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PubMed2003

Reactive oxygen species are involved in arsenic trioxide inhibition of pyruvate dehydrogenase activity.

Arsenite was shown to inhibit pyruvate dehydrogenase (PDH) activity through binding to vicinal dithiols in pure enzyme and tissue extract. However, no data are available on how arsenite inhibits PDH activity in human cells. The IC(50) values for arsenic trioxide (As(2)O(3)) to inhibit the PDH activity in porcine heart pure enzyme preparation and in human leukemia cell line HL60 cells were estimated to be 182 and 2 microM, respectively. Thus, As(2)O(3) inactivation of PDH activity was about 90 times more potent in HL60 cells than in purified enzyme preparation. The IC(50) values for As(2)O(3) and phenylarsine oxide to reduce the vicinal thiol content in HL60 cells were estimated to be 81.7 and 1.9 microM, respectively. Thus, As(2)O(3) is a potent PDH inhibitor but a weak vicinal thiol reacting agent in HL60 cells. Antioxidants but not dithiol compounds suppressed As(2)O(3) inhibition of PDH activity in HL60 cells. Conversely, dithiol compounds but not antioxidants suppressed the inhibition of PDH activity by phenylarsine oxide. As(2)O(3) increased H(2)O(2) level in HL60 cells, but this was not observed for phenylarsine oxide. Mitochondrial respiration inhibitors suppressed the As(2)O(3)-induced H(2)O(2) production and As(2)O(3) inhibition of PDH activity. Moreover, metal chelators ameliorated whereas Fenton metals aggravated As(2)O(3) inhibition of PDH activity. Treatment with H(2)O(2) plus Fenton metals also decreased the PDH activity in HL60 cells. Therefore, it seems that As(2)O(3) elevates H(2)O(2) production in mitochondria and this may produce hydroxyl through the Fenton reaction and result in oxidative damage to the protein of PDH. The present results suggest that arsenite may cause protein oxidation to inactivate an enzyme and this can occur at a much lower concentration than arsenite binding directly to the critical thiols.

Animals↗

PubMed1998

Expression of pyruvate dehydrogenase isoforms during the aerobic/anaerobic transition in the development of the parasitic nematode Ascaris suum: altered stoichiometry of phosphorylation/inactivation.

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic metabolism of the parasitic nematode Ascaris suum. Two isoforms of the alpha-subunit of pyruvate dehydrogenase (E1) have been identified: alpha I is most abundant in anaerobic adult muscle and alpha II in aerobic larvae. Both isoforms have been expressed as alpha 2 beta 2 tetramers with a muscle-specific beta-subunit, purified to apparent homogeneity, reconstituted with E1-deficient adult A. suum muscle PDC, and assayed for PDC and E1 kinase activity. Recombinant alpha II is a poor substrate for the adult E1 kinase, but its stoichiometry of phosphorylation/inactivation is similar to that reported for the human E1. Initially, inactivation parallels the incorporation of about 1 mol 32P/mol E1 and at maximal phosphorylation about 2.4 32P/mol E1 is incorporated. In contrast, recombinant alpha I (r alpha I) is phosphorylated rapidly, and substantially more phosphorylation accompanies inactivation. To examine this altered pattern of phosphorylation, the two phosphorylation sites in each E1 alpha subunit of the r alpha I (site 1 and site 2) were changed either individually or together from Ser to Ala by site-directed mutagenesis. Site 1 was phosphorylated more rapidly than site 2, but the phosphorylation of either site resulted in inactivation, and the phosphorylation of only a single E1 alpha subunit of the tetramer was necessary for inactivation. However, both E1 alpha subunits of the tetramer were phosphorylated, based on the incorporation of about 3.5 mol 32P/mol E1 at maximal phosphorylation and the altered mobility of most of the E1 alpha subunits during SDS-PAGE. These observations suggest that the regulation of both E1 isoforms is modified to maintain PDC activity during the transition to anaerobiosis.

Aerobiosis↗

PubMedDate not supplied

[Effect of thiamine on the activity of the pyruvate dehydrogenase complex in rat liver following stimulation of lipogenesis].

