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PubMed1983

Ruthenium Red inhibits the activation of pyruvate dehydrogenase caused by positive inotropic agents in the perfused rat heart.

The increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase caused by positive inotropic agents (from a control value of about 10%, to 40% of total enzyme) in the perfused rat heart could be completely blocked by prior perfusion with 2.5 micrograms of Ruthenium Red/ml. A similar increase caused by 5 mM-pyruvate was not blocked. This concentration of Ruthenium Red caused a 25% decrease in contractile force of hearts perfused in the absence of positive inotropic agents; however, in their presence the contractile force reached the same value in the absence or presence of Ruthenium Red. Neither control nor stimulated phosphorylase a content was affected by Ruthenium Red. Verapamil (0.1 microM) also decreased control contraction (by 40%), but did not block the activation of pyruvate dehydrogenase caused by a rise in extracellular [Ca2+]. The results support the hypothesis that positive inotropic agents activate pyruvate dehydrogenase in rat heart by increasing intramitochondrial [Ca2+].

Animals↗

PubMed2003

Organization of the cores of the mammalian pyruvate dehydrogenase complex formed by E2 and E2 plus the E3-binding protein and their capacities to bind the E1 and E3 components.

The subunits of the dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex can form a 60-mer via association of the C-terminal I domain of E2 at the vertices of a dodecahedron. Exterior to this inner core structure, E2 has a pyruvate dehydrogenase component (E1)-binding domain followed by two lipoyl domains, all connected by mobile linker regions. The assembled core structure of mammalian pyruvate dehydrogenase complex also includes the dihydrolipoyl dehydrogenase (E3)-binding protein (E3BP) that binds the I domain of E2 by its C-terminal I' domain. E3BP similarly has linker regions connecting an E3-binding domain and a lipoyl domain. The composition of E2.E3BP was thought to be 60 E2 plus approximately 12 E3BP. We have prepared homogenous human components. E2 and E2.E3BP have s(20,w) values of 36 S and 31.8 S, respectively. Equilibrium sedimentation and small angle x-ray scattering studies indicate that E2.E3BP has lower total mass than E2, and small angle x-ray scattering showed that E3 binds to E2.E3BP outside the central dodecahedron. In the presence of saturating levels of E1, E2 bound approximately 60 E1 and maximally sedimented 64.4 +/- 1.5 S faster than E2, whereas E1-saturated E2.E3BP maximally sedimented 49.5 +/- 1.4 S faster than E2.E3BP. Based on the impact on sedimentation rates by bound E1, we estimate fewer E1 (approximately 12) were bound by E2.E3BP than by E2. The findings of a smaller E2.E3BP mass and a lower capacity to bind E1 support the smaller E3BP substituting for E2 subunits rather than adding to the 60-mer. We describe a substitution model in which 12 I' domains of E3BP replace 12 I domains of E2 by forming 6 dimer edges that are symmetrically located in the dodecahedron structure. Twelve E3 dimers were bound per E248.E3BP12 mass, which is consistent with this model.

Dihydrolipoamide Dehydrogenase↗

PubMed1998

Pyruvate dehydrogenase complex deficiency and absence of subunit X.

The pyruvate dehydrogenase complex (PDHc) is a multienzyme complex consisting of three catalytic and two regulatory enzymes, as well as a less well defined subunit called protein X. PDHc deficiency is a common cause of congenital lactic acidosis. Most patients with PDH deficiency have a mutation in the alpha chain of the PDH E1 enzyme. Very few patients have been described in whom the basic defect of a PDH deficiency is situated in the X protein. We studied a boy with severe lactic acidosis and developmental delay in whom a deficiency of PDH activity led to further investigations. Immunochemical analysis with anti-PDHc antibodies demonstrated an absence of the X component. This report is the fourth family in which an abnormal protein X has been found. In cases with PDH deficiency where no mutation of the PDHE1 alpha gene is found, further investigations by means of immunoblotting with specific antibodies against the different subunits should be performed.

