SEARCH · PubMed Health

Results for “Pyruvate Dehydrogenase Complex”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 433 records · Page 24Linked to original sources

PubMed1993

Restructuring an interdomain linker in the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex of Escherichia coli.

The lipoyl, subunit-binding and catalytic domains of the dihydrolipoamide acetyltransferase subunits (E2p) of the Escherichia coli pyruvate dehydrogenase complex are connected by linker sequences which are characteristically rich in alanine and proline residues. By facilitating domain movement these linkers are thought to promote interactions between the three types of active site that participate in the catalytic cycle of the complex. To investigate functional constraints associated with linker composition and sequence, the natural linker of an E2p subunit containing one lipoyl domain was replaced by shorter sequences containing: mixtures of alanine plus proline residues; mainly alanine; mainly proline; and mainly charged residues. Each artificial linker possessed a central histidine residue for assessing linker flexibility by 1H-NMR spectroscopy. The resultant complexes exhibited 181% (proline), 74-79% (alanine plus proline), 63% (alanine) and 7% (charged residues) of parental activity compared with a value of 75% expected for a complex with a comparably shortened linker. The 1H-NMR spectra showed that the alanine plus proline linkers are flexible but the alanine linker and the proline linker are relatively inflexible. Substantial variations in linker sequence and composition were tolerated without loss of function, and the enhanced activity conferred by the proline linker was attributed to the combined effects of length and relative inflexibility.

Acetyltransferases↗

PubMed1998

Pyruvate dehydrogenase complex deficiency and altered respiratory chain function in a patient with Kearns-Sayre/MELAS overlap syndrome and A3243G mtDNA mutation.

Combined alteration of the pyruvate dehydrogenase complex and respiratory chain function is described in a 21 year-old male patient with overlapping MELAS (mitochondrial encephalomyopathy, lactic acidosis, and 'stroke-like' episodes) and Kearns-Sayre syndrome. Progressive external ophthalmoplegia, pigmentary retinopathy and right bundle branch block were present when he experienced the first 'stroke-like' episode at 18 years old. The A>G tRNALeu(UUR) point mutation at nucleotide 3243 of the mitochondrial DNA was predominant in muscle tissue (79%) and present, but at lower levels in fibroblasts (49%) and blood cells (37%). Biochemical analysis revealed diminished activities of pyruvate dehydrogenase (23%) and respiratory chain complexes I and IV (57%, respectively) in muscle, but normal activities in the fibroblasts. Immunochemical studies of the muscular pyruvate dehydrogenase components showed normal content of E1alpha, E1beta and E2 protein. Molecular screening of the E1alpha gene did not indicate a nuclear mutation. These observations suggest that mitochondrial DNA defects may be associated with altered nuclear encoded enzymes which are actively imported into mitochondria and constitute components of the mitochondrial matrix. Biochemical workup of mitochondrial disorders should not be restricted to the respiratory chain even if mitochondrial DNA mutations are present.

Adult↗

PubMed1980

Protein mobility inside pyruvate dehydrogenase complexes as reflected by laser-pulse fluorometry. A new approach to multi-enzyme catalysis.

The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three-component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme-free complex is compared with complex to which Mg2+ and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11 degrees C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipo-amide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.

Azotobacter↗

PubMed2000

Separation of intact pyruvate dehydrogenase complex using blue native agarose gel electrophoresis.

We show that the blue native gel polyacrylamide electrophoresis system (BN-PAGE) can be applied to pyruvate dehydrogenase complex (PDC). BN-PAGE has been used extensively to study the multisubunit enzymes of oxidative phosphorylation, as nondenaturing separation in the first dimension maintains holoenzyme integrity. However, the standard protocol was inappropriate for PDC as, at 10 MDa, it is approximately ten times larger than the largest respiratory chain enzyme complex. Therefore, agarose was substituted for polyacrylamide. Moreover, a substantial decrease in salt concentration was necessary to prevent dissociation of PDC. As with standard BN-PAGE, immunoblots of second-dimensional sodium dodecyl sulfate-PAGE (SDS-PAGE) provided more detailed information on specific subunits and subcomplexes. The method was applied to human heart mitochondrial fragments, control cultured human cells, rho0 cells that lack mitochondrial DNA, and two cell lines derived from patients with PDC deficiency. The PDC deficient cell lines showed a clear correlation between amount of PDC holoenzyme and disease severity. In cells lacking mitochondrial DNA, synthesis and assembly of all PDC subunits (all nuclearly encoded) appeared normal, suggesting that respiratory function has no regulatory role in PDC biogenesis. Blue native agarose gel electrophoresis coupled with standard second-dimensional SDS-PAGE provides a new tool to be used in conjunction with biochemical assays and immunoblots of one-dimensional SDS-PAGE to further elucidate the nature of PDC in normal and disease states. Furthermore, other cellular protein complexes of 1 MDa or more can be analysed by this method.