It is shown that thiamine administration to rats (250 micrograms per 100 g of mass) who were given high-carbohydrate diet (lipogenesis intensification) after fasting inhibits an increase in the pyruvate dehydrogenase activity in the liver homogenate and mitochondria usual under these conditions. This is observed when determining total activity of the pyruvate dehydrogenase complex and activity of its first component--pyruvate dehydrogenase estimated from the ferricyanide reduction and [1-14C] CO2 formation from [1-14C] pyruvate. Fasting animals and animals whom thiamine was administered against a background of lipogenesis intensification revealed a higher ability of the liver tissue to synthesize acetoin as compared with the control group and animals with the intensified lipogenesis without thiamine administration.

Acetoin↗

PubMed1998

Expression of genes encoding the E2 and E3 components of the Bacillus stearothermophilus pyruvate dehydrogenase complex and the stoichiometry of subunit interaction in assembly in vitro.

Genes encoding the dihydrolipoyl acetyltransferase (E2) and dihydrolipoyl dehydrogenase (E3) components of the pyruvate dehydrogenase (PDH) multienzyme complex from Bacillus stearothermophilus were overexpressed in Escherichia coli. The E2 component was purified as a large soluble aggregate (molecular mass > 1 x 10(6) Da) with the characteristic 532 symmetry of an icosahedral (60-mer) structure, and the E3 as a homodimer with a molecular mass of 110 kDa. The recombinant E2 component in vitro was capable of binding either 60 E3(alpha2) dimers or 60 heterotetramers (alpha2beta2) of the pyruvate decarboxylase (E1) component (also the product of B. stearothermophilus genes overexpressed in E. coli). Assembling the E2 polypeptide chain into the icosahedral E2 core did not impose any restriction on the binding of E1 or E3 to the peripheral subunit-binding domain in each E2 chain. This has important consequences for the stoichiometry of the assembled complex in vivo. The lipoyl domain of the recombinant E2 protein was found to be unlipoylated, but it could be correctly post-translationally modified in vitro using a recombinant lipoate protein ligase from E. coli. The lipoylated E2 component was able to bind recombinant E1 and E3 components in vitro to generate a PDH complex with a catalytic activity comparable with that of the wild-type enzyme. Reversible unfolding of the recombinant E2 and E3 components in 6 M guanidine hydrochloride was possible in the absence of chaperonins, with recoveries of enzymic activities of 95% and 85%, respectively. However, only 26% of the E1 enzyme activity was recovered under the same conditions as a result of irreversible denaturation of both E1alpha and E1beta. This represents the first complete post-translational modification and assembly of a fully active PDH complex from recombinant proteins in vitro.

Genes, Bacterial↗

PubMed1973

Kinetic properties of the partially purified pyruvate dehydrogenase complex of ox brain.

The properties of a purified preparation of the pyruvate dehydrogenase complex from ox brain have been compared with those of a similar preparation from ox kidney. A broad pH optimum around 7.8, similar dependence on ionic strength, and independence of the nature of the buffer anions or cations characterized preparations from both tissues. Michaelis constants for the binding of pyruvate, thiamin pyrophosphate, NAD(+) and CoA were also similar. Enzyme from both tissues was inhibited by NADH, by copper and other heavy metals, by high concentrations of tricarboxylic acid-cycle intermediates, and by preincubation with ATP. Acetyl-CoA itself did not appear to inhibit these preparations, although some commercial preparations of acetyl-CoA did contain an inhibitor. Although oxaloacetate and alpha-oxobutyrate were weak inhibitors, a number of other alpha-oxo acids including phenylpyruvate did not inhibit. The properties of the pyruvate dehydrogenase complex from brain and kidney appeared similar.

Adenosine Triphosphate↗

PubMed2004

Pyruvate dehydrogenase deficiency presenting as dystonia in childhood.

