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PubMed2002

Diagnosis and molecular analysis of three male patients with thiamine-responsive pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex (PDHC) deficiency is a major cause of congenital lactic acidemia in children. PDHC catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment was effective for some patients with PDHC deficiency. We reexamined 30 patients with congenital lactic acidemia of unknown origin who had normal PDHC activity in their cultured fibroblasts using a routine assay with a high (0.4 mM) thiamine pyrophosphate (TPP) concentration. We measured the activity of PDHC in the presence of a low (1x10(-4) mM) TPP concentration, and analyzed for mutations in the E1alpha subunit gene. Three males had low PDHC activity in the presence of 1x10(-4) mM TPP. The DNA sequence of these three patients' X-linked E1alpha subunit revealed a substitution of alanine for valine at position 71 (V71A) in exon 3, phenylalanine for cysteine at position 101 (C101F) in exon 4, and glycine for arginine at position 263 (R263G) in exon 8, respectively. Thiamine treatment was effective in these three patients. Therefore, they had a thiamine-responsive PDHC deficiency due to a point mutation in the E1alpha subunit gene. PDHC activity should be measured at a low TPP concentration to detect thiamine-responsive PDHC deficiency so that thiamine treatment can be initiated as soon as possible.

Acidosis, Lactic↗
PubMed1985

Relationship between activation state of pyruvate dehydrogenase complex and rate of pyruvate oxidation in isolated cerebro-cortical mitochondria: effects of potassium ions and adenine nucleotides.

The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [1-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.

Acetyltransferases↗
PubMed2000

Effects of low-level lead on glycolytic enzymes and pyruvate dehydrogenase of rat brain in vitro: relevance to sporadic Alzheimer's disease?

Lead is known to be a potent inhibitor of many enzymes working in the brain, thus possibly inducing functional problems in the brain under pathophysiological conditions. Among such enzymes are those involved in glucose metabolism and energy production. We investigated the inhibitory effects of low-level lead on brain hexokinase (HK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK) and pyruvate dehydrogenase complex (PDHc) with rat brain homogenate. PDHc was distinctively inhibited when low-dose lead acetate was added last of all (IC50 = 5 microM) to the reaction mixture. The other enzymes were completely resistant to 5 microM of lead acetate. When the homogenate was preincubated with lead acetate HK was dramatically inhibited by low-level lead acetate (1-5 microM), in a manner dependent on both preincubation time and lead concentration. However, the inhibitory effect was abolished by coincubation with its substrates, glucose or ATP. The results suggest that exposure to low levels of lead may increase the risk of cerebral hypometabolism caused by direct inhibition of specific glucose-utilizing enzymes. In this context, lead might be regarded as a risk factor in the abnormal glucose metabolism seen in some kinds of neurodegenerative disorders such as sporadic Alzheimer's disease.

Adenosine Triphosphate↗
PubMed2005

Gibberellin regulates mitochondrial pyruvate dehydrogenase activity in rice.

Pyruvate dehydrogenase kinase (PDK) is a negative regulator of the mitochondrial pyruvate dehydrogenase complex (mtPDC) that plays a key role in intermediary metabolism. OsPDK1 was identified as a gibberellin-up-regulated gene using a cDNA microarray. The full-length cDNA for OsPDK1 was 1498 bp and encoded a predicted polypeptide of 363 amino acids. Genomic DNA analysis showed the presence of another isoform of PDK, OsPDK2, in rice. Reverse transcriptase-PCR analysis revealed differential expression of the two isoforms. OsPDK1 was expressed in leaf blade and leaf sheath but not in callus and root, while OsPDK2 was expressed constitutively in all tissues examined. Maximum expression of OsPDK1 in leaf sheath was detected by Northern blot analysis when seedlings were treated with 5 microM GA3 for 24 h. OsPDK1 expression was up-regulated by GA3, and there was little effect of other plant hormones. Mitochondrial pyruvate dehydrogenase (PDH) activity was reduced compared with control plants in 2-week-old seedlings treated with GA3. The beta-glucuronidase (GUS) reporter gene, driven by a 2,067 bp OsPDK1 promoter region fragment, was mainly expressed in the aleurone layer of germinating seed and leaf sheath. Transgenic rice expressing PDK1 RNAi had altered vegetative growth with reduced accumulation of vegetative tissues. These results suggest that gibberellin modulates the activity of mtPDC by regulating OsPDK1 expression and subsequently controlling plant growth and development.