Electrophoresis, Agar Gel↗

PubMed1990

Cloning and cDNA sequence of the beta-subunit component of human pyruvate dehydrogenase complex.

Two cDNA clones (lambda E1 beta 1, 1469 bp; lambda E1 beta 12, 1437 bp) encoding the beta-subunit of the pyruvate dehydrogenase (E1) component of the human pyruvate dehydrogenase complex were isolated from a human liver lambda gt11 cDNA library. The composite human liver E1 beta cDNA encoded the entire mature E1 beta [329 amino acids (aa)] as well as a portion (26 aa) of the E1 beta leader peptide. Significant discrepancies were identified between the nucleotide and deduced aa sequences of human liver E1 beta cDNAs and the corresponding sequences of a previously reported cultured human foreskin fibroblast E1 beta cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45]. The composite human liver E1 beta cDNA generated in this study provides a reference sequence for investigating the structure-function relationship of human E1 beta and for characterizing genetic mutations in patients with E1 deficiency.

Amino Acid Sequence↗

PubMed2004

Covalent modification as a mechanism for the breakdown of immune tolerance to pyruvate dehydrogenase complex in the mouse.

The autoimmune liver disease primary biliary cirrhosis (PBC) is characterized by the breakdown of normal immune self tolerance to pyruvate dehydrogenase complex (PDC). How tolerance is broken to such a central and highly conserved self antigen in the initiation of autoimmunity remains unclear. One postulated mechanism is that reactivity arises to an altered form of self antigen with subsequent cross-reactivity to native self. In this murine study, we set out to examine whether sensitization with a covalently modified form of self PDC can give rise to the pattern of breakdown of B-cell and T-cell tolerance to self PDC seen in PBC patients. The notion that altered self can lead to tolerance breakdown was studied by sensitizing SJL/J mice with a covalently modified (biotinylated) preparation of self murine PDC (mP/O-B). Subsequently, antibody and T-cell reactivities to unmodified self mP/O were studied. Sensitization with mP/O-B elicited high-titre, high-affinity antibody responses reactive with both the mP/O-B immunogen and, importantly, native mP/O. In addition, significant MHC class II restricted splenic T-cell responses to native mP/O (i.e., true autoimmune responses) were seen in mP/O-B sensitized animals. The breakdown of T-cell self tolerance to mP/O was not seen in animals sensitized with irrelevant biotinylated antigens. In conclusion, this study provides evidence to support the concept that exposure to covalently modified self PDC can, in the correct proimmune environment, replicate the full breakdown of B-cell and T-cell immune tolerance to PDC seen in PBC. One potential etiological pathway in PBC therefore could be the breakdown of tolerance to self PDC occurring after exposure to self antigen covalently modified in the metabolically active environment of the liver.

Animals↗

PubMed1998

Is absence of pyruvate dehydrogenase complex in mitochondria a possible explanation of significant aerobic glycolysis by normal human leukocytes?

The oxygen consumption of leukocyte mitochondria of both healthy donors and leukemic patients was tested by using different respiratory substrates. The results indicate that pyruvate could not be utilized by mitochondria of normal leukocytes, whereas mitochondria of leukemic leukocytes could use pyruvate as a good respiratory substrate. A search for the possible presence of pyruvate dehydrogenase complex (PDC) in leukocytes indicates that this enzyme is apparently absent in mitochondria of normal leukocytes but is quite active in mitochondria of leukemic leukocytes. The absence of PDC in normal leukocyte mitochondria can explain the phenomenon of significant aerobic glycolysis that has been observed in normal leukocytes.

Acetyl Coenzyme A↗

PubMed2002

Investigation of a mechanism for accelerated breakdown of immune tolerance to the primary biliary cirrhosis-associated autoantigen, pyruvate dehydrogenase complex.