Female↗

PubMed1988

A deficiency of both subunits of pyruvate dehydrogenase which is not expressed in fibroblasts.

Pyruvate dehydrogenase complex (PDC) deficiency usually has been detected by decreased activity in cultured skin fibroblasts. We investigated two brothers in whom PDC activity was less than 10% of controls in lymphocytes but normal in skin fibroblasts. They both had abnormal neuromuscular development and lactic acidosis which was aggravated by ingestion of carbohydrate. One brother died at age 3 yr and tissues were obtained at autopsy soon after death. The brain was swollen with diffuse acute hemorrhages but without the lesions characteristic of Leigh's disease. PDC activity was virtually undetectable in mitochondria or homogenates of liver, skeletal muscle, and heart, but was about 30% of controls in kidney. The activity of the first component E1 was not detectable in mitochondria from liver, whereas the activities of the second and third components were normal; the activities of all components were normal in fibroblasts. Western immunoblot analysis showed absent to trace amounts of both the E1 alpha and E1 beta subunits in liver, skeletal muscle, and heart, with normal amounts of the second and third components. About one-fourth of control amounts of E1 alpha and E1 beta were present in kidney and normal levels were present in fibroblasts. PDC activity in lymphocytes from the mother was 35% of controls; she had normal PDC activity in her fibroblasts. PDC activity was normal in lymphocytes from the brothers' sister, father, and maternal grandparents and great-grandmother. The mode of inheritance was not established. In conclusion, PDC deficiency may not be detected in skin fibroblasts in some cases; the mechanism of variable tissue expression of E1 remains to be delineated.

Child, Preschool↗

PubMed2000

Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues.

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.

Alanine↗

PubMed1980

Halothane anaesthesia can block insulin stimulation of pyruvate dehydrogenase activity in mammary glands of 24-hour starved lactating rats.

The effect of halothane anaesthesia on the activity of the mitochondrial enzyme pyruvate dehydrogenase was studied in starved lactating rats. Extracts of freeze-clamped mammary gland and liver were assayed for pyruvate dehydrogenase activity. The fraction of the enzyme in the phosphorylated inactive form was increased greatly by starvation or by streptozotocin diabetes, and halothane anaesthesia did not disturb this effect. In starved animals not exposed to halothane, injection of insulin led to a rapid increase in the active fraction of the enzyme in mammary gland but not in liver. In animals under halothane anaesthesia this effect of insulin was largely abolished. The combination of starvation and halothane anaesthesia may impair mitochondrial accumulation of calcium which may be involved in the stimulation of pyruvate dehydrogenase by insulin.

Anesthesia, Inhalation↗

PubMed2004

Pyruvate dehydrogenase kinase isoform 2 activity limited and further inhibited by slowing down the rate of dissociation of ADP.

Pyruvate dehydrogenase kinase 2 (PDK2) activity is enhanced by the dihydrolipoyl acetyltransferase core (E2 60mer) that binds PDK2 and a large number of its pyruvate dehydrogenase (E1) substrate. With E2-activated PDK2, K(+) at approximately 90 mM and Cl(-) at approximately 60 mM decreased the K(m) of PDK2 for ATP and competitive K(i) for ADP by approximately 3-fold and enhanced pyruvate inhibition. Comparing PDK2 catalysis +/- E2, E2 increased the K(m) of PDK2 for ATP by nearly 8-fold (from 5 to 39 microM), increased k(cat) by approximately 4-fold, and decreased the requirement for E1 by at least 400-fold. ATP binding, measured by a cold-trapping technique, occurred at two active sites with a K(d) of 5 microM, which equals the K(m) and K(d) of PDK2 for ATP measured in the absence of E2. During E2-aided catalysis, PDK2 had approximately 3 times more ADP than ATP bound at its active site, and the pyruvate analogue, dichloroacetate, led to 16-fold more ADP than ATP being bound (no added ADP). Pyruvate functioned as an uncompetitive inhibitor versus ATP, and inclusion of ADP transformed pyruvate inhibition to noncompetitive. At high pyruvate levels, pyruvate was a partial inhibitor but also induced substrate inhibition at high ATP levels. Our results indicate that, at physiological salt levels, ADP dissociation is a limiting step in E2-activated PDK2 catalysis, that PDK2.[ADP or ATP].pyruvate complexes form, and that PDK2.ATP.pyruvate.E1 reacts with PDK2.ADP.pyruvate accumulating.

