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PubMed1994

The dependence of electrophoretic and spectroscopic properties of the pyruvate dehydrogenase complex on mono- and divalent ions.

The effects of various mono- and divalent ions on the pyruvate dehydrogenase complex (PDC) were investigated. To determine the radius of PDC under various conditions a two-dimensional agarose gel electrophoresis technique was used. The radius of PDC cross-linked with glutaraldehyde at ionic strength 0.04 M was calculated to be 22.0 +/- 0.1 nm. The presence of K+, Na+ or HPO4(2-) prevented changes in electromobility and of the calculated radius of PDC induced by alteration in ionic strength. The fluorescence emission spectra of PDC depended on the ionic strength and monovalent cations. The fluorescence intensity of PDC increased in the presence of 80 mM K+, and decreased in the presence of 80 mM Na+ with no shift in the emission maximum wavelength. Changes in the ionic strength to which PDC was exposed resulted in alteration of the UV absorption spectra in the 230 nm region. These alterations were prevented by HPO4(2-), whereas Na+ or K+ ions had no effect on the UV absorption spectrum of PDC.

Electrophoresis, Agar Gel↗
PubMed1995

Peripheral B lymphocyte repertoire to mitochondrial antigen in primary biliary cirrhosis--positive correlation between the disease activity and the frequency of circulating B lymphocytes specific for pyruvate dehydrogenase complex.

B lymphocytes committed to the production of IgG antibodies (Abs) to mitochondrial antigen such as pyruvate dehydrogenase complex(PDC) were quantitated in the peripheral blood of patients with primary biliary cirrhosis(PBC) using Epstein-Barr virus as a polyclonal activator of human B lymphocytes. B lymphocytes committed to the production of IgG Abs to PDC were found in high frequency in patients with PBC(0.54 +/- 0.16%, mean value +/- SE, of total IgG-producing B lymphocytes) in contrast to type C chronic hepatitis and healthy subjects in which they were less than 0.01%. The frequency of these B lymphocytes specific for PDC increased in parallel to the progression of the Scheuer's stage from I to II (stage I: 0.35 +/- 0.23%, stage II: 1.04 +/- 0.32%), but decreased with further progression to stage IV (stage III: 0.39 +/- 0.21%, stage IV: 0.07 +/- 0.06%). In addition, B lymphocytes specific for PDC decreased in the peripheral blood during the administration of cyclosporin A; this was accompanied by an improvement of lymphocyte infiltration severity in the liver. These data indicate that B lymphocytes specific for PDC are present in the peripheral blood of patients with PBC and their frequency reflects the degree of the lymphocyte infiltration in the liver.

Adult↗
PubMed1993

Diurnal patterns of cardiac and hepatic pyruvate dehydrogenase complex activity in gold-thioglucose-obese mice.

The diurnal pattern of the activity of the pyruvate dehydrogenase complex (PDHC) was studied in the heart and liver of gold-thioglucose (GTG)-obese mice and age-matched controls. The diurnal pattern of lipogenesis was also measured in the liver. Both lean and obese mice had one main eating period, from 20:00 to 24:00 h. Eating produced no change in serum glucose of control mice but there was a significant rise in serum insulin and triacylglycerols. There was also a 3-fold increase in cardiac PDHC activity and a 3-fold increase in hepatic lipogenesis in the control mice, but little change in hepatic PDHC activity. GTG-obese mice were hyperglycaemic, hyperinsulinaemic and hypertriglyceridaemic at all times studied, with significant increases in these parameters being seen in response to eating. Eating produced little change in cardiac PDHC activity, but there was a 5-fold increase in hepatic PDHC activity, paralleled by a 10-fold increase in hepatic lipogenesis. Hepatic PDHC activity was significantly higher in GTG-obese mice at all times except 16:00 h. The simultaneous rise of hepatic PDHC activity, lipogenesis and serum triacylglycerols in GTG-obese mice suggests an increased utilization of glucose for lipogenesis. The lack of change in heart PDHC activity in GTG-obese mice over 24 h suggests that a general decrease in PDHC activity may contribute to the development of the glucose intolerance and insulin resistance of obesity and non-insulin-dependent diabetes. However, it appears that a different level of metabolic control allows hepatic PDHC activity of the same obese animals to increase in response to hyperinsulinaemia and contribute to the higher rates of lipogenesis seen in obese mice.

Animals↗
PubMed2002

Molecular cloning, and characterization and expression of dihydrolipoamide acetyltransferase component of murine pyruvate dehydrogenase complex in bile duct cancer cells.