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by autoreactive T- and B-cell responses to the highly conserved enzyme pyruvate dehydrogenase complex (PDC). In this study we have examined the breakdown of T-cell tolerance to self-PDC using a mouse model. Female SJL/J mice were sensitized intraperitoneally with foreign-PDC (bovine) and/or self-PDC (murine) in complete Freund's adjuvant, and serum, spleen, and liver tissue was taken 8 weeks later. Animals sensitized with foreign-PDC produced IgG antibodies that were reactive with both foreign and self-PDC, but splenic T cells from these animals only responded to stimulation with foreign PDC. Sensitization with self-PDC elicited neither antibodies nor reactive T cells. Significantly, cosensitization with mixed self-PDC and foreign-PDC resulted in a full breakdown of self-tolerance, with generation of both antibody and T-cell responses to self-PDC of the type seen exclusively in human PBC patients. Mild bile duct lesions deficient in CD8(+) T cells were seen 8 weeks after sensitization with either foreign or self-PDC. However, after sensitization with mixed self-PDC and foreign-PDC, these lesions were significantly larger and heavily infiltrated by CD8(+) T cells. Liver-infiltrating T cells derived from the self-PDC and foreign-PDC cosensitized but not from control animals showed reactivity with self-PDC, suggesting a possible role for autoreactive PDC-specific T-cell responses in the pathogenesis of the observed histologic changes. It is likely that B-cell cross-reactivity between foreign and self-PDC enhances the potential for breakdown of T-cell self-tolerance by allowing efficient presentation of self-antigens in the inoculum. This model may provide a useful system for investigating the etiology and treatment of PBC.

Animals↗

PubMed1992

Combined deficiencies of the pyruvate dehydrogenase complex and enzymes of the respiratory chain in mitochondrial myopathies.

In six patients with mitochondrial (encephalo-) myopathy investigations of skeletal muscle revealed a defect of pyruvate dehydrogenase complex (PDHC) in combination with one or more respiratory chain complex deficiencies. A combination of defects of this kind has not been reported previously. Five of the six patients presented within the 1st year of life and had a severe clinical course. Intrafamilial variability of the clinical course in dizygotic twins both suffering from a cytochrome c oxidase deficiency and one of them also from a PDHC deficiency suggests an additional effect of PDHC deficiency on the clinical symptoms. Immunoblot studies of PDHC in five of the patients revealed no abnormalities in their subunit pattern, rendering a defect of mitochondrial protein import or assembly unlikely. The finding of a combined PDHC and respiratory chain deficiency has implications for the diagnostic approach, for therapy and genetic counselling. The exact pathogenetic mechanism of this combination of defects remains to be elucidated.

Female↗

PubMed1980

Pyruvate dehydrogenase complex from germinating castor bean endosperm.

Subcellular organelles from castor bean (Ricinus communis) endosperm were isolated on discontinuous sucrose gradients from germinating seeds which were 1 to 7 days postimbibition. Marker enzyme activities of the organelles were measured (fumarase, catalase, and triose phosphate isomerase) and the homogeneity of the organelle fractions was examined by electron microscopy. Pyruvate dehydrogenase complex activity was measured only in the mitochondrial fraction and attempts to activate or release the enzyme from the proplastid were not successful. A pathway is proposed for the most efficient use of endosperm carbon for de novo fatty acid biosynthesis that does not require the presence of the pyruvate dehydrogenase complex in the proplastid to provide acetyl-coenzymeA.

Journal Article↗

PubMed1993

Characterization of cDNAs encoding the rat testis-specific E1 alpha subunit of the pyruvate dehydrogenase complex: comparison of expression of the corresponding mRNA with that of the somatic E1 alpha subunit.

cDNA clones encoding the testis-specific form of the rat pyruvate dehydrogenase complex E1 alpha subunit have been isolated. Comparison of the predicted amino acid sequence with those of the somatic and testis-specific E1 alpha forms of man and mouse and the somatic E1 alpha form of rat indicates the change of a serine residue, believed to be phosphorylated in vivo by pyruvate dehydrogenase E1 alpha-specific kinase, to an alanine at position 233. The implications of this change are discussed. Northern blot analysis and RNase protection assays indicate that the expression of mRNA encoding testis-specific E1 alpha subunit is restricted to testis whereas mRNA for the somatic form is found in all tissues analyzed, albeit in very small amounts in testis.

Amino Acid Sequence↗

PubMed1983

Inactivation of the 2-ketoglutarate and pyruvate dehydrogenase complexes of beef heart by branched chain keto acids.