Acetyl Coenzyme A↗

PubMed1998

Molecular cloning, functional expression, and characterization of pyruvate dehydrogenase kinase from anaerobic muscle of the parasitic nematode Ascaris suum.

The pyruvate dehydrogenase complex (PDC) plays a key role in the anaerobic mitochondrial metabolism of the parasitic nematode Ascaris suum. A cDNA coding for an A. suum pyruvate dehydrogenase kinase (APDK) has been cloned and sequenced from poly(A)+ RNA isolated from adult A. suum muscle.2 APDK exhibited significant sequence identity to mammalian PDKs. Nucleotide sequence analysis of the APDK cDNA revealed a 22-nucleotide spliced leader, characteristic of many nematode mRNAs, a 5'-UTR of 6 nucleotides, an open reading frame of 1197 nucleotides, and a 3'-UTR of 101 nucleotides that included a putative polyadenylation signal. The open reading frame predicted a protein of 399 amino acids with a molecular weight of 45,402 that included a putative 18-aminoacid leader peptide. Recombinant APDK (rAPDK) was functionally expressed in Escherichia coli with a his tag at its N-terminus and purified to apparent homogeneity on Ni-NTA-agarose. Recombinant APDK was a dimer and was not autophosphorylated and its activity was stimulated in the presence of APDK-deficient adult A. suum muscle PDC presumably by the binding of APDK to the dihydrolipoyl transacetylase (E2) core of the complex. After binding to the core, rAPDK activity was stimulated by elevated NADH/NAD+ and acetyl CoA/CoA ratios within the same ranges as observed for the native APDK. Immunoblotting suggested that native APDK focused as a series of 43-kDa spots (pI 6.1-6.8) on two-dimensional gels of the purified adult A. suum muscle PDC.

Amino Acid Sequence↗

PubMed1993

Regulation of pyruvate dehydrogenase activity in rat fat pads and isolated hepatocytes by levamisole.

The effect of levamisole on the catalytic activity of the pyruvate dehydrogenase (PDH) complex in rat adipose tissue segments and isolated hepatocytes was studied. Levamisole, an anthelmintic and immunotherapeutic agent with concentration-dependent oxidant/antioxidant properties, increased the catalytic activity of the pyruvate dehydrogenase complex in these systems. The activity of this enzyme complex may be regulated in part by protein-thiol modification. The treatment of rat adipose tissue segments and isolated hepatocytes with and without levamisole (0.1-3.0 mM) for 30 min resulted in a reversible increase in the catalytic activity of this enzyme complex. Levamisole at 1.5 mM and 2.0 mM were as potent as insulin (1.0 mU/ml) in adipose tissue and as dichloroacetate (2.0 mM) in isolated hepatocytes, respectively, in increasing the catalytic activity of the PDH complex. The stimulatory effects seen with levamisole were not accompanied by decreases in adenosine triphosphate (ATP) levels. The results of the present investigation suggest that the pyruvate dehydrogenase complex in adipose tissue and liver may be useful models for studying the mechanism of action of levamisole.

Adipose Tissue↗

PubMed1998

Plant mitochondrial pyruvate dehydrogenase complex: purification and identification of catalytic components in potato.

