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At least 343 records · Page 19Linked to original sources
PubMed2000

Carbohydrate ingestion prior to exercise augments the exercise-induced activation of the pyruvate dehydrogenase complex in human skeletal muscle.

This study examined the effect of pre-exercise carbohydrate (CHO) ingestion on pyruvate dehydrogenase complex (PDC) activation, acetyl group availability and substrate level phosphorylation (glycogenolysis and phosphocreatine (PCr) hydrolysis) in human skeletal muscle during the transition from rest to steady-state exercise. Seven male subjects performed two 10 min treadmill runs at 70 % maximum oxygen uptake (VO2,max), 1 week apart. Each subject ingested 8 ml (kg body mass (BM))-1 of either a placebo solution (CON trial) or a 5.5 % CHO solution (CHO trial) 10 min before each run. Muscle biopsy samples were obtained from the vastus lateralis at rest and immediately after each trial. Muscle PDC activity was higher at the end of exercise in the CHO trial compared with the CON trial (1.78+/-0.18 and 1.27+/-0.16 mmol min(-1) (kg wet matter (WM))(-1), respectively; P 0.05) and this was accompanied by lower acetylcarnitine (7.1+/-1.2 and 9.1+/-1.1 mmol kg(-1) (dry matter (DM))(-1) in CHO and CON, respectively; P<0.05) and citrate concentrations (0.73+/-0.05 and 0.91+/-0.10 mmol (kg DM)(-1) in CHO and CON, respectively; P<0.05). No difference was observed between trials in the rates of muscle glycogen and PCr breakdown and lactate accumulation. This is the first study to demonstrate that CHO ingestion prior to exercise augments the exercise-induced activation of muscle PDC and reduces acetylcarnitine accumulation during the transition from rest to steady-state exercise. However, those changes did not affect the contribution of substrate level phosphorylation to ATP resynthesis.

Acetylcarnitine↗
PubMed2002

Molecular architecture and mechanism of an icosahedral pyruvate dehydrogenase complex: a multifunctional catalytic machine.

Electron cryo-microscopy of 'single particles' is a powerful method to determine the three-dimensional (3D) architectures of complex cellular assemblies. The pyruvate dehydrogenase multi-enzyme complex couples the activity of three component enzymes (E1, E2 and E3) in the oxidative decarboxylation of pyruvate to generate acetyl-CoA, linking glycolysis and the tricarboxylic acid cycle. We report here a 3D model for an 11 MDa, icosahedral pyruvate dehydrogenase sub-complex, obtained by combining a 28 A structure derived from electron cryo-microscopy with previously determined atomic coordinates of the individual E1 and E2 components. A key feature is that the E1 molecules are located on the periphery of the assembly in an orientation that allows each of the 60 mobile lipoyl domains tethered to the inner E2 core to access multiple E1 and E2 active sites from inside the icosahedral complex. This unexpected architecture provides a highly efficient mechanism for active site coupling and catalytic rate enhancement by the motion of the lipoyl domains in the restricted annular region between the inner core and outer shell of the complex.

Catalysis↗
PubMed1994

The pyruvate dehydrogenase complex: cloning of the rat somatic E1 alpha subunit and its coordinate expression with the mRNAs for the E1 beta, E2, and E3 catalytic subunits in developing rat brain.

We report the isolation of cDNA clones encoding the somatic form of the E1 alpha subunit of the pyruvate dehydrogenase complex of rat. The deduced amino acid sequence has 99.5, 98, and 97% identity, respectively, with the orthologous proteins of mouse, human, and pig and 98.5% identity with a rat E1 alpha sequence reported previously. The cDNAs isolated in this and earlier studies predict different E1 alpha subunit mRNA sizes and amino acid sequences. These differences have been investigated by PCR, northern blot hybridization, and RNase protection. We have used our E1 alpha cDNA, in conjunction with cDNA probes to the E1 beta, E2, and E3 catalytic subunits of rat pyruvate dehydrogenase complex and also to rat citrate synthase, to perform RNase protection assays of developing rat whole brain RNA. The results show a 2.5-fold increase in the concentration of each of the subunit mRNAs and a 1.2-fold increase in citrate synthase mRNA from late foetal stage to 5 days post partum. Thereafter, the mRNA levels remained constant. These data indicate that the respective six- and threefold increases in the amounts of pyruvate dehydrogenase complex and citrate synthase found to occur in rat brain between birth and adulthood are mediated principally by translational and/or posttranslational mechanisms.

