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PubMed2006

Characterization of testis-specific isoenzyme of human pyruvate dehydrogenase.

Pyruvate dehydrogenase (PDH), the first component of the human pyruvate dehydrogenase complex, has two isoenzymes, somatic cell-specific PDH1 and testis-specific PDH2 with 87% sequence identity in the alpha subunit of alpha(2) beta(2) PDH. The presence of functional testis-specific PDH2 is important for sperm cells generating nearly all their energy from carbohydrates via pyruvate oxidation. Kinetic and regulatory properties of recombinant human PDH2 and PDH1 were compared in this study. Site-specific phosphorylation/dephosphorylation of the three phosphorylation sites by four PDH kinases (PDK1-4) and two PDH phosphatases (PDP1-2) were investigated by substituting serines with alanine or glutamate in PDHs. PDH2 was found to be very similar to PDH1 as follows: (i) in specific activities and kinetic parameters as determined by the pyruvate dehydrogenase complex assay; (ii) in thermostability at 37 degrees C; (iii) in the mechanism of inactivation by phosphorylation of three sites; and (iv) in the phosphorylation of sites 1 and 2 by PDK3. In contrast, the differences for PDH2 were indicated as follows: (i) by a 2.4-fold increase in binding affinity for the PDH-binding domain of dihydrolipoamide acetyltransferase as measured by surface plasmon resonance; (ii) by possible involvement of Ser-264 (site 1) of PDH2 in catalysis as evident by its kinetic behavior; and (iii) by the lower activities of PDK1, PDK2, and PDK4 as well as PDP1 and PDP2 toward PDH2. These differences between PDH2 and PDH1 are less than expected from substitution of 47 amino acids in each PDH2 alpha subunit. The multiple substitutions may have compensated for any drastic alterations in PDH2 structure thereby preserving its kinetic and regulatory characteristics largely similar to that of PDH1.

Amino Acid Sequence↗
PubMed1976

Transition state analogs for thiamin pyrophosphate-dependent enzymes.

Many of the transition states that are formed from thiamin pyrophosphate in enzymic reactions are expected to have structures in which the thiazolium ring of thiamin pyrophosphate has lost most of its positive charge. We have synthesized thiamin thiazolone pyrophosphate from the unphosphorylated compound. The sulphur-containing ring of thiamin thiazolone pyrophosphate is uncharged, and thus the compound resembles these transition states. In agreement with the prediction from the transition state theory of reaction rates, thiamin thiazolone pyrophosphate binds to Escherichia coli pyruvate dehydrogenase complex (EC 1.2.7.1) much more strongly than thiamin pyrophosphate itself. An upper limit for the value of the dissociation constant, calculated from the extent of inactivation of the enzyme by a low concentration of thiamin thiazolone pyrophosphate, is 5 X 10(-10) M at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. The dissociation constant for thiamin pyrophosphate under similar conditions is about 10(-5) M. The kinetics of inactivation of pyruvate dehydrogenase complex by thiamin thiazolone pyrophosphate are first order with respect to both enzyme and thiamin thiazolone pyrophosphate; the value of the second order rate constant is 5.7 X 10(5) M-1 min-1 at 3 degrees in 0.5 mM MgCl2/10 mM potassium phosphate, pH 6.6. An analysis of the decrease in the rates of inactivation caused by thiamin pyrophosphate indicates that thiamin thiazolone pyrophosphate binds at the coenzyme sites. We have also synthesized thiamin thiothiazolone pyrophosphate and obtained very similar results with this compound.

Alkaline Phosphatase↗
PubMed2003

Pyruvate ameliorates post ischemic injury of rat astrocytes and protects them against PARP mediated cell death.

This in vitro study was designed to examine the efficacy of exogenous pyruvate and glucose as a fuel substrate to protect rat astrocytes from post-ischemic injury. Astrocytes were incubated in Kreb's buffer deprived of oxygen and glucose for 6 h (ischemia) followed by incubation with added pyruvate or glucose and normoxia for the next 6 h (reperfusion). The transformation of reactive astrocytes in response to various treatments was examined by immunostaining with glial fibrillary acidic protein. The extent of cell damage was evaluated in terms of lactate dehydrogenase leakage from the cells and altered intracellular redox status. The mechanism of cell death was determined by immunoblotting with cytochrome C, caspase-3 and PARP antibodies. The mechanism of the action of pyruvate was determined by measuring the activity of pyruvate dehydrogenase complex, and cellular metabolic status by measuring ATP levels. In comparison to glucose, supply of exogenous pyruvate restored the morphological integrity of post-ischemic astrocytes and prevented gliosis. Pyruvate prevented the cell death of post-ischemic astrocytes by inhibiting the leakage of lactate dehydrogenase, decreasing the redox ratio and restraining the activation of apoptotic events such as release of mitochondrial cytochrome c and fragmentation of caspase-3 and PARP. This study also suggests that pyruvate may accelerate its own metabolism by increasing the activity of pyruvate dehydrogenase and thus restores the cellular ATP levels in post-ischemic astrocytes. Use of pyruvate as an alternate fuel substrate may provide a possibility for the novel therapeutic approach to the treatment of cerebral ischemia.