The pyruvate dehydrogenase complex (mPDC) from potato (Solanum tuberosum cv. Romano) tuber mitochondria was purified 40-fold to a specific activity of 5.60 micromol/min per mg of protein. The activity of the complex depended on pyruvate, divalent cations, NAD+ and CoA and was competitively inhibited by both NADH and acetyl-CoA. SDS/PAGE revealed the complex consisted of seven polypeptide bands with apparent molecular masses of 78, 60, 58, 55, 43, 41 and 37 kDa. N-terminal sequencing revealed that the 78 kDa protein was dihydrolipoamide transacetylase (E2), the 58 kDa protein was dihydrolipoamide dehydrogenase (E3), the 43 and 41 kDa proteins were alpha subunits of pyruvate dehydrogenase, and the 37 kDa protein was the beta subunit of pyruvate dehydrogenase. N-terminal sequencing of the 55 kDa protein band yielded two protein sequences: one was another E3; the other was similar to the sequence of E2 from plant and yeast sources but was distinctly different from the sequence of the 78 kDa protein. Incubation of the mPDC with [2-14C]pyruvate resulted in the acetylation of both the 78 and 55 kDa proteins.

Acetylation↗

PubMed2002

Thiamine-responsive pyruvate dehydrogenase deficiency in two patients caused by a point mutation (F205L and L216F) within the thiamine pyrophosphate binding region.

The human pyruvate dehydrogenase complex (PDHC) catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment is very effective for some patients with PDHC deficiency. Among these patients, five mutations of the pyruvate dehydrogenase (E1)alpha subunit have been reported previously: H44R, R88S, G89S, R263G, and V389fs. All five mutations are in a region outside the thiamine pyrophosphate (TPP)-binding region of the E1alpha subunit. We report the biochemical and molecular analysis of two patients with clinically thiamine-responsive lactic acidemia. The PDHC activity was assayed using two different concentrations of TPP. These two patients displayed very low PDHC activity in the presence of a low (1 x 10(-4) mM) TPP concentration, but their PDHC activity significantly increased at a high (0.4 mM) TPP concentration. Therefore, the PDHC deficiency in these two patients was due to a decreased affinity of PDHC for TPP. Treatment of both patients with thiamine resulted in a reduction in the serum lactate concentration and clinical improvement, suggesting that these two patients have a thiamine-responsive PDHC deficiency. The DNA sequence of these two male patients' X-linked E1alpha subunit revealed a point mutation (F205L and L216F) within the TPP-binding region in exon 7.

Binding Sites↗

PubMed1984

Apparent identity of alpha-subunit of pyruvate dehydrogenase and the protein phosphorylated in the presence of glutamate in P2-fractions of rat cerebral cortex.

Addition of L-glutamate or several citric acid cycle intermediates stimulated the phosphorylation of a protein with apparent molecular weight of 43,000 ( P43 ) in P2-fractions from rat cerebral cortex, and this phosphorylation was inhibited by dichloroacetic acid, a specific inhibitor of pyruvate dehydrogenase kinase. Comparison of several molecular properties of phosphorylated P43 and the phosphorylated alpha-subunit of pyruvate dehydrogenase indicated that both proteins are extracted by a similar procedure and have an identical apparent molecular weight and isoelectric point. Furthermore, digestion of both phosphorylated proteins by several different proteases in the presence of SDS yielded a similar pattern of phosphorylated peptides indicating that these proteins have a considerable sequence homology. Thus, various pieces of evidence indicate that P43 and the alpha-chain of pyruvate dehydrogenase are very similar if not identical. The possible implication of a glutamate stimulated phosphorylation of pyruvate dehydrogenase for long term potentiation and epilepsy is discussed.

Adenosine Diphosphate↗

PubMedDate not supplied

Metabolic control analysis of eucaryotic pyruvate dehydrogenase multienzyme complex.

