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At least 667 records · Page 37Linked to original sources
PubMed2006

Acute flaccid paralysis as initial symptom in 4 patients with novel E1alpha mutations of the pyruvate dehydrogenase complex.

We report on 4 boys from 3 families presenting initially in infancy with an acute onset of flaccid tetraparesis and areflexia, resembling Guillain-Barré syndrome (GBS). However, the cerebrospinal fluid (CSF) protein was normal, while serum and CSF lactate were elevated. All patients had recurrent similar episodes, usually associated with infections. Brain MRI showed T (2) hyperintensities in the basal ganglia in two boys, in one of them at the first clinical presentation; the other one had a normal brain MRI during the first episode. A third boy had a normal MRI twice but an increased lactate peak in the basal ganglia in (1)H-MR spectroscopy. Motor nerve conduction velocities (NCV) were normal in all patients. Biochemical analyses of muscle tissue, performed in two patients, revealed a deficiency of the pyruvate dehydrogenase (PDH). Molecular genetic analysis of the X-chromosomal E1alpha subunit of PDH showed three new mutations in phylogenetically conserved areas of the protein: Glu358Lys in patient 1; Arg88Lys in patient 2 and 3 (brothers); and Leu216Ser in patient 4. In conclusion, children with "atypical GBS" should be evaluated for a mitochondrial disorder, including pyruvate dehydrogenase deficiency, even after a first episode.

Adolescent↗
PubMed1995

The pyruvate dehydrogenase complex: nutrient control and the pathogenesis of insulin resistance.

This review examines the molecular mechanisms underlying substrate competition between glucose and lipid in starvation and in insulin-resistant states. We demonstrate that lipid-derived substrates are oxidized in preference to glucose by skeletal muscle in vivo during prolonged starvation. An accelerated and exaggerated lipolytic and ketogenic response to starvation in late pregnancy is associated with more rapid suppression of glucose oxidation by the maternal skeletal-muscle mass. These benign adaptations to changes in lipid availability (which occur secondarily to changes in carbohydrate supply and demand) contrast with the well-documented detrimental effects to health of an inappropriately high supply of dietary lipid. We present results that indicate that the prolonged consumption of a diet high in saturated fat is associated with a stable enhancement of pyruvate dehydrogenase (PDH) kinase activity at least in two oxidative tissues--liver and heart. This long-term enhancement of PDH kinase activity is concomitant with the development of whole-body insulin resistance and adds a new dimension to the potential role of dietary composition in the pathogenesis of insulin resistance.

Animals↗
PubMed2001

Thermally induced changes of lipoate acetyltransferase inner core isolated from the Bacillus stearothermophilus pyruvate dehydrogenase complex.

Incubation at 70 degrees C converted the Bacillus stearothermophilus lipoate acetyltransferase inner core into an unidentified active molecular form, X, yielding an inactive aggregate. The core and X showed similar thermostabilities, but they were different in the recovery of enzyme activity after incubation with 1.2-2.0 M guanidine hydrochloride and its subsequent removal; the core was hardly recovered, but X was well recovered.

Acetyltransferases↗
PubMed1998

Expression of normal and mutant pyruvate dehydrogenase complex E1 alpha cDNAs in cultured human lymphoblasts.

Transfection of PDH E1 alpha cDNAs into human normal (3781) and PDH-deficient (4787) lymphoblast cell lines was performed to study the expression of different E1 alpha cDNAs. Transfection of normal human E1 alpha cDNA into a severely PDH-deficient cell line with 10% residual activity resulted in a fivefold increase in residual PDH complex activity. Transfection of the normal cDNA into the normal cell line did not affect the residual enzyme activity. Transfection of three known human PDH E1 alpha mutations (A875T, C787G, and a 13-bp insertion at nucleotide 981) into the normal cell line resulted in a decrease of PDH complex activity. Expression of these same mutations in the deficient cell line resulted in an increase of PDH complex activity, with the C787G mutation causing the greatest increase in enzyme activity. The increase in activity seen with A875T expressed in the mutant cell line suggested that interallelic complementation had occurred.

Cells, Cultured↗
PubMed1989

Domain structures of the dihydrolipoyl transacetylase and the protein X components of mammalian pyruvate dehydrogenase complex. Selective cleavage by protease Arg C.