Adenosine Triphosphate↗
PubMed2001

Adaptive increase in pyruvate dehydrogenase kinase 4 during starvation is mediated by peroxisome proliferator-activated receptor alpha.

Pyruvate dehydrogenase kinase isoform 4 (PDK4) is upregulated by starvation in many tissues of the body during starvation. This causes inactivation of the pyruvate dehydrogenase complex which blocks pyruvate oxidation and conserves lactate and alanine for gluconeogenesis. Enhanced PDK4 expression may be caused by the increase in free fatty acids that occurs during starvation. Free fatty acids can activate peroxisome proliferator-activated receptor alpha (PPARalpha), and activation of PPARalpha can promote PDK4 expression. This model is supported by the findings reported here that WY-14,643, a synthetic PPARalpha activator, increases PDK4 expression in wild-type mice but not in PPARalpha-null mice. Starvation likewise increases the expression of PDK4 in tissues of wild-type mice but not in tissues of PPARalpha-null mice. These findings document the functional importance of PPARalpha for PDK4 expression during starvation and suggest an important role for elevated free fatty acids in the induction.

Animals↗
PubMed1985

Differences in some of the metabolic properties of mitochondria isolated from cerebral cortex and olfactory bulb of the rat.

The metabolic properties of mitochondria from rat cerebral cortex and olfactory bulb were investigated. The pyruvate-supported oxygen uptake rates by olfactory bulb mitochondria were significantly lower than those by cerebrocortical mitochondria. This is consistent with the differences in pyruvate dehydrogenase complex activities between these mitochondrial preparations. Pyruvate dehydrogenase kinase, NAD-linked isocitrate dehydrogenase, and hexokinase activities in olfactory bulb mitochondria were significantly lower than those in cerebrocortical mitochondria. However, NADP-linked isocitrate dehydrogenase, and NAD-linked and NADP-linked glutamate dehydrogenase activities in olfactory bulb mitochondria were significantly higher than those in cerebrocortical mitochondria. The differences between these two mitochondrial preparations in terms of the activities of these energy-metabolizing enzymes reflect the differences detected in the homogenates of these regions.

Animals↗
PubMed2004

Regulation of expression of tissue plasminogen activator and plasminogen activator inhibitor-1 by dichloroacetic acid in human fibroblasts from normal peritoneum and adhesions.

OBJECTIVE: As part of our ongoing studies to understand the biologic mechanisms of wound repair that lead to postoperative adhesions, we have identified characteristics of an adhesion phenotype that differs between fibroblasts that are obtained from human normal peritoneum and adhesions. In this study, we sought to examine whether stimulation of aerobic metabolism would alter differential expression of tissue plasminogen activator and plasminogen activator inhibitor-1, thereby creating a milieu likely to be less favorable to postoperative adhesion development. To examine this issue, we used a compound, dichloroacetic acid, that stimulates the pyruvate dehydrogenase complex, which causes pyruvate to be metabolized in the Kreb's cycle rather than being converted into lactate, thereby switching anaerobic to aerobic metabolism. STUDY DESIGN: Human fibroblasts from normal peritoneum and adhesions were cultured in the absence or presence of dichloroacetic acid (100 microg/mL) for 24 hours, under normal and hypoxic (2% O(2)) conditions. Real-time reverse transcriptase-polymerase chain reaction of tissue plasminogen activator, plasminogen activator inhibitor-1, and a housekeeping gene beta-actin was performed with messenger RNA that was extracted from all treatment points. RESULTS: Dichloroacetic acid stimulated normal peritoneal fibroblast tissue plasminogen activator messenger RNA expression under hypoxic conditions. In adhesion fibroblasts, dichloroacetic acid treatment enhanced tissue plasminogen activator messenger RNA expression under both normoxic and hypoxic conditions. Plasminogen activator inhibitor-1 messenger RNA expression was unaltered by dichloroacetic acid in normoxic normal peritoneal fibroblasts; but during culture under hypoxic conditions, dichloroacetic acid reduced plasminogen activator inhibitor-1 messenger RNA expression. Similarly, in adhesion fibroblasts, dichloroacetic acid reduced plasminogen activator inhibitor-1 messenger RNA expression under both normoxic and hypoxic conditions. As a result, in normal peritoneal fibroblasts under hypoxic conditions and in adhesion fibroblasts under normoxic and hypoxic conditions, dichloroacetic acid greatly increased the tissue plasminogen activator/plasminogen activator inhibitor-1 ratios. CONCLUSION: These findings confirm that fibroblasts from adhesions are characterized by reduced tissue plasminogen activator and increased plasminogen activator inhibitor-1 production. These observations are extended to show the stimulation of oxidative metabolism by dichloroacetic acid increases tissue plasminogen activator expression under hypoxic conditions. Dichloroacetic acid reduces plasminogen activator inhibitor-1 production by hypoxic normal peritoneal fibroblasts and adhesion fibroblasts under hypoxic conditions. The resultant increases in the tissue plasminogen activator/plasminogen activator inhibitor-1 ratios would favor the development of a fibrinolytic milieu, which would be expected potentially to limit postoperative adhesion development. Thus, regulation of metabolic activity of peritoneal cells may provide a target for future interventions for the reduction of the development of postoperative adhesions, particularly as intervention relates to the healing of peritoneal sites that previously had adhesions. (eg, sites of potential adhesion reformation).