Metabolic control analysis (MCA) of pyruvate dehydrogenase multienzyme (PDH) complex of eucaryotic cells has been carried out using both in vitro and in vivo mechanistic models. Flux control coefficients (FCC) for the sensitivity of pyruvate decarboxylation rate to activities of various PDH complex reactions are determined. FCCs are shown to be strong functions of both pyruvate levels and various components of PDH complex. With the in vitro model, FCCs are shown to be sensitive to only the E1 component of the PDH complex at low pyruvate concentrations. At high pyruvate concentrations, the control is shared by all of the components, with E1 having a negative influence while the other three components, E2, X, and K, exert a positive control over the pyruvate decarboxylation rate. An unusual behavior of deactivation of the E1 component leading to higher net PDH activity is shown to be linked to the combined effect of protein X acylation and E1 deactivation. The steady-state analysis of the in vivo model reveals multiple steady state behavior of pyruvate metabolism with two stable and one unstable steady-states branches. FCCs also display multiplicity, showing completely different control distribution exerted by pyruvate and PDH components on three branches. At low pyruvate concentrations, pyruvate supply dominates the decarboxylation rate and PDH components do not exert any significant control. Reverse control distribution is observed at high pyruvate concentration. The effect of dilution due to cell growth on pyruvate metabolism is investigated in detail. While pyruvate dilution effects are shown to be negligible under all conditions, significant PDH complex dilution effects are observed under certain conditions. Comparison of in vitro and in vivo models shows that PDH components exert different degrees of control outside and inside the cells. At high pyruvate levels, PDH components are shown to exert a higher degree of control when reactions are taking place inside the cells as compared to the in vitro situation.

Cell Division↗

PubMed1999

Sepsis alters pyruvate dehydrogenase kinase activity in skeletal muscle.

Chronic sepsis promotes a stable increase in pyruvate dehydrogenase kinase (PDHK) activity in skeletal muscle. PDHK is found tightly bound to the pyruvate dehydrogenase (PDH) complex and as free kinase. We investigated the ability of sepsis to modify the activity of the PDHK intrinsic to the PDH and free PDHK. Sepsis was induced by the intraabdominal introduction of a fecal-agar pellet infected with E. coli and B. fragilis. Five days later, mitochondria were isolated from skeletal muscle and PDHK measured in mitochondrial extracts. Sepsis caused an approximate 2-fold stimulation of PDHK. The mitochondrial extracts from control and septic rats were fractionated by gel chromatography on Sephacryl S-300 to separate PDHK intrinsic to PDH complex and free PDHK. PDH complex eluted at void volume and was assayed for PDHK intrinsic to the complex. The activity of PDHK intrinsic to PDH complex was a significantly increased 3 fold during sepsis. Free PDHK activity eluted after the PDH complex and its activity was enhanced by 70% during sepsis. Incubation of PDHK intrinsic to PDH with dichloroactate, an uncompetitive inhibitor of PDHK, showed the PDHK from septic rats relatively less sensitive to inhibition than controls. These results indicate that sepsis induces stable changes in PDHK in skeletal muscle.

Adenosine Triphosphate↗

PubMed1978

Regulation of brain pyruvate dehydrogenase multienzyme complex.

A number of excellent and comprehensive reviews on various aspects of pyruvate dehydrogenase multienzyme complex have been written recently. The purpose of the present review is to summarize briefly the reaction mechanism and the regulation of this enzyme. Emphasis is put on the most recent literature not covered by previous reviews. Particular attention is also paid to the regulation of brain pyruvate dehydrogenase multienzyme complex, since a number of patients with neuromuscular diseases, such as Friedreich's ataxia, show a decreased rate of pyruvate oxidation.

Animals↗

PubMed1985

Mechanism of activation of bovine kidney pyruvate dehydrogenase a kinase by malonyl-CoA and enzyme-catalyzed decarboxylation of malonyl-CoA.