Blotting, Northern↗
PubMed2003

Immunocapture and microplate-based activity measurement of mammalian pyruvate dehydrogenase complex.

Altered pyruvate dehydrogenase (PDH) functioning occurs in primary PDH deficiencies and in diabetes, starvation, sepsis, and possibly Alzheimer's disease. Currently, the activity of the enzyme complex is difficult to measure in a rapid high-throughput format. Here we describe the use of a monoclonal antibody raised against the E2 subunit to immunocapture the intact PDH complex still active when bound to 96-well plates. Enzyme turnover was measured by following NADH production spectrophotometrically or by a fluorescence assay on mitochondrial protein preparations in the range of 0.4 to 5.0 micro g per well. Activity is sensitive to known PDH inhibitors and remains regulated by phosphorylation and dephosphorylation after immunopurification because of the presence of bound PDH kinase(s) and phosphatase(s). It is shown that the immunocapture assay can be used to detect PDH deficiency in cell extracts of cultured fibroblasts from patients, making it useful in patient screens, as well as in the high-throughput format for discovery of new modulators of PDH functioning.

Adenosine Triphosphate↗
PubMed2001

Gender-specific occurrence of West syndrome in patients with pyruvate dehydrogenase complex deficiency.

Pyruvate dehydrogenase complex (PDHC) deficiency, a major cause of congenital lactic acidemia in children, usually is complicated by seizures, and, in some patients, West syndrome has occurred. We diagnosed 60 patients with PDHC deficiency, including equal numbers of affected males and females. We studied the clinical features in 10 patients with West syndrome caused by PDHC deficiency, and examined the relation to the mutation of the E(1)alpha subunit, representing the great majority of PDHC deficiencies. Among 30 boys and 30 girls with PDHC deficiency,1 boy and 9 girls had West syndrome, even though overall West syndrome shows a slight male preponderance. Therefore, West syndrome associated with PDHC deficiency occurred in 9 of 30 female patients (33%), but in only 1 of 30 male patients (3%). The frequency of West syndrome in patients with PDHC deficiency was significantly higher in females than in males(p<0.05). Lactate concentrations in blood and CSF should be measured in female patients with West syndrome as a screening test for PDHC deficiency, because of gender-specific occurrence of West syndrome caused by PDHC deficiency.

Acidosis, Lactic↗
PubMed1987

Isolated tumoral pyruvate dehydrogenase can synthesize acetoin which inhibits pyruvate oxidation as well as other aldehydes.

Oxidation of 1 mM pyruvate by Ehrlich and AS30-D tumor mitochondria is inhibited by acetoin, an unusual and important metabolite of pyruvate utilization by cancer cells, by acetaldehyde, methylglyoxal and excess pyruvate. The respiratory inhibition is reversed by other substrates added to pyruvate and also by 0.5 mM ATP. Kinetic properties of pyruvate dehydrogenase complex isolated from these tumor mitochondria have been studied. This complex appears to be able to synthesize acetoin from acetaldehyde plus pyruvate and is competitively inhibited by acetoin. The role of a new regulatory pattern for tumoral pyruvate dehydrogenase is presented.

Acetoin↗
PubMed1991

Characterization of the mutations in three patients with pyruvate dehydrogenase E1 alpha deficiency.