Aging↗
PubMed1994

Mapping the lipoyl groups of the pyruvate dehydrogenase complex by use of gold cluster labels and scanning transmission electron microscopy.

This paper describes the organization of lipoyl moieties within the pyruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM). The PDH complex is a multienzyme complex consisting of E1, pyruvate dehydrogenase, E2, dihydrolipoyl transacetylase, and E3, dihydrolipoyl dehydrogenase. The core of the complex is the cubic 24-subunit E2 component, which contains the lipoyl moieties bonded to lipoyl-bearing domains. E1 and E3 are associated along the edges (E1) and on the faces (E3) of the core. The lipoyl moieties were reduced with NADH and alkylated with a p-maleimidobenzoyl undecagold cluster complex. The gold labels were found to be bound very nearly specifically by dihydrolipoyl transacetylase (E2). Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis. The mass of the E2E3 subcomplex is about half that of the PDH complex. The PDH complex and GC-PDH are both about 420 A in diameter, as determined by radial mass analysis, and the E2E3 subcomplex and GC-E2E3 are 320 and 350 A, respectively. The outer boundary of the E2E3 subcomplex was clearly shown in STEM micrographs by the undecagold labels in GC-E2E3. Data obtained from radial mass analysis of GC-E2E3 and the unlabeled E2E3 subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surrounding the central E2 core. The capabilities of lipoyl moieties to undergo translocation over long distances through structural mobility in the lipoyl-bearing domains was confirmed by the observation that many of the lipoyl groups in E2E3 subcomplexes relax outward into space vacated by the removal of E1 during the preparation of the subcomplex from PDH complex. Radial mass analysis of the PDH complex and GC-PDH indicates that lipoyl groups are distributed over a large region of the PDH complex, extending from the central core to 170-180 A from the center of the complex, with the highest density at about 75 A from the particle centers, near the interface between E2 and the associated components E1 and E3.

Escherichia coli↗
PubMed1977

Plant Pyruvate Dehydrogenase Complex: II. ATP-Dependent Inactivation and Phosphorylation.

ATP inactivated plant pyruvate dehydrogenase complex (PDC) from broccoli (Brassica oleracea) mitochondria. ATP inactivation of the complex was time-dependent and proportional to the ATP concentration. Time-dependent incorporation of (32)P from [gamma(32)P]ATP into trichloroacetic acid-precipitable protein corresponded to the inactivation of the PDC. It is concluded that plant PDC is phosphorylated and inactivated by a PDC kinase.

Journal Article↗
PubMed2003

Disruption of plE2, the gene for the E2 subunit of the plastid pyruvate dehydrogenase complex, in Arabidopsis causes an early embryo lethal phenotype.

The pyruvate dehydrogenase multi-enzyme complex is the main source of acetyl-CoA formation in the plastids of plants and is composed of multiple copies of four different subunits, E1alpha, E1beta, E2, and E3. A T-DNA insertion into the gene for the plastidic E2 (dihydrolipoyl acetyltransferase) subunit, plE2, of the complex in Arabidopsis destroys the expression of that gene. The resulting mutation has no apparent phenotype in the heterozygous state, but the homozygous mutation is lethal. Haploid sperm and eggs that contain only the disrupted plE2 gene function normally resulting in the formation of an embryo that is homozygous for the mutation. This embryo only develops to an early stage before the development arrests resulting in an early embryo-lethal phenotype. While the mutation could not be complemented with the cDNA for the plE2 gene under control of the 35S, the AtSERK1, or the napin promoter, it could be complemented using the endogenous plE2 promoter to drive expression of the plE2 cDNA. This verifies the essential nature of the plastidic pyruvate dehydrogenase complex and its role in embryo formation.

Arabidopsis↗
PubMed1987

Muscle involvement in pyruvate dehydrogenase complex (PDHC) deficiency.