The activity of the pyruvate dehydrogenase kinase, which phosphorylates and thereby inactivates the pyruvate dehydrogenase complex, was stimulated by malonyl-CoA. Treatment with [2-14C]malonyl-CoA resulted in acylation of sites in the complex. Both acylation and activation of kinase activity increased in a time-dependent manner with a parallel increase in those activities when the malonyl-CoA:CoA ratio was varied. Protein-bound acyl groups were labilized by performic acid treatment indicating their attachment to protein at thiol residues; however, the product released was volatile, which is not characteristic of malonic acid. While malonyl-CoA was initially free of acetyl-CoA, stimulation of kinase activity and acylation of sites in the complex by malonyl-CoA were shown to be contingent upon enzyme-catalyzed decarboxylation. Decarboxylation appeared to be catalyzed by a trace contaminant present in highly purified preparations of both the pyruvate and 2-oxoglutarate dehydrogenase complexes. Under conditions in which both free CoA was removed (by conversion to succinyl-CoA) and then, after various periods, free acetyl-CoA was removed (by enzymic conversion to acetyl phosphate), both acetylation of sites in the complex and activation of kinase activity increased in a time-dependent manner. Concomitantly there was a decrease in the concentration dependence for activation of the kinase by malonyl-CoA. Our results strongly support the conclusion that activation of kinase activity is associated with acylation of sites in the complex, and that, in the case of malonyl-CoA, those processes depend on enzyme-catalyzed decarboxylation.

Acetyl Coenzyme A↗

PubMed1990

Isolation, characterization and chromosomal localization of cDNA clones for the E1 beta subunit of the pyruvate dehydrogenase complex.

A full-length cDNA clone for the E1 beta subunit of the human pyruvate dehydrogenase (PyrDH) complex was isolated from a human skin fibroblast cDNA library. When sequenced, it showed differences from the nucleotide sequence already published [Koike, K., Ohta, S., Urata, Y., Kagawa, Y. & Koike, M. (1988) Proc. Natl Acad. Sci. USA 85, 41-45], such that 19 amino acids were different in the translated open reading frame. Northern blotting of human fibroblast cell lines revealed a major mRNA species of 1.6 kb and a weaker band of 5.5 kb. In a series of nine PyrDH-complex-deficient cell lines from patients with this deficiency, no patients had severely reduced amounts of mRNA, but there was one patient cell line with an increased amount of abnormal-size mRNA. Chromosome localization carried out with DNA blots from man-mouse hybrid cell lines indicated that the E1 beta subunit of pyruvate dehydrogenase is located on chromosome 3. A motif AXGXXXXGL(R/K)X15(D/E)Q was found in common with a variety of other oxo-acid oxidoreductases, but its function is not known.

Amino Acid Sequence↗

PubMed1988

Effect of a deficiency of thiamine on brain pyruvate dehydrogenase: enzyme assay by three different methods.

A simple and rapid method based on the NADH-linked reduction of a tetrazolium dye was described for the determination of pyruvate dehydrogenase activity in rat brain homogenates. The method (method 3) gave a value of 36.06 +/- 1.24 nmol of pyruvate utilised/min/mg of whole brain protein. This value was higher than that obtained by measurement of the rate of decarboxylation of [1-14C]pyruvate (15.10 +/- 0.88 nmol/min/mg of protein; method 1) and was comparable with the rate of transfer of acetyl groups to an arylamine (39.04 +/- 1.32 nmol/min/mg of protein; method 2). A critique of the values reported by others by different methods was given. The pyruvate dehydrogenase activity in the mitochondria isolated from rat brain was in the "active" (nonphosphorylated) form. A deficiency of thiamine in rats was produced by treatment with pyrithiamine, an antagonist of thiamine. This treatment resulted in abnormal neurological signs, such as ataxia and convulsions. The measurement of the total activity of pyruvate dehydrogenase in the brain by all three methods showed no significant change in the enzymic activity in thiamine-deficient rats after treatment with pyrithiamine. The activities of the enzyme in the brains of pair-fed animals were similar to those in the controls.

Animals↗