Two individuals with pyruvate dehydrogenase (PDH) deficiency due to missense mutations in the gene for the E1alpha subunit (PDHA1) presented during childhood with dystonia. The first patient, a male, presented at age 4 years with dystonia affecting the lower limbs, which responded to treatment with combined carbidopa and levodopa. The second patient, a female, was first investigated at age 6 years because of a dystonic gait disorder. In both patients, the main clue to the biochemical diagnosis was a raised concentration of lactate in the cerebrospinal fluid. PDH activity was significantly reduced in cultured fibroblasts in both cases. Dystonia is a previously unrecognized major manifestation of PDH deficiency and is of particular interest as the mutations in the PDHA1 gene in these patients have both been identified previously in individuals with typical presentations of the condition.

Child↗

PubMed2001

Inactivation of the murine pyruvate dehydrogenase (Pdha1) gene and its effect on early embryonic development.

A deficiency of pyruvate dehydrogenase complex (PDC) in humans results in lactic acidosis and neurological dysfunction that frequently results in death during infancy. Using gene targeting technology, a silent mutation was introduced into the murine X-linked Pdha1 gene that encodes the alpha subunit of the pyruvate dehydrogenase or E1 component of the complex. Two loxP sequences were introduced into intronic sequences flanking exon 8 to generate the Pdha1(flox8) allele. In vitro studies in embryonic stem cells demonstrated that deletion of exon 8 ablated PDC activity. Homozygous Pdha1(flox8) females were bred with male mice carrying a wild-type Pdha1 allele and a transgene that ubiquitously expresses the Cre recombinase to produce progeny with a deletion in exon 8, Pdha1(Deltaex8). The majority of progeny were found to be mosaic with the presence of both the flox and deleted alleles, and there were no apparent phenotypic effects associated with the null allele. The mosaic mice were interbred to increase the degree of mosaicism for the Pdha1(Deltaex8) allele in the subsequent generation, resulting in a significantly smaller litter size (54% reduction). Embryos carrying predominantly the Pdha1(Deltaex8) allele were found to be globally delayed in development by 9.5 days postcoitus, with resorption occurring over the following several days. These findings demonstrate an essential role for oxidative metabolism of glucose during the early postimplantation period of prenatal development.

Alleles↗

PubMed1990

Interactions of lipoyl domains with the E1p subunits of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Equilibrium binding experiments were carried out with lipoyl domains and the pyruvate decarboxylase [pyruvate dehydrogenase (lipoamide), E1p, EC 1.2.4.1)] component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The dissociation constant (Ks) was estimated to be not less than 0.3 mM, exceeding the Km value (33 microM) for reductive acetylation of the domains by an order of magnitude. Thus, the lipoyl domain, which is required to promote reductive acetylation of the lipoyl group, does not appear to do this simply by enhancing the binding to E1p. The difference between Ks and Km suggests that the formation and release of reductively acetylated lipoyl domains from the enzyme may be a relatively rapid step in the mechanism.

Escherichia coli↗

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↗

PubMed2002

Glucose-stimulated insulin secretion does not require activation of pyruvate dehydrogenase: impact of adenovirus-mediated overexpression of PDH kinase and PDH phosphate phosphatase in pancreatic islets.

Glucose-stimulated increases in mitochondrial metabolism are generally thought to be important for the activation of insulin secretion. Pyruvate dehydrogenase (PDH) is a key regulatory enzyme, believed to govern the rate of pyruvate entry into the citrate cycle. We show here that elevated glucose concentrations (16 or 30 vs 3 mM) cause an increase in PDH activity in both isolated rat islets, and in a clonal beta-cell line (MIN6). However, increases in PDH activity elicited with either dichloroacetate, or by adenoviral expression of the catalytic subunit of pyruvate dehydrogenase phosphatase, were without effect on glucose-induced increases in mitochondrial pyridine nucleotide levels, or cytosolic ATP concentration, in MIN6 cells, and insulin secretion from isolated rat islets. Similarly, the above parameters were unaffected by blockade of the glucose-induced increase in PDH activity by adenovirus-mediated over-expression of PDH kinase (PDK). Thus, activation of the PDH complex plays an unexpectedly minor role in stimulating glucose metabolism and in triggering insulin release.