Muscle biopsies from a 13-month-old female infant with a delay in developmental milestones, lactic acidosis and visual disturbance, and a 6 year-old female with frequent epileptic fits are described. Biochemical studies of biopsied muscles and skin fibroblasts demonstrated markedly decreased pyruvate dehydrogenase complex (PDHC) activity to about 16% of normal value. Muscle histochemistry in both patients showed disorganized intermyofibrillar networks containing large diformazan granules on NADH-TR, small angulated fibers with high nonspecific esterase (NSE) activity and basophilic fibers. Ragged-red fibers and increased lipid droplet accumulation were absent. Patient 1 had increased numbers of type 2C fibers (11.3%) and mild fiber type grouping. On electron microscopy, most mitochondria were nearly normal. There were focal aggregates of mildly enlarged mitochondria in the subsarcolemmal areas in both patients. Morphometric study showed that the mean mitochondrial size and the mitochondrial percentage of fiber volume were not significantly different amongst patients and normal controls.

Child↗
PubMed1988

Carnitine stimulation of pyruvate dehydrogenase complex (PDHC) in isolated human skeletal muscle mitochondria.

L-carnitine stimulated CO2 production from 1-14C pyruvate in mitochondria from human skeletal muscle nearly twofold. A comparable increase in the pyruvate dehydrogenase complex (PDHC) activity was seen. Moreover, in the presence of L-carnitine and at pyruvate concentration greater than 0.25 mM, this effect was associated with a marked increase of acetylcarnitine synthesis. Deoxycarnitine, an inhibitor of carnitine acetyltransferase (CAT), partially reversed the effect of carnitine on PDHC activity. The stimulatory effect of carnitine on PDHC activity in human mitochondria is mediated by the modulation of the intramitochondrial acetyl-CoA/CoASH ratio.

Acetyl Coenzyme A↗
PubMed1981

Rotational diffusion of eosin-labeled pyruvate dehydrogenase complex of Escherichia coli.

The enzymatically reduced lipoyl residues of the transacetylase component of the pyruvate dehydrogenase complex from Escherichia coli were labeled with eosin maleimide. Using eosin as triplet probe, triplet-triplet absorption dichroism measurements were performed to obtain rotational correlation times of the complex in the microsecond time domain. It was found that the hydrodynamic properties determined from the correlation times are in very good agreement with those obtained with other methods of different origin. The results can be fully explained by eosin molecules rotating with the whole complex, which consists of a mixture of heavy (60 S) and light (20 S) particles. Since no independent mobility could be detected it is suggested that the (charged) chromophoric group is folded against the protein surface. Labeling with excess eosin maleimide tends to destabilize the complex, since the longer correlation time (60 S) decreases and the contribution of the shorter correlation time (20 S) becomes more significant upon labeling.

Coloring Agents↗
PubMed1988

Biosynthesis, import and processing of precursor polypeptides of mammalian mitochondrial pyruvate dehydrogenase complex.

An immunological analysis has been conducted of early events in the biosynthesis, import and assembly of the mammalian pyruvate dehydrogenase complex (PDC). For this purpose, monospecific polyclonal antisera were produced against the intact assembly from ox heart, Mr 8.5 x 10(6), and each of its component polypeptides, E1 alpha, E1 beta, E2, E3 and protein X. Optimal detergent-based incubation mixtures were developed for obtaining clean immunoprecipitation of PDC polypeptides and their precursors from [35S]methionine-labelled extracts of PK-15 (pig kidney), NBL-1 (bovine kidney) and BRL (Buffalo Rat liver) cells. In PK-15 cells, independent higher Mr species, corresponding to precursors of the E2, E1 alpha and E1 beta subunits of PDC, could be detected by immune precipitation and fluorography after incubation of intact cells for 4 h with [35S]methionine and 1-2 mM-2,4-dinitrophenol or 10-15 microM-carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Similar precursor states could be observed in uncoupler-treated BRL or NBL-1 cells. Pre-E1 alpha, pre-E1 beta and also pre-E3, have signal sequences in the Mr range 1500-3000 while pre-E2 contains a long additional segment of Mr 7000-9000. All of these forms exhibit similar kinetics of processing to the mature subunits with a transit time of 10-12 min. In NBL-1 cells, E3 is present in the immune complexes formed with anti-PDC serum whereas this is not the case in PK-15 cells. Thus, there are significant variations in the affinity of lipoamide dehydrogenase (E3) for the E2 core structure in different species. Pre-E1 alpha accumulates only poorly in PK-15 cells and is aberrantly processed on removal of uncoupler. This precursor is markedly more stable in NBL-1 and BRL cells. The lack of detection of a precursor form of component X is also discussed.