BACKGROUND: The association between the dihydrolipoamide acetyltransferase component (E2) of pyruvate dehydrogenase complex (PDC) and primary biliary cirrhosis (PBC) is clinically established. However, the detailed pathological function of the PDC-E2 gene is as yet unclear. In order to study the gene function in knockout and transgenic mouse models, we cloned and characterized the mouse PDC-E2 (mPDC-E2) gene. Because the expression level of PDC-E2 was elevated in PBC bile duct cells, we tried to construct a bile duct carcinoma cell line that overexpressed PDC-E2 as a PBC cell model. METHODS: The mPDC-E2 cDNA was obtained by the 3'Race method. We overexpressed this gene in KMBC cells, using a retrovirus vector. The transcript and translated protein of mPDC-E2 were detected by Northern blot and Western blot, respectively. RESULTS: The deduced amino-acid sequence from the cloned cDNA indicated that the fully mature protein consisted of 557 amino-acid residues, with a calculated molecular mass of 59kD. This mature protein was highly consistent with those of previously reported rat and human PDC-E2, which possessed three structurally identifiable regions: the lipoyl-bearing domain, the E3-binding site, and the catalytic domain. Mouse fibroblast NIH3T3 cells expressed one species of mPDC-E2 mRNA, 3.5kb in length. We also successfully constructed a stable KMBC cell line overexpressing the PDC-E2. CONCLUSIONS: This is the first report of the mPDC-E2 sequence and is valuable for further investigation of PDC-E2 gene function in transgenic or knockout mouse models. The PDC-E2 overexpressing KMBC cell line can be used to study alterations in signal transduction or gene expression profiles in PBC bile duct.

Acetyltransferases↗
PubMed1992

Incorporation of the enantiomers of lipoic acid into the pyruvate dehydrogenase complex from Escherichia coli in vivo.

The uptake of 35S-labelled enantiomers of lipoic acid into cells from Escherichia coli was studied. The R-enantiomer was taken up by a factor of two more efficiently than the S-form. Autoradiography of polyacrylamide gels of partially purified pyruvate dehydrogenase complex from these cells showed that only the R-lipoic acid was covalently incorporated as a cofactor into the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex.

Autoradiography↗
PubMed2004

Downregulation of the skeletal muscle pyruvate dehydrogenase complex in the Otsuka Long-Evans Tokushima Fatty rat both before and after the onset of diabetes mellitus.

The pyruvate dehydrogenase complex (PDC) catalyzes the irreversible oxidative decarboxylation of pyruvate in mitochondria. The PDC activity is regulated by a phosphorylation/dephosphorylation cycle catalyzed by specific kinases (PDK) and phosphatases (PDP). In this study, the regulatory mechanisms of PDC were examined in skeletal muscle of the spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rat before and after the onset of diabetes. The Long-Evans Tokushima Otsuka (LETO) rat was used as control. Plasma glucose and insulin concentrations were at normal levels in both groups at 8 weeks of age but were significantly higher in OLETF than in LETO rats at 25 weeks of age (1.2-fold for glucose and 15-fold for insulin), indicating development of diabetes in the former. Plasma free fatty acids were 1.6-fold concentrated and the skeletal muscle PDC activity state was significantly lower in OLETF than in LETO rats at both ages, suggesting suppression of pyruvate oxidation in OLETF rats even before the onset of diabetes. The PDK activity and the abundance of the PDK isoform 4 protein as well as mRNA were greater in OLETF rats at both ages. Conversely, the abundance of the PDP isoform 1 protein and mRNA was less in OLETF than in LETO rats at both ages. These results suggest that concomitant greater PDK4 and less PDP1 expression in skeletal muscle of OLETF rats before the onset of diabetes are responsible for the lowering of the PDC activity and may be related with the development of diabetes mellitus.

Age Factors↗
PubMed1984

Proportion of active dephosphorylated pyruvate dehydrogenase complex in heart and isolated heart mitochondria is decreased in obese hyperinsulinaemic mice.

The proportion of active, dephosphorylated, pyruvate dehydrogenase complex was decreased in the mouse heart by obesity (by 56%), and this decrease in enzyme activity persisted during preparation and extraction of heart mitochondria. Phosphorylation and inactivation of pyruvate dehydrogenase may be a major factor in mediating the inhibitory effects of obesity on glucose oxidation in muscle, and this may represent an important mechanism in the development and/or expression of cellular insulin-resistance.

Animals↗
PubMed1988

Regulation of the phosphorylation of mitochondrial pyruvate dehydrogenase complex in situ: effects of respiratory substrates and calcium.