Treatment of the dihydrolipoyl transacetylase-protein X-kinase subcomplex (E2-X-KcKb) with protease Arg C selectively converted protein X into an inner domain fragment (Mr approximately equal to 35,000) and an outer (lipoyl-bearing) domain fragment (Mr approximately equal to 15,500). These fragments were larger and much smaller, respectively, than the inner domain and outer domain fragments derived from the E2 component, supporting the conclusion that protein X is distinct from the E2 component. Protease Arg C cleaved the Kb subunit more slowly than protein X. An increase in kinase activity correlated with this cleavage of the Kb subunits. An even slower cleavage of E2 subunits generated an inner domain fragment (Mr approximately equal to 31,500) and a lipoyl-bearing domain fragment (Mr approximately equal to 49,000) which had Mr values at least 3,000 and 10,000 larger, respectively, than the corresponding E2 fragments generated by trypsin treatment of the subcomplex. Following various extents of cleavage with protease Arg C or trypsin, residual oligomeric subcomplexes were isolated and characterized. We found that selective removal of the lipoyl-bearing domain of protein X did not alter lipoyl-mediated regulation of the kinase indicating that the lipoyl residues bound to E2 subunits are effective, that the inner domain of protein X remained associated with the inner domain of E2 subunits following the complete removal of the outer domains of both E2 and protein X, that, with only 10% of the E2 subunits intact, nearly half of the catalytic (Kc) subunits of the kinase were bound by the residual subcomplex, and that removal of the remaining outer domains from E2 subunits released the Kc subunits. Thus, protein X is unique among the subunits of the complex in binding tightly to the oligomeric inner domain of the transacetylase, and the outer domain of the transacetylase serves to bind to and facilitate the regulation of the catalytic subunit of the kinase.

Acyltransferases↗
PubMed1988

Time-resolved fluorescence studies on the dihydrolipoyl transacetylase (E2) component of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

The dihydrolipoyl transacetylase (E2) component of A. vinelandii PDC and its lipoyl domain shows similar dynamic properties as revealed with fluorescence anisotropy decay of lipoyl-bound IAANS. The lipoyl domain (32.6 kDa), containing three almost identical subdomains shows a mode of rotation characteristic for a protein of about 30 kDa. A similar rotation is found in E2, indicating an independent rotational mobility of the whole domain in the multimeric E2 core (1.6 MDa). No independent rotation of a single lipoyl subdomain (10 kDa) is observed. The E1 component, in contrast to the E3 component, shows interaction with the lipoyl domain.

Acetyltransferases↗
PubMed1989

Determination of partial amino acid sequence of lipoate acetyltransferase of rat pyruvate dehydrogenase complex.

The partial amino acid sequence of rat lipoate acetyltransferase was determined using the intact protein and the peptides derived from a digest with Achromobacter protease I. The results showed the amino-terminal sequence of the mature enzyme to be (N) Ser-Leu-Pro-Pro-His-Gln-Lys-Val-Pro-Leu-Pro-Ser- Leu-Ser-Pro-Thr-Met-Gln-Ala-Gly-Thr-Ile-Ala-Arg-Trp-Glu-Lys. In addition, the sequences of two possible lipoyl-binding sites in the subunit, which are very similar to each other, were established.

Acetyltransferases↗
PubMedDate not supplied

Effect of insulin on the pyruvate dehydrogenase complex in the rat brain.

The level of PDHa and PDHt is substantially reduced in the rat brain 24 hours after alloxan administration. Effects are almost completely reversed by insulin administration. PDHa and PDHt from alloxan rat brains are remarkably activated when assayed on samples obtained by combining and preincubating at 30 degrees C for 30 min a homogenate from fresh unfrozen brains of alloxan rats, with a similarly treated preparation from fresh unfrozen brains of normal or insulin rats. On the contrary, no activation at all is obtained if the preincubation is carried out on homogenates from frozen and thawed brains. In alloxan rats, brain acetyl CoA level decreases remarkably whereas plasma free fatty acid concentration increases. Such changes disappear after insulin administration. The oxygen uptake, the respiratory control index and the ADP/O ratio in mitochondrial preparations obtained from brains of alloxan rats show no modifications at all.

Animals↗
PubMed1993

An improvement in the pyruvate dehydrogenase complex assay: a high-yield method for purifying arylamine acetyltransferase.

A new method for purifying arylamine acetyltransferase (AAT) has been devised using polyethylene glycol fractionation and hydroxylapatite chromatography. The new procedure gives a final yield of approximately 70% based on activity in crude homogenates and can be performed in a single day. This represents a threefold higher yield than previous methods. The procedure may be used to purify AAT from pigeon, chicken, and duck livers with equivalent yield of the final enzyme. However, the preparation from pigeon liver is preferred because of the sevenfold higher activity in this tissue. Nevertheless, if fresh pigeon livers cannot be acquired, calculations reveal that the preparation from chicken liver is comparable to that from pigeon liver acetone powder with respect to total activity obtained per gram wet weight starting material. One can also calculate that the chicken liver preparation is approximately 40 times cheaper than that from pigeon liver acetone powder, making the preparation of AAT from fresh chicken livers a good alternative when pigeon livers are not available.