2,4-Dinitrophenol↗
PubMed1994

Expression, purification, and characterization of the dihydrolipoamide dehydrogenase-binding protein of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae.

Genes encoding dihydrolipoamide dehydrogenase (E3) and the E3-binding protein (E3BP, protein X), components of the Saccharomyces cerevisiae pyruvate dehydrogenase (PDH) complex, were coexpressed in Escherichia coli to produce an E3BP-E3 complex, thereby minimizing proteolysis of E3BP and facilitating its purification. The 2 genes were linked into a single transcriptional unit separated by a 31-nucleotide segment containing a ribosome-binding sequence. The E3BP-E3 complex was highly purified and then separated into E3 and E3BP by chromatography on hydroxylapatite in the presence of 5 M urea. The E3BP-E3 complex combined rapidly with a pyruvate dehydrogenase (E1)-dihydrolipoamide acetyltransferase (E2) subcomplex (E1-E2 subcomplex) to reconstitute a functional PDH complex, with pyruvate oxidation activity similar to that of PDH complex from bakers' yeast. The stoichiometry of binding of E3BP and E3BP-E3 complex to the 60-subunit pentagonal dodecahedron-like E2 was determined with a truncated form of E2 (tE2, residues 206-454) lacking the lipoyl domain and the E1-binding domain, and with E1-E2 subcomplex, which contains intact E2. Mixtures containing tE2 or E1-E2 subcomplex and excess E3BP or E3BP-E3 complex were subjected to ultracentrifugation to separate the large complexes from unbound E3BP or E3BP-E3, and the complexes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After staining with Coomassie brilliant blue and destaining, the gels were analyzed with a video area densitometer. The results showed that the E1-E2 subcomplex binds about 12 E3BP monomers attached to 12 E3 homodimers. Similar results were obtained by analysis of highly purified PDH complex from bakers' yeast.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

The relationships between transketolase, yeast pyruvate decarboxylase and pyruvate dehydrogenase of the pyruvate dehydrogenase complex.

The amino acid sequences of four thiamine pyrophosphate-requiring enzymes were aligned with the published amino acid sequence of the transketolase of Hansenula polymorpha. Sequences of the combined alpha and beta subunits of the E1 enzyme of the pyruvate dehydrogenase complexes of Homo sapiens and Bacillus stearothermophilus aligned well with the transketolase while the E1 of the pyruvate dehydrogenase complex of Escherichia coli aligned easily provided a non-aligning segment of 77 amino acids was omitted. The non-acetylating pyruvate decarboxylase of Saccharomyces cerevisiae could only be aligned if the sequence was cut in two with the C-terminus corresponding to the N-terminus of the other TPP-dependent enzymes. Using the published 2.5 A resolution of the X-ray crystal structure of Saccharomyces cerevisiae transketolase as a template we show that a hydrophobic region of the beta-subunit of the PDH E1 alpha beta enzymes likely contains a binding site for the thiazolium ring of TPP and key motifs are retained in common by all the TPP-dependent enzymes considered, which are essential for catalysis.

Amino Acid Sequence↗
PubMed1995

Metabolic engineering in Escherichia coli: lowering the lipoyl domain content of the pyruvate dehydrogenase complex adversely affects the growth rate and yield.