Adenoviridae↗

PubMed2000

Selective modification of the pyruvate dehydrogenase kinase isoform profile in skeletal muscle in hyperthyroidism: implications for the regulatory impact of glucose on fatty acid oxidation.

The pyruvate dehydrogenase kinases (PDK1-4) regulate glucose oxidation through inhibitory phosphorylation of the pyruvate dehydrogenase complex (PDC). Immunoblot analysis with antibodies raised against recombinant PDK isoforms demonstrated changes in PDK isoform expression in response to experimental hyperthyroidism (100 microg/100 g body weight; 3 days) that was selective for fast-twitch vs slow-twitch skeletal muscle in that PDK2 expression was increased in the fast-twitch skeletal muscle (the anterior tibialis) (by 1. 6-fold; P<0.05) but not in the slow-twitch muscle (the soleus). PDK4 protein expression was increased by experimental hyperthyroidism in both muscle types, there being a greater response in the anterior tibialis (4.2-fold increase; P<0.05) than in the soleus (3.2-fold increase; P<0.05). The hyperthyroidism-associated up-regulation of PDK4 expression was observed in conjunction with suppression of skeletal-muscle PDC activity, but not suppression of glucose uptake/phosphorylation, as measured in vivo in conscious unrestrained rats (using the 2-[(3)H]deoxyglucose technique). We propose that increased PDK isoform expression contributes to the pathology of hyperthyroidism and to PDC inactivation by facilitating the operation of the glucose --> lactate --> glucose (Cori) and glucose --> alanine --> glucose cycles. We also propose that enhanced relative expression of the pyruvate-insensitive PDK isoform (PDK4) in skeletal muscle in hyperthyroidism uncouples glycolytic flux from pyruvate oxidation, sparing pyruvate for non-oxidative entry into the tricarboxylic acid (TCA) cycle, and thereby supporting entry of acetyl-CoA (derived from fatty acid oxidation) into the TCA cycle.

Animals↗

PubMed1976

Studies on the effects of coenzyme A-SH: acetyl coenzyme A, nicotinamide adenine dinucleotide: reduced nicotinamide adenine dinucleotide, and adenosine diphosphate: adenosine triphosphate ratios on the interconversion of active and inactive pyruvate dehydrogenase in isolated rat heart mitochondria.

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DL-carnitine and acetyl-DL-carnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl2. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.

Acetyl Coenzyme A↗

PubMed1984

Central hypoventilation syndrome in pyruvate dehydrogenase complex deficiency.

The presentation and treatment of a central hypoventilation syndrome in a boy with pyruvate dehydrogenase complex (PDHC) deficiency are reported. Dephosphorylated PDHC was assayed in disrupted fibroblasts after pretreatment with dichloroacetate, a pyruvate dehydrogenase kinase inhibitor. Maximal specific activity of activated patient PDHC was 10% to 30% of control values. Patient PDHC activity was not increased by alterations in concentrations of pyruvate or cofactors (thiamine pyrophosphate [TPP], coenzyme A [CoA], oxidized form of nicotinamide adenine dinucleotide [NAD+]). Clinically, normalization of plasma lactate by a high-lipid diet did not prevent slowly progressive neurologic decline. The patient manifested intermittent ataxia, episodic profound weakness, moderate psychomotor retardation, ophthalmoplegia, and retinal pigment epithelial changes. A true central hypoventilation syndrome was documented on the basis of rigorous radiologic, electrophysiologic, and pulmonary function criteria. Theophylline, progesterone, and ritalin neither altered ventilatory response to CO2 nor permitted weaning from the ventilator. In contrast, peripheral chemoreceptor stimulants (intravenous doxapram; oral almitrine) effected an acute doubling of minute ventilation with appropriate decreases in PaCO2. However, a positive response to long-term therapy with almitrine could not be unequivocally shown. It was concluded that measurement of disrupted fibroblast PDHC following dichloroacetate activation constitutes an accurate assay for PDHC deficiency. PDHC deficiency must be considered in the differential diagnosis of the central hypoventilation syndrome; this appears to be the first report of such an association. Finally, a therapeutic trial of a peripheral chemoreceptor agonist is warranted in the management of central hypoventilation syndrome.