Incubation of 2-ketoglutarate dehydrogenase complex with 2-ketoisovalerate, 2-keto-4-methylvalerate, or 2-keto-3-methylvalerate leads to the appearance of a lag phase and of a progressive loss of activity in subsequent measurements of the initial rate of oxidation of 2-ketoglutarate. In the case of 2-ketoisovalerate these effects are shown to be due to the formation of an isobutyryllipoate derivative of the enzyme, as a result of the very slow oxidation of 2-ketoisovalerate by the enzyme complex (Vmax congruent to 0.15% of that for 2-ketoglutarate). Incubation of the enzyme complex with 2-keto[14C]isovalerate or 2-keto[14C]glutarate results in comparable incorporation of radioactivity, amounting to 3.5 to 5.3 nmol of isobutyryl or succinyl residues per mg of protein in the complex. Isobutyryl residues are also incorporated in the enzyme during the simultaneous oxidation of both of these substrates. During the early phase of incubation of the complex with 2-ketoisovalerate the incorporation of isobutyryl residues is much faster than the loss of enzyme activity. This observation seems to support the suggestion that each 2-ketoglutarate decarboxylase subunit of the complex may catalyze the succinylation of more than one lipoate succinyltransferase subunit. Results are also presented showing the inactivation of pyruvate dehydrogenase complex on preincubation with 2-ketoisovalerate and of 2-ketoglutarate dehydrogenase complex with methylenecyclopropylpyruvate, the keto acid corresponding to the toxic amino acid hypoglycin. The relevance of covalent modifications of the two keto acid dehydrogenase complexes to the pathological manifestations of maple syrup urine disease are discussed.

Animals↗

PubMed1995

Pyruvate dehydrogenase complex from the primitive insect trypanosomatid, Crithidia fasciculata: dihydrolipoyl dehydrogenase-binding protein has multiple lipoyl domains.

The pyruvate dehydrogenase complex (PDC) has been purified to apparent homogeneity from the insect trypanosomatid, Crithidia fasciculata, a member of the most primitive eukaryotic group to contain mitochondria. Separation of the purified PDC by SDS-PAGE yielded five bands of 70 (p70), 60 (p60), 55, 46 and 36.5 kDa, which appeared to correspond to dihydrolipoyl dehydrogenase binding protein (E3BP), dihydrolipoyl transacetylase (E2), E3, E1 alpha and E1 beta, respectively. The purified complex did not exhibit endogenous PDHa kinase activity. p70 was much less abundant than p60. Polyclonal antisera raised against p70 did not cross-react with p60, and antisera raised against p60 did not cross-react with p70, suggesting that p60 did not arise from p70 by proteolysis. Both p70 and p60 contained similar amino terminal sequences. Both sequences contained the MPALSP motif similar to sequences present in both E3BP and E2 from other sources. Incubation of the purified PDC with [2-14C]pyruvate in the absence of CoA resulted in the acetylation of both p70 and p60, suggesting that both proteins contained lipoyl domains, but the specific incorporation of label into p70 was significantly greater than for p60. Limited proteolysis of the acetylated complex with trypsin yielded two major fragments derived from p60 of 35 and 30 kDa, corresponding to E2L and E2I, and one major acetylated fragment of 58 kDa derived from p70. Therefore, these results suggest that p70 is an E3BP and given its apparent M(r) and degree of acetylation, it contains multiple lipoyl domains.

Amino Acid Sequence↗

PubMed1993

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: implications for their activity in vivo.

The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecalis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.

Aerobiosis↗

PubMed1988

The relationship between changes in lipid fuel availability and tissue fructose 2,6-bisphosphate concentrations and pyruvate dehydrogenase complex activities in the fed state.

An elevated concentration of non-esterified fatty acids in the fed state elicited inhibition of cardiac, but not hepatic, pyruvate dehydrogenase complex (PDH). There was a modest decline in fructose 2,6-bisphosphate (Fru-2,6-P2) concentration in heart, and, to a lesser extent, in liver. Surgical stress decreased PDH activities and Fru-2,6-P2 concentrations in both heart and liver. Only the former response was abolished if postoperative lipolysis was inhibited. Surgery also decreased the [Fru-2,6-P2] in gastrocnemius: this response was abolished if lipolysis was inhibited.

Animals↗

PubMed2005

E1 enzyme of the pyruvate dehydrogenase complex in Corynebacterium glutamicum: molecular analysis of the gene and phylogenetic aspects.