The human pyruvate dehydrogenase complex catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA. Defects in several of the seven subunits have been reported, but the majority of mutations affect the E1 component and especially the E1 alpha subunit. However, the clinical presentation of patients with pyruvate dehydrogenase E1 alpha deficiency is extremely variable. Dependency of the brain on pyruvate dehydrogenase activity and localization of the gene for the somatic form of the pyruvate dehydrogenase E1 alpha subunit to the X chromosome provide the basis for a better understanding of the variation in the clinical manifestations. Further understanding of the function and interaction of subunits and the pathophysiology of pyruvate dehydrogenase deficiency necessitates the characterization of mutations in the pyruvate dehydrogenase complex. We report the analysis of three patients with pyruvate dehydrogenase E1 alpha deficiency. One female has a three base pair deletion which affects dephosphorylation of the subunit. Of two males analysed, one has a two base pair deletion causing a shift in the reading frame. The other has a base change, resulting in an Arg to His substitution. All three mutations are located near the carboxyl terminus of the subunit.

Amino Acid Sequence↗
PubMed1996

Role of the regulatory subunit of bovine pyruvate dehydrogenase phosphatase.

Bovine pyruvate dehydrogenase phosphatase (PDP) is a Mg2+-dependent and Ca2+-stimulated heterodimer that is a member of the protein phosphatase 2C family and is localized to mitochondria. Insight into the function of the regulatory subunit of PDP (PDPr) has been gained. It decreases the sensitivity of the catalytic subunit of PDP (PDPc) to Mg2+. The apparent Km of PDPc for Mg2+ is increased about 5-fold, from about 0.35 mM to 1.6 mM. The polyamine spermine increases the sensitivity of PDP but not PDPc to Mg2+, apparently by interacting with PDPr. PDPc but not PDP can use the phosphopeptide RRAT(P)VA as a substrate. These observations are interpreted to indicate that PDPr blocks or distorts the active site of PDPc and that spermine produces a conformational change in PDPr that reverses its inhibitory effect. These findings suggest that PDPr may be involved in the insulin-induced activation of the mitochondrial PDP in adipose tissue, which is characterized by a decrease in its apparent Km for Mg2+.

Adipose Tissue↗
PubMed1977

[Study of the kinetic mechanism of the pyruvate-2,6-dichlorophenolindophenol reductase activity of muscle pyruvate dehydrogenase].

The mechanism of pyruvate-2,6-dichlorophenol-indophenol (2,6-CPI) reductase reaction catalyzed by the pyruvate dehydrogenase complex from pigeon breast muscle and by its pyruvate dehydrogenase component was studied. The K'm values for 2,6-DCPI in both cases were found equal to 1.3--1.4-10(-5) M. The double reverse values plots obtained at a fixed concentration of the first substrate and a variable concentration of the second one were linear and had a constant K'm/V'max ratio. The substitution of thiamine pyrophosphate and pyruvate by the substrate decarboxylation product, i.e. 2-oxyethyl thiamine pyrophosphate under similar conditions resulted in kinetic plots, typical for the "ping-pong" mechanism of enzymatic reactions. A mechanism of the pyruvate 2,6-DCPI reductase reaction, providing for the interaction of 2-oxyethyl thiamine pyrophosphate after its binding to the apoenzyme with a certain protein group of the pyruvate dehydrogenase active centre, was postulated. The reaction was shown to result in the production of acetyl-substituted reduced form of the enzyme. Regeneration of free enzyme required the presence of 2,6-DCPI as oxidizing agent.

2,6-Dichloroindophenol↗
PubMed1976

Regulation of the human pyruvate dehydrogenase complex.

1. The pyruvate dehydrogenase complex from human heart has been partially purified and shown to be regulated by a phosphorylation-dephosphorylation cycle similar to that previously found for other mammalian tissues. 2. Incubation of the complex with ATP (2 mmol/1) led to its inactivation associated with the concomitant incorporation into the protein of 32P from the terminal phosphate group of the ATP. Pyruvate, ADP, thiamin pyrophosphate and dichloroacetate diminished the rate of inactivation by ATP. 3. Pyruvate dehydrogenase phosphatase from human heart requires Mg2+ for activity and is sensitive to Ca2+ at concentrations of a few mumol/1. Similar ionic requirements of the skeletal muscle phosphatase have been demonstrated in a crude tissue extract. 4. The activity of pyruvate dehydrogenase in human adipose tissue was less than 10% of typical values in rats. This could be due to the high level of dietary fat consumed by humans, which is known to repress the enzyme activity in rats.