Aniline Compounds↗
PubMed1989

Molecular biology of the human pyruvate dehydrogenase complex: structural aspects of the E2 and E3 components.

The availability of the primary amino acid sequences of the E2 of PDC, alpha-KGDC and BCKADC from several prokaryotic and eukaryotic species has allowed us to compare the structural aspects of human PDC-E2 with those of the E2 components from the other complexes. The PDC-E2 components from all the species examined so far contain three structurally identifiable regions: the lipoyl-bearing domain, the E3-binding site, and the catalytic domain. The primary structure of the lipoyl-bearing domain shows considerable variation in its size, ranging from one to three repeating units of approximately 110 amino acids, but essentially preserving its function in the E2 components. In contrast, the sizes of the E3-binding site and the catalytic domain of PDC-E2 from several species are essentially similar and show considerable conservation of specific amino acid residues. Obviously, additional studies are warranted to better understand the structure-function relationships of these domains and the evolutionary conservation of PDC-E2 in different species. Similarly, the availability of the primary amino acid sequences of E3 from several prokaryotes and eukaryotes has also permitted comparison of the structural domains of these proteins with that of the known structure of human GR, a flavoprotein member of the pyridine nucleotide-disulfide oxidoreductase family. Four structural domains (FAD, NAD+, central, and interface domains) have been identified in the E3 components. On the basis of the comparison of the secondary structural elements of GR and E3, the core structure of these two proteins are shown to be similar. It is hoped that further analysis of E3 using site-directed mutagenesis and determination of its crystal structure will provide better insight into its structure-function relationships.

Amino Acid Sequence↗
PubMed2001

Tissue-specific and developmental-specific expression of an Arabidopsis thaliana gene encoding the lipoamide dehydrogenase component of the plastid pyruvate dehydrogenase complex.

We describe an Arabidopsis thaliana gene, ptlpd2, which codes for a protein with high amino acid similarity to lipoamide dehydrogenases (LPDs) from diverse species. Ptlpd2 codes for a precursor protein possessing an N-terminal extension predicted to be a plastid-targeting signal. Expression of the ptlpd2 cDNA in Escherichia coli showed the encoded protein possessed the predicted LPD activity. PTLPD2 protein, synthesized in vitro, was efficiently imported into isolated chloroplasts of Pisum sativum and shown to be located in the stroma. In addition, fusion proteins containing the predicted transit peptide of PTLPD2 or the entire protein fused at the N-terminus with the green fluorescent protein (GFP), showed accumulation in vivo in chloroplasts but not in mitochondria of A. thaliana. Expression of ptlpd2 was investigated by introducing ptlpd2 promoter-beta-glucuronidase (GUS) gene fusions into Nicotiana tabacum. GUS expression was observed in seeds, flowers, root tips and young leaves. GUS activity was highest in mature seeds, decreased on germination and increased again in young leaves. Expression was also found to be temporally regulated in pollen grains where it was highest in mature grains at dehiscence. Database searches on ptlpd2 sequences identified a second A. thaliana gene encoding a putative plastidial LPD and two genes encoding proteins with high similarity to the mitochondrial LPD of P. sativum.

Amino Acid Sequence↗
PubMed1982

Limited proteolysis and proton NMR spectroscopy of Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.

The pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus was treated with chymotrypsin at pH 7 and 0 degrees C. Loss of the overall catalytic activity lagged behind the rapid cleavage of the lipoate acetyltransferase polypeptide chains, whose apparent Mr fell from 57 000 to 45 000 as judged by sodium dodecylsulphate/polyacrylamide gel electrophoresis. The inactive chymotrypsin-treated enzyme had lost the lipoic-acid-containing regions of the lipoate acetyltransferase chains, yet remained a highly assembled structure. Treatment of this chymotryptic core complex with trypsin at pH 7.0 and 0 degrees C caused a further shortening of the lipoate acetyltransferase polypeptide chains to an apparent Mr of 28 000 and was accompanied by disassembly of the complex. The lipoic-acid-containing regions are therefore likely to be physically exposed in the intact complex, protruding from the structural core formed by the lipoate acetyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy demonstrated that the enzyme complex contains large regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after excision of the lipoic-acid-containing regions with chymotrypsin. It is likely that the highly mobile regions are in the lipoate acetyltransferase component and facilitate movement of the lipoic acid residues. Such polypeptide chain mobility provides the molecular basis of a novel system of active-site coupling in the 2-oxo acid dehydrogenase multienzyme complexes.

Bacterial Proteins↗
PubMed1995

Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis.

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.

Adult↗