The E1p enzyme is an essential part of the pyruvate dehydrogenase complex (PDHC) and catalyzes the oxidative decarboxylation of pyruvate with concomitant acetylation of the E2p enzyme within the complex. We analyzed the Corynebacterium glutamicum aceE gene, encoding the E1p enzyme, and constructed and characterized an E1p-deficient mutant. Sequence analysis of the C. glutamicum aceE gene and adjacent regions revealed that aceE is not flanked by genes encoding other enzymes of the PDHC. Transcriptional analysis revealed that aceE from C. glutamicum is monocistronic and that its transcription is initiated 121 nucleotides upstream of the translational start site. Inactivation of the chromosomal aceE gene led to the inability to grow on glucose and to the absence of PDHC and E1p activities, indicating that only a single E1p enzyme is present in C. glutamicum and that the PDHC is essential for the growth of this organism on carbohydrate substrates. Surprisingly, the E1p enzyme of C. glutamicum showed up to 51% identity to homodimeric E1p proteins from gram-negative bacteria but no similarity to E1 alpha- or beta-subunits of heterotetrameric E1p enzymes which are generally assumed to be typical for gram-positives. To investigate the distribution of E1p enzymes in bacteria, we compiled and analyzed the phylogeny of 46 homodimeric E1p proteins and of 58 alpha-subunits of heterotetrameric E1p proteins deposited in public databases. The results revealed that the distribution of homodimeric and heterotetrameric E1p subunits in bacteria is not in accordance with the rRNA-based phylogeny of bacteria and is more heterogeneous than previously assumed.

Amino Acid Sequence↗

PubMed1991

The catalytic domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli. Expression, purification, properties and preliminary X-ray analysis.

Partial sequences of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over-expressed in E. coli TG2. A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine-proline-rich sequence between a putative binding region for the peripheral components and the catalytic domain (apa-4). Most of the catalytic domain from A. vinelandii without the apa-4 sequence was degraded intracellularly, probably due to incorrect folding. Fusion proteins of six amino acids from beta-galactosidase, the apa-4 region and the catalytic domains of A. vinelandii or E. coli E2p could be highly purified. Both catalytic domains were assembled in 24-subunit structures with a molecular mass of approximately 670 kDa. The expression of catalytic domain from A. vinelandii E2p is more than twice as high as found for wild-type E2p. This can be explained by intracellular degradation of over-expressed wild-type E2p, whereas the catalytic domains are stable against proteolysis in vivo and in vitro. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose-6 and sedimentation velocity experiments. No binding of either E1p or E3 to the catalytic domain of either organism was detectable. Crystals of the catalytic domain of A. vinelandii E2p could be grown to a maximum size of 0.6 x 0.6 x 0.4 mm. They diffract up to a resolution of 0.28 nm.

Acetyltransferases↗

PubMed1998

Pyruvate dehydrogenase complex activation status and acetyl group availability as a site of interchange between anaerobic and oxidative metabolism during intense exercise.

During high intensity muscular contraction ATP is supplied at near maximal rates by PCr degradation and glycolysis. However, as exercise duration increases, the contribution of anaerobic ATP turnover to energy delivery declines due to the depletion of PCr stores and a reduction in the rate of glycogenolysis, which together may be responsible for the parallel reduction in muscle force production and power output. The importance of oxidative phosphorylation to total ATP production during intense muscle contraction has been underestimated to date. Recent studies have, however, demonstrated that the reduction in work production during repeated bouts of maximal exercise is less than the reduction observed in anaerobic energy provision. This observation has been suggested to reflect an increased contribution from oxidative phosphorylation to total energy production; but the mechanism responsible for this increased contribution is poorly understood. Recent evidence has pointed to the activation status of the pyruvate dehydrogenase complex and/or acetyl group availability as being focal in dictating temporal changes in ADP flux at the onset of intense exercise and, hence, the relative contribution made by anaerobic and oxidative ATP regenerating pathways under these conditions. As might be expected, therefore, maximising the contribution from oxidative ATP regeneration at the onset of exercise (by pharmacologically activating the pyruvate dehydrogenase complex prior to exercise) has been shown to have substantial functional benefits during high intensity contraction. This body of work has also illustrated that, contrary to popular theory, a large proportion of muscle lactate accumulation at the onset of exercise is associated with a lag in the activation of oxidative ATP production rather than with a lag in oxygen delivery.

Anaerobiosis↗