Adipose Tissue↗
PubMed1995

The effect of phosphorylation on pyruvate dehydrogenase.

Phosphorylation of the pyruvate dehydrogenase component (E1) of the muscle pyruvate dehydrogenase complex (PDC) by E1-kinase inhibits substrate conversion both in oxidative and non-oxidative reactions. Circular dichroism spectra were used to monitor the effect of phosphorylation on the following stages of the process: holoform formation from apo-E1 and thiamine pyrophosphate (TPP), substrate binding and active site deacetylation. It has been shown that phosphorylation of E1 reduces its affinity for TPP and prevents holo-E1 interaction with pyruvate. Phosphorylated and dephosphorylated PDC convert 2-hydroxyethyl-TPP in similar ways involving half of their active sites; all active sites of E1 function in the presence of deacetylating agents. The data obtained suggest that the phosphorylation and substrate binding sites interact with each other.

Adenosine Triphosphate↗
PubMed1979

Purification and properties of the Ascaris pyruvate dehydrogenase complex.

The pyruvate dehyhdrogenase complex (pyruvate:lipoate oxidoreductase (decarboxylating and acceptor-acetylating), EC 1.2.4.1) has been isolated from Ascaris muscle mitochondria and purified to near homogeneity by differential centrifugation, (NH4)2SO4 fractionation and calcium phosphate gel-cellulose chromatography. It is similar in shape, size and physical characteristics to pyruvate dehydrogenase complexes isolated from mammalian sources. It has an absolute dependence on CoA, NAD+ and pyruvate for activity and is competitively inhibited by acetyl-CoA and NADH. However, much higher NADH/NAD+ ratios are necessary to inhibit activity, suggesting regulation by the more reduced state of the pyridine nucleotide pool in Ascaris mitochondria.

Acetyl Coenzyme A↗
PubMed1969

Alpha-keto acid dehydrogenase complexes. X. Regulation of the activity of the pyruvate dehydrogenase complex from beef kidney mitochondria by phosphorylation and dephosphorylation.

This paper reports the discovery that the activity of the multienzyme pyruvate dehydrogenase complex from beef kidney mitochondria is regulated by a phosphorylation-dephosphorylation reaction sequence. The site of this regulation is the pyruvate dehydrogenase component of the complex. Phosphorylation and concomitant inactivation of pyruvate dehydrogenase are catalyzed by an ATP-specific kinase (i.e., a pyruvate dehydrogenase kinase), and dephosphorylation and concomitant reactivation are catalyzed by a phosphatase (i.e., a pyruvate dehydrogenase phosphatase). The kinase and the phosphatase appear to be regulatory subunits of the pyruvate dehydrogenase complex.

Animals↗
PubMed1982

Purification and properties of the pyruvate dehydrogenase complex from Salmonella typhimurium and formation of hybrids with the enzyme complex from Escherichia coli.

The pyruvate dehydrogenase (Pyruvate:lipoamide oxidoreductase (decarboxylating and acceptor acetylating), EC 1.2.4.1) complex from Salmonella typhimurium was purified, characterized and compared to the enzyme complex from Escherichia coli. No difference could be found in the molecular weights of the native enzyme complexes or in the single polypeptide chains of the enzymes of the two organisms. Values of 100 000, 87 000 and 56 000 were obtained for the polypeptide chains of the pyruvate dehydrogenase, the dihydrolipoamide transacetylase (acetyl-CoA:dihydrolipoamide S-acetyltransferase, EC 2.3.1.12) and the dihydrolipoamide dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) components, respectively. Complete cross-reactivity was found with antibodies directed against the pyruvate dehydrogenase complex from E. coli and electron micrographs of both enzyme complexes reveal identical structures. A high Michaelis constant for pyruvate with a Km = 6 . 10(-4) M and a somewhat weaker cooperativity as compared to the enzyme from E. coli reflect some minor differences, while the binding of the cofactor thiamine diphosphate (Km = 1 . 10(-6) M) is identical for both enzyme complexes. Reassociation to a fully active complex molecule works with equal facility between the pyruvate dehydrogenase component and a dihydrolipoamide transacetylase: dihydrolipoamide dehydrogenase subcomplex from either organism in all possible combinations.

