase complex regulatory enzymes (kinase and phosphatase) was studied in experiments with isolated enzyme preparations. It is shown that ThDP caused a pronounced activation of pyruvate dehydrogenase phosphatase (Ka is equal to 65.0 nM). ThTP inhibits phosphatase competitively against the substrate--the phosphorylated pyruvate dehydrogenase complex. The both thiamine phosphates inhibit the pyruvate dehydrogenase kinase activity almost similarly in concentrations exceeding 10 microM. The physiological significance of the antagonistic action of ThDP and ThTP on the pyruvate dehydrogenase phosphatase activity is discussed.

Animals↗
PubMed1982

Pyruvate-dehydrogenase complex in ataxic patients: enzyme deficiency in ataxic encephalopathy plus lactic acidosis and normal activity in Friedreich ataxia.

Pyruvate dehydrogenase complex (PDHC) activity was measured in cultured fibroblasts from 12 patients with Friedreich's ataxia (FA), and in 1 patient with lactic acidosis and ataxia. The activities obtained after extraction of PDHC by different methods were compared. Triton-X-100 extraction yielded enzyme activities 5 to 10 times greater than those obtained with the older methods. With this sensitive technique, PDHC activity was markedly deficient in fibroblasts from the patient with lactic acidosis and ataxia but it was normal in the fibroblasts from FA patients. Mg++ activation of the PDHC in FA fibroblasts was normal.

Acidosis↗
PubMed2004

A family with pyruvate dehydrogenase complex deficiency due to a novel C>T substitution at nucleotide position 407 in exon 4 of the X-linked Epsilon1alpha gene.

UNLABELLED: The pyruvate dehydrogenase complex (PDHc; McKusick 312170), localised in the mitochondrial matrix, is a multienzyme complex which converts pyruvate to acetyl-CoA. A deficiency of PDHc leads to inadequate removal of pyruvate and lactate resulting in lactic acidaemia and insufficient energy production. The major cause of PDHc deficiency is a defect in the E1alpha component. The gene of this component is localised to Xp22.1. We describe two brothers with a relatively mild clinical phenotype of PDHc deficiency. Onset of disease was associated with muscle weakness and swallowing difficulties in both. At follow-up, the older brother developed encephalopathic features consistent with Leigh syndrome. Lactate to pyruvate ratios were low, consistent with a PDHc deficiency which was confirmed by measurements of PDHc activity in thrombocytes. A 407C>T change in exon 4 of the E1alpha gene was found in both brothers and their mother. This substitution predicts a replacement of a conserved alanine at position 136 by valine. CONCLUSION: Due to the X-linked inheritance pattern combined with the overall results of clinical investigations, molecular genetic findings and a corresponding functional deficiency of the gene product we believe that this substitution in the pyruvate dehydrogenase E1alpha gene is a mutation leading to pyruvate dehydrogenase complex deficiency in this family.

Amino Acid Substitution↗
PubMed1987

Immunochemical characterization of the pyruvate dehydrogenase complex in adult Ascaris suum and its developing larvae.

Polyclonal antibody was prepared against the pyruvate dehydrogenase complex purified from adult Ascaris suum body wall muscle. The antibody reacted with the E2, X, alpha E1 and beta E1 subunits of the complex in immunoblots of mitochondrial supernatant fractions and homogenates of adult muscle. In addition, the same subunits were observed in immunoblots of homogenates of L3 and L4 ascarid larvae, suggesting that a similar enzyme complex was present in all developmental stages despite their marked differences in energy metabolism. The phosphorylated and dephosphorylated alpha E1 peptides migrated differently during sodium dodecylsulfate polyacrylamide gel electrophoresis and both forms of the enzyme were recognized by the antibody. These results and those obtained with ELISA suggest that both phosphorylated and dephosphorylated forms of the alpha E1 subunit react equally well with the antibody. In immunoblots of adult body wall muscle, the phosphorylated alpha E1 peptide predominated, while immunoblots of L3 larvae contained predominantly the dephosphorylated form. These results reflect the in vivo activity state of the pyruvate dehydrogenase complex in these two stages and suggest that this technique may be useful for determining the activity state of enzyme complex directly from immunoblots of homogenates A. suum and other helminths.

Aerobiosis↗
PubMed1985

Hybrid pyruvate dehydrogenase complexes reconstituted from components of the complexes from Escherichia coli and Azotobacter vinelandii.

The pyruvate dehydrogenase complex of Escherichia coli was isolated in a simple three-step procedure. Its chain stoichiometry, determined by trinitrobenzoate modification was found to be 1.4 E1:1 E2:0.6 E3. It was reproducible within 10% from preparation to preparation. The E. coli complex was resolved by chromatography on activated thiol Sepharose. Reconstitution of activity yielded a stoichiometry of 1.0 E1:1 E2:0.5 E3. The optimum binding stoichiometry of E1E2 and E2E3 subcomplexes was determined by sedimentation experiments and found to be 2.0 E1:1 E2 and 2.5 E3:1 E2, respectively. Competition between E1 and E3 was observed in the binding experiments, but not in the kinetic experiments. Hybrid active complexes could be reconstituted from either an E1E2 subcomplex from Azotobacter vinelandii and the E3 component from E. coli or from E2E3 subcomplex from E. coli and the E1 component from A. vinelandii. Low activity and weak binding was observed when E1 from E. coli was recombined with an E2E3 subcomplex from A. vinelandii or when E3 from A. vinelandii was recombined with an E1E2 subcomplex from E. coli. The association behaviour and stoichiometry of the reconstituted complexes is determined by the nature of the E2 component. The formation of hybrid complexes indicates a considerable structural similarity between the complexes from both sources, despite the differences in size and stoichiometry.

Azotobacter↗
PubMed1984

[Inhibition of kinase activity of the adrenal pyruvate dehydrogenase complex by the mitochondrial component].

A component inhibiting the phosphorylation-linked inactivation of the adrenal pyruvate dehydrogenase complex in the presence of ATP was revealed during purification of the complex from bovine adrenal mitochondria. The degree of the kinase activity inhibition is greater at lower concentrations of ATP. It was assumed that the mitochondrial component screens the kinase active site or the phosphorylation sites of pyruvate dehydrogenase, thus limiting the ATP access to them. Proteins and lipids are incorporated into the component at a ratio 2:1, which is suggestive of its lipoprotein nature. The effect of the mitochondrial component on the kinase activity of the pyruvate dehydrogenase complex is somewhat specific and is unaffected by bovine serum albumin or blood serum lipoproteins.

Adenosine Triphosphate↗