The activity of the pyruvate dehydrogenase complex (PDC), as controlled by reversible phosphorylation, was studied in situ with mitochondria oxidizing dfifferent substrates. PDCs from both plant and animal tissues were inactivated when pyruvate became limiting. The PDC did not inactivate in the presence of saturating levels of pyruvate. Calcium stimulated reactivation of PDC in chicken heart but not pea (Pisum sativum L.) leaf mitochondria. With pea leaf mitochondria oxidizing malate, inactivation of PDC was pH dependent corresponding to the production of pyruvate via malic enzyme. When pea leaf mitochondria oxidized succinate or glycine, PDC was inactivated. This inactivation was reversed by the addition of pyruvate. Reactivation by pyruvate was enhanced by the addition of thiamine pyrophosphate, as previously observed with nonrespiring mitochondria. These results indicate a major role for pyruvate in regulating the covalent modification of the PDC.

Journal Article↗
PubMed1989

Effect of starvation and insulin in vivo on the activity of the pyruvate dehydrogenase complex in rat skeletal muscles.

The in vivo responses of pyruvate dehydrogenase (PDH) complex to starvation and insulin was assessed in heart, diaphragm and red quadriceps muscle. PDH complex activity was decreased by starvation (3.4-10.2-fold), the magnitude of change depending on muscle type. Insulin increased PDH activity in all muscle types. In fed rats, this effect was relatively small (1.25-1.29-fold). In starved rats there were effects in heart (4.3-fold) and red quadriceps (1.7-fold) but no effect in diaphragm. These results demonstrate that PDH complex in different groups of muscle has different insulin sensitivity (particularly in tissues from starved animals).

Adipose Tissue, Brown↗
PubMed2001

Direct evidence for the size and conformational variability of the pyruvate dehydrogenase complex revealed by three-dimensional electron microscopy. The "breathing" core and its functional relationship to protein dynamics.

Structural studies by three-dimensional electron microscopy of the Saccharomyces cerevisiae truncated dihydrolipoamide acetyltransferase (tE(2)) component of the pyruvate dehydrogenase complex reveal an extraordinary example of protein dynamics. The tE(2) forms a 60-subunit core with the morphology of a pentagonal dodecahedron and consists of 20 cone-shaped trimers interconnected by 30 bridges. Frozen-hydrated and stained molecules of tE(2) in the same field vary in size approximately 20%. Analyses of the data show that the size distribution is bell-shaped, and there is an approximately 40-A difference in the diameter of the smallest and largest structures that corresponds to approximately 14 A of variation in the length of the bridge between interconnected trimers. Companion studies of mature E(2) show that the complex of the intact subunit exhibits a similar size variation. The x-ray structure of Bacillus stearothermophilus tE(2) shows that there is an approximately 10-A gap between adjacent trimers and that the trimers are interconnected by the potentially flexible C-terminal ends of two adjacent subunits. We propose that this springlike feature is involved in a thermally driven expansion and contraction of the core and, since it appears to be a common feature in the phylogeny of pyruvate dehydrogenase complexes, protein dynamics is an integral component of the function of these multienzyme complexes.

Acetyltransferases↗
PubMed1989

Role of protein X in the function of the mammalian pyruvate dehydrogenase complex.

Two lipoyl-bearing subunits--the dihydrolipoyl transacetylase and protein X--form the core of the mammalian pyruvate dehydrogenase complex. Selective removal of the lipoyl domain of protein X results in loss in the activity of the complex with a relationship suggesting the involvement of the lipoyl domain of protein X in a key but not rate limiting step. The dihydrolipoyl dehydrogenase component markedly reduces both the cleavage of protein X and the loss in activity. Using a microplate binding assay, we demonstrate that the lipoyl domain of protein X and the transacetylase component contribute to the binding of the dihydrolipoyl dehydrogenase component. These roles of protein X in the catalytic function and organization of the complex require new reactions and afford an explanation for the unusual stoichiometry of dihydrolipoyl dehydrogenase dimers in the complex.

Animals↗
PubMed1999

Cloning and characterization of the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) from Arabidopsis.

An Arabidopsis cDNA encoding the dihydrolipoamide S-acetyltransferase subunit of the plastid pyruvate dehydrogenase complex (E2) was isolated from a lambdaPRL2 library. The cDNA is 1709 bp in length, with a continuous open reading frame of 1440 bp encoding a protein of 480 amino acids with a calculated molecular mass of 50,079 D. Southern analysis suggests that a single gene encodes plastid E2. The amino acid sequence has characteristic features of an acetyltransferase, namely, distinct lipoyl, subunit-binding, and catalytic domains, although it is unusual in having only a single lipoyl domain. The in vitro synthesized plastid E2 precursor protein has a relative molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon incubation of the precursor with pea (Pisum sativum) chloroplasts, it was imported and processed to a mature-sized relative molecular weight of 60,000. The imported protein was located in the chloroplast stroma, associated with the endogenous pyruvate dehydrogenase. Catalytically active recombinant plastid E2 was purified as a glutathione S-transferase fusion protein. Analysis of plastid E2 mRNA by reverse transcriptase-polymerase chain reaction showed highest expression in flowers, followed by leaves, siliques, and roots. The results of immunoblot analysis indicate that protein expression was similar in roots and flowers, less similar in leaves, and even less similar in siliques. This is the first report, to our knowledge, describing a plastid E2.