Almitrine↗

PubMed1993

Insulin-induced activation of pyruvate dehydrogenase complex in skeletal muscle of diabetic rats.

The pyruvate dehydrogenase (PDH) complex undergoes reversible phosphorylation catalyzed by a PDH kinase (inactivating) and a PDH phosphatase (activating). In skeletal muscle, a decreased proportion of active PDH (PDHa) complex limits glucose oxidation in insulin-deficient states. The time-course for reactivation of the PDH complex by insulin in skeletal muscle of diabetic rats is important to understanding the potential mode of the action of insulin in regulating glucose metabolism. A single injection of insulin (1 U/kg) completely reversed the effects of alloxan-diabetes on PDHa activity within 1 hour. The normalization of the effects of diabetes on PDHa activity by insulin was maintained for a minimum of 6 hours. The increase in PDHa activity occurred before an insulin-induced decrease in plasma free fatty acids levels, demonstrating a dissociation between the antilipolytic effects of insulin and its ability to activate the PDH complex. PDH kinase activity was not normalized to control values following a single injection of insulin. Therefore, acute (1 to 6 hours) insulin-mediated activation of the PDH complex does not result from a decrease in PDH kinase activity. However, longer-term insulin therapy (1 U/kg body weight; twice daily) restored both PDHa and PDH kinase activities. The results are consistent with the hypothesis that activation of the PDH complex immediately following insulin administration is not mediated by a decreased PDH kinase activity. However, with daily insulin therapy in diabetes, activation of the PDH complex results from decreased PDH kinase activity.

Animals↗

PubMed2006

Increased expression of hepatic pyruvate dehydrogenase kinases 2 and 4 in young and middle-aged Otsuka Long-Evans Tokushima Fatty rats: induction by elevated levels of free fatty acids.

The activity of the pyruvate dehydrogenase complex (PDC) is regulated by covalent modification of its E1 component, which is catalyzed by specific pyruvate dehydrogenase kinases (PDKs) and phosphatases. In the liver, PDK2 and PDK4 are the most abundant PDK isoforms, which are responsible for inactivation of PDC when glucose availability is scarce in the body. In the present study, regulatory mechanisms of hepatic PDC were examined before and after the onset of type 2 diabetes mellitus in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, using Long-Evans Tokushima Otsuka (LETO) rats as controls. Plasma glucose and insulin concentrations were at normal levels in rats aged 8 weeks, but were significantly higher in OLETF than in LETO rats aged 25 weeks, indicating insulin resistance in OLETF rats. Plasma free fatty acids (FFAs) were 1.6-fold concentrated, and the liver PDC activity was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidative decarboxylation in OLETF rats before and after the onset of diabetes. Pyruvate dehydrogenase kinase activity and abundance of PDK2 and PDK4 proteins, as well as mRNAs, were greater in OLETF rats at both ages. These results suggest that persistently elevated levels of circulating free fatty acid in normal and diabetic OLETF rats play an important role in stimulating PDK2 and PDK4 expression in liver.

Age Factors↗

PubMed1983

The catalytic mechanism of transketolase. Thiamin pyrophosphate-derived transition states for transketolase and pyruvate dehydrogenase are not identical.