Acetyltransferases↗
PubMed1999

Cytosolic redox state mediates postischemic response to pyruvate dehydrogenase stimulation.

Augmented pyruvate oxidation via pharmacological stimulation of pyruvate dehydrogenase (PDH) during reperfusion has been related to improved recovery of postischemic hearts independent of glycolytic activity. This study examined recovery of postischemic rabbit hearts during activation of PDH with dichloroacetate (DCA) in the presence of lactate, as a source of pyruvate, to determine the response to substrate-dependent changes in cytosolic redox state. After 10 min of ischemia, isolated hearts were reperfused with either 2.5 mM or 0. 5 mM pyruvate (Pyr) or 2.5 mM lactate (Lac), with or without 5 mM DCA. (13)C-enriched substrates allowed NMR assessment of metabolic perturbations. During normal perfusion, Pyr and Lac supported similar mechanical work. Increasing Pyr oxidation restored postischemic rate-pressure product to 82 +/- 4 and 88 +/- 6% of preischemic values during reperfusion with 2.5 and 0.5 mM Pyr, respectively, vs. 61 +/- 6 and 45 +/- 14% for untreated 2.5 and 0.5 mM Pyr, respectively (P < 0.05). In contrast, increasing Lac oxidation did not benefit recovery of RPP in untreated (44 +/- 7%) vs. DCA-treated 36 +/- 4% hearts. Thus the benefit of PDH activation for contractile recovery of postischemic hearts is mediated by the source of pyruvate, which also influences cytosolic redox state.

Animals↗
PubMed1976

Energy-linked regulation of glucose and pyruvate oxidation in isolated perfused rat heart. Role of pyruvate dehydrogenase.

1. The regulation of glycolysis and pyruvate oxidation under varying conditions of ATP and oxygen consumption was studied in isolated perfused rat hearts. Potassium-induced arrest was employed to inhibit the ATP consumption of the heart. 2. Under the experimental conditions, the beating heart used solely glucose as the oxidisable substrate. The glycolytic flux through the aldolase step decreased in pace with the decreasing oxygen consumption during the potassium-induced arrest of the heart. The decrease in glucose oxidation was larger than the inhibition of the oxygen consumption, suggesting that the arrested heart switches to fatty acid oxidation. The time course and percentage changes of the inhibition of pyruvate oxidation and the decrease in the amount of the active form of pyruvate dehydrogenase suggest that the amount of active pyruvate dehydrogenase is the main regulator of pyruvate oxidation in the perfused heart. 3. To test the relative significance of the possible mechanisms regulating covalent interconversions of pyruvate dehydrogenase, the following parameters were measured in response to the potassium-induced cardiac arrest: concentrations of pyruvate, acetyl-CoA, CoA-SH, citrate, alpha-oxoglutarate, ATP, ADP, AMP, creatine, creatine phosphate and inorganic phosphate and the mitochondrial NADH/NAD+ ratio. In cardiac tissue the adenylate system is not a good indicator of the energy state of the mitochondrion, even when the concentrations of AMP and free cytosolic ADP are calculated from the adenylate kinase and creatine kinase equilibria. Only creatine phosphate and inorganic phosphate undergo significant changes, but evidence of the participation of the latter compounds in the regulation of the pyruvate dehydrogenase interconversions is lacking. The potassium-induced arrest of the heart resulted in a decrease in pyruvate, a slight increase in acetyl-CoA, a large increase in the concentration of citrate and an increase in the mitochondrial NADH/NAD+. The results can be interpreted as showing that in the heart, the pyruvate dehydrogenase interconversions are mainly regulated by the pyruvate concentration and the mitochondrial redox state. Concentrations of all the regulators tested shifted to directions which one would expect to result in a decrease in the amount of active pyruvate dehydrogenase, but the changes were quite small. Therefore, the energy-linked regulation of pyruvate dehydrogenase in intact tissue is possibly mediated by the equilibrium relations between the cellular redox state and the phosphorylation potential recently confirmed in cardiac tissue.

Animals↗