Acetyltransferases↗
PubMed1997

Defects in the mitochondrial energy metabolism outside the respiratory chain and the pyruvate dehydrogenase complex.

Disturbances in substrate oxidations in muscle mitochondria from patients with a suspicion of a mitochondrial myopathy may arise from a deficiency of one or more of the complexes of the respiratory chain or of the pyruvate dehydrogenase complex. However, we found no clear-cut defect in a substantial part of such patients. In this report we discuss some of the other possibilities which could account for the disturbed substrate oxidation rates. Special attention will be paid to defects which are localized outside the respiratory chain, such as defects in post-respiratory chain enzymes, defects in transport mechanisms of the mitochondrial inner or outer membrane, deficiency of cofactors and deficiency of heat-shock protein.

Electron Transport↗
PubMed1987

Subgenes expressing single lipoyl domains of the pyruvate dehydrogenase complex of Escherichia coli.

Subgenes encoding the lipoyl domains from the acetyltransferase components of two types of pyruvate dehydrogenase complex of Escherichia coli were made by site-specific oligonucleotide-directed nonsense mutagenesis of the corresponding aceF genes. One of the domains is capable of binding lipoic acid whereas the other is not. The subgenes were cloned into an expression vector under the transcriptional control of the lambda PL and lambda PR promoters and a temperature-sensitive lambda repressor. Under non-permissive conditions expression of the lipoyl domains was not detected, but 6 h after thermo-induction the domains were amplified by at least 35-50-fold relative to the normal amounts of each type of covalently bound domain.

Base Sequence↗
PubMed1986

Activities of cardiac and hepatic pyruvate dehydrogenase complex are decreased after surgical stress.

The work investigated the effects of surgical stress on the activities of cardiac and hepatic pyruvate dehydrogenase complex (active form, PDHa) in fed rats. PDHa activities in heart and liver were decreased within 4h of surgery with maximum inhibition at 24h after surgery. PDHa activities remained low until the fourth (liver) and eighth (heart) post-operative days. The decreased activities found at 4h and 24h after surgery were associated with increased plasma fatty acid concentrations, and inhibition of lipolysis resulted in reactivation of the enzyme complex. The results are discussed with reference to the control of pyruvate dehydrogenase activities by the oxidation of fat fuels and multisite phosphorylation in stress states, and its possible importance in glucose conservation after surgery and trauma.

Animals↗
PubMed1989

The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. A reconsideration.

After limited proteolysis of the dihydrolipoyl transacetylase component (E2) of Azotobacter vinelandii pyruvate dehydrogenase complex (PDC), a C-terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl-containing N-terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C-terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C-terminus of E2. Together with the previously determined N-terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24-meric structure is concluded instead of the 32-meric structure proposed previously. From the effect of guanidine hydrochloride on the light-scattering of intact E2 it was concluded that dissociation occurs in a two-step reaction resulting in particles with an average mass 1/6 that of the original mass before the N----D transition takes place. Cross-linking experiments with the catalytic domain indicated that the multimeric E2 is built from tetramers and that the tetramers are arranged as a dimer of dimers. A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A. vinelandii.

Acetyltransferases↗
PubMed1998

Heterologously expressed inner lipoyl domain of dihydrolipoyl acetyltransferase inhibits ATP-dependent inactivation of pyruvate dehydrogenase complex. Identification of important amino acid residues.

The activity of the pyruvate dehydrogenase multienzyme complex (PDC), which catalyses the oxidation of pyruvate to acetyl-CoA within the mitochondrion, is diminished in animal models of diabetes. Studies with purified PDC components have suggested that the kinases responsible for inactivating the decarboxylase catalytic subunits of the complex are most efficient in their regulatory role when they are bound to dihydrolipoyl acetyltransferase (E2) subunits, which form the structural core of the complex. We report that the addition of an exogenous E2 subdomain (inner lipoyl domain) to an intact PDC inhibits ATP-dependent inactivation of the complex. By combining molecular modelling, site-directed mutagenesis and biophysical characterizations, we have also identified two amino acid residues in this subdomain (Ile229 and Phe231) that largely determine the magnitude of this effect.

Acetyltransferases↗