Isogenic strains of Escherichia coli W3110 containing pyruvate dehydrogenase complexes with three (wild-type), two or one lipoyl domains per lipoate acetyltransferase (E2p) chain, were constructed. The maximum growth rates (mumax) for batch cultures growing in minimal medium containing different carbon sources showed that reducing the number of lipoyl domains adversely mumax value of the mutant containing one lipoyl domain per E2p chain was restored by the presence of compatible multicopy plasmids encoding PDH complexes with either one or three lipoyl domains per E2p chain. In glucose-limited chemostat cultures the protein contents of all strains were similar and substrate carbon was totally accounted for in the biomass and CO2 produced. However, the carbon efficiencies (percentage carbon conversion to biomass) were significantly lower when the lipoyl domain content of the E2p subunit was reduced from three to one. Similarly, the cellular maintenance energy (m(e)) and the maximum growth yield (Ymax) were lower in bacteria containing PDH complexes with fewer than three lipoyl domains per E2p chain. Wild-type values were restored by supplementing the medium with either casamino acids (0.01%) or acetate (up to 0.1 mM). The lower growth efficiencies of the mutants were further confirmed in competition experiments where equal numbers of genetically marked (NalR) mutant and wild-type bacteria were used to inoculate glucose-limited chemostat cultures (dilution rate 0.075 h-1). The mutants with one or two lipoyl domains per E2p chain were washed out, whereas in controls, the initial ratio of wild-type (Nals) to reconstructed wild-type (NalR) bacteria was maintained over 50 generations.

Acetyltransferases↗
PubMed1995

A library of monoclonal antibodies to Escherichia coli K-12 pyruvate dehydrogenase complex. Competitive epitope mapping studies.

Presented here are competitive epitope mapping studies on a monoclonal antibody library to K-12 Escherichia coli pyruvate dehydrogenase complex (PDHc) and its pyruvate decarboxylating (EC1.2.4.1) subunit (E1). Several of the monoclonal antibodies had been found to inhibit PDHc from 0 to 98%. Of the 10 monoclonal antibodies that showed the greatest inhibition of PDHc, 4 were elicited by PDHc and 6 by E1. Surface plasmon resonance was used for competitive epitope mapping and revealed that these 10 monoclonal antibodies had at least 6 separate binding regions on the PDHc. The three monoclonal antibodies that demonstrated the strongest inhibition appeared to bind the same region on the PDHc. Mapping studies with the E1 antigen using an additional five monoclonal antibodies demonstrated that the two strongest inhibitory monoclonal antibodies (18A9 and 21C3) shared the same binding region on E1, whereas the third strongest inhibitor (15A9) displayed an epitope region that overlapped the previous two on the E1 subunit. Antibody 15A9 had been shown to counteract GTP regulation of PDHc. Simultaneous multiple site binding experiments confirmed that the defined epitope regions were indeed independent. Limited competitive epitope binding experiments using radiolabeled E1 confirmed the surface plasmon resonance results.

Animals↗
PubMed1993

Autoreactive liver-infiltrating T cells in primary biliary cirrhosis recognize inner mitochondrial epitopes and the pyruvate dehydrogenase complex.

Primary biliary cirrhosis (PBC) is characterized by lymphoid infiltrates in the portal tracts of the liver and the occurrence of antimitochondrial autoantibodies in serum directed against components of the pyruvate dehydrogenase complex and the other alpha-keto acid dehydrogenase complexes. These enzymes are located on the inner mitochondrial membrane. The destruction of the biliary tract in PBC is thought to be mediated by autoreactive liver-infiltrating T cells exerting cytotoxic activity or releasing certain lymphokines. In this study the reactivity of liver infiltrating T cells was shown to a bovine pyruvate dehydrogenase complex (PDH), a purified E2 subunit (PDH-E2) and a crude preparation of human liver mitoplasts (HLM), i.e. mitochondria depleted of their outer membranes. Peripheral blood lymphocytes (PBL) from 11 of 15 patients (73.3%) with PBC showed a HLA class II-restricted proliferative response to the PDH complex whereas PBL from patients with chronic viral hepatitis, autoimmune hepatitis or extrahepatic cholestatic icterus (n = 20) and healthy controls (n = 5) did not. In addition 13 of 15 PBL from patients with PBC (86.6%) and three of nine PBL from patients with autoimmune hepatitis (33.3%) reacted with the crude HLM preparation whereas no reactivity was found with PBL from eight patients with chronic viral hepatitis, three patients with extrahepatic cholestasis or five healthy controls. Clonal analysis of 115 liver-infiltrating T cells derived from two diagnostic liver biopsies of patients with PBC revealed a predominance of activated CD4+CD8- T helper cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