Thiamin thiazolone pyrophosphate (TTPP) has been reported to be an effective transition state analogue for the thiamin pyrophosphate-dependent partial reaction of pyruvate dehydrogenase (Gutowski, J. A., and Lienhard, G. E. (1976) J. Biol. Chem. 251, 2863-2866). The kinetics of the interaction of TTPP with transketolase are reported here. TTPP is a competitive inhibitor, with respect to thiamin pyrophosphate, of bakers' yeast transketolase but it is neither a tight binding inhibitor nor a slow binding inhibitor. TTPP decreases the kinetically observed negative cooperativity seen for thiamin pyrophosphate and also decreases the rate constant for the hysteretic activation of the enzyme by thiamin pyrophosphate. We conclude that thiamin thiazolone pyrophosphate is not an effective transition state analogue for the reaction catalyzed by bakers' yeast transketolase. This difference between transketolase and pyruvate dehydrogenase may be related to differences in the polarity of the active sites of the enzymes. It is conceivable that the active sites of the pyruvate decarboxylase subunit of pyruvate dehydrogenase is hydrophobic, by analogy with the known hydrophobicity of the active site of brewers' yeast pyruvate decarboxylase. This hydrophobicity would stabilize a transition state with no charge on the thiazole portion of the coenzyme, similar to the "uncharged" thiazole portion of TTPP. In contrast, the active site of bakers' yeast transketolase, which is known to contain charged amino acid side chains, should be less favorable for such an uncharged transition state. A charge-separated canonical form related to TTPP could be preferentially stabilized in the active site of transketolase.

Kinetics↗

PubMed1996

Sepsis-induced alterations in pyruvate dehydrogenase complex activity in rat skeletal muscle: effects on plasma lactate.

The pyruvate dehydrogenase (PDH) complex undergoes reversible phosphorylation catalyzed by a PDH kinase (inactivating) and a PDH phosphatase (activating). In skeletal muscle, a decreased proportion of PDH complex in the active, nonphosphorylated form (PDHa) limits glucose oxidation and promotes the conversion of pyruvate to lactate. Increased lactate formation with the accompanying hyperlactatemia is a frequent metabolic complication of sepsis. The time course for inactivation of the PDH complex in skeletal muscle during sepsis was contrasted with changes in PDHa during sterile inflammation 3,7, or 14 days following the implantation of the foreign body nidus. Total PDH complex activity was not altered in any of the conditions examined. Sepsis, but not sterile inflammation, caused a reduction in the muscle PDHa measured 3 or 7 days following induction of sepsis. The inhibition of the muscle PDHa during sepsis was associated with a sustained hyperlactatemia. PDH kinase activity measured in extracts of mitochondria was enhanced twofold during this period. Fourteen days after induction of sepsis, there were no differences in the PDHa or plasma lactate concentrations in septic rats compared with either control or sterile inflammation. Furthermore, the PDH kinase activity was decreased to values observed in control values. The results are consistent with the hypothesis that a reduced PDHa in skeletal muscle during sepsis is responsible, in part, for the hyperlactatemia characteristic of septic hypermetabolism. Furthermore, the results provide evidence that the decrease in PDHa results from a stable stimulation of PDH kinase activity.

Abdominal Abscess↗

PubMed1999

Characterization of the dihydrolipoamide acetyltransferase of the mitochondrial pyruvate dehydrogenase complex from potato and comparisons with similar enzymes in diverse plant species.

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) can be disassociated in 1 M NaCl and 0.1 M glycine into a large dihydrolipoamide acetyltransferase (E2) complex and smaller pyruvate dehydrogenase (E1) and dihydrolipoamide dehydrogenase (E3) complexes. The E2 complex consists of 55 and 78-kDa polypeptides which are reversibly radiolabelled to a similar degree in the intact mPDC by [2-14C]pyruvate. Affinity-purified antibodies against the 55-kDa protein do not cross-react with the 78-kDa protein and the two proteins show different peptide patterns following partial proteolysis. The 78 and 55-kDa proteins are present in approximately equal abundance in the E2 complex and incorporate a similar amount of [14C] on incubation with [2-14C]pyruvate. Native mPDC and the E2 complex have sedimentation coefficients of 50S and 30S, respectively. Titration of electro-eluted polypeptides against the intact mPDC and E2 complex revealed that each mg of mPDC contains 0.4 mg of E1, 0.4 mg of E2 and 0.2 mg of E3. Labelling of partially purified mPDC from potato, pea, cauliflower, maize and barley, with [2-14C]pyruvate, suggest that a 78-kDa acetylatable protein is only found in the dicotyledonous species, while all plant species tested contained a smaller 52-60 kDa acetylatable protein.

Acetylation↗