Cell Hypoxia↗
PubMed2000

Autoimmunity and primary biliary cirrhosis.

The history of primary biliary cirrhosis (PBC) began in 1851, with autoimmunity introduced in 1958 and expanded from the 1960s on. In PBC, autoantibodies are present to mitochondria-located antigens (AMA) and to nuclear-located antigens (ANA). The AMA react with E2 subunits of three members of the 2-oxoacid dehydrogenase complex family, but most frequently with pyruvate dehydrogenase complex (PDC); the inner lipoyl domain of PDC-E2 contains a major B- and T-cell epitope. The ANA react with three nuclear components, centromeric proteins, nuclear dot proteins and nuclear pore complex. Autoimmune diseases including PBC reflect a failure in mechanisms of self-tolerance which is developed in central lymphoid tissues in embryonic life by deletion of self-reactive lymphocytes, and maintained in peripheral tissues in post-natal life by regulatory processes. Primary biliary cirrhosis has not yet been identified with failure in any one particular tolerance mechanism. Genetic influences are revealed by familial occurrences and by associations with HLA alleles, and environmental influences by epidemiological data. A lead to pathogenesis is the accumulation uniquely in PBC of PDC-E2-like material at the plasma membrance of biliary epithelial cells (BECs). Although the origin of this accumulation of PDC-E2 at the surface of BECs is uncertain, it provides a credible 'tissue-specific' target for an autoimmune attack by T and B lymphocytes at the site of the actual pathology.

Autoantibodies↗
PubMed2001

New splicing-site mutations in the SURF1 gene in Leigh syndrome patients.

The gene SURF1 encodes a factor involved in the biogenesis of cytochrome c oxidase, the last complex in the respiratory chain. Mutations of the SURF1 gene result in Leigh syndrome and severe cytochrome c oxidase deficiency. Analysis of seven unrelated patients with cytochrome c oxidase deficiency and typical Leigh syndrome revealed different SURF1 mutations in four of them. Only these four cases had associated demyelinating neuropathy. Three mutations were novel splicing-site mutations that lead to the excision of exon 6. Two different novel heterozygous mutations were found at the same guanine residue at the donor splice site of intron 6; one was a deletion, whereas the other was a transition [588+1G>A]. The third novel splicing-site mutation was a homozygous [516-2_516-1delAG] in intron 5. One patient only had a homozygous polymorphism in the middle of the intron 8 [835+25C>T]. Western blot analysis showed that Surf1 protein was absent in all four patients harboring mutations. Our studies confirm that the SURF1 gene is an important nuclear gene involved in the cytochrome c oxidase deficiency. We also show that Surf1 protein is not implicated in the assembly of other respiratory chain complexes or the pyruvate dehydrogenase complex.

Base Sequence↗
PubMed1988

Energy metabolism in disorders of the nervous system.

"Energy metabolism" is deranged in a wide variety of disorders of the nervous system. This term refers rather loosely to the pathways responsible for the utilization of the major substrates of brain. Primary disorders of energy metabolism are those in which the primary insult affects the cellular machinery required for energy metabolism. A typical example would be a defect in a gene coding for a mitochondrial protein. Biochemically, defects which appear to be hereditary and which lead to disease of the central nervous system have been described in each of the pathways of energy metabolism: glycogenolysis (the break-down of glycogen to glucose); glycolysis (the break down of glucose to pyruvate and lactate); the pyruvate dehydrogenase complex (which oxidizes pyruvate to enter the Krebs tricarboxylic acid cycle); the tricarboxylic acid cycle itself (which completes the oxidation of carbohydrates and other substrates to carbon dioxide); electron transport (which carries out their oxidation to water); the pentose phosphate pathway (an alternate pathway for glucose oxidation); and several "minor" mitochondrial pathways. Clinically, the spectrum of syndromes associated with primary disorders of energy metabolism is wide. Common manifestations include psychomotor retardation, with associated lactic acidosis and/or hypoglycemia. The laboratory abnormalities may be intermittent. Syndromes which have been culled out include congenital lactic acidosis, Leigh disease, intermittent ataxia, Kearns-Sayre-Shy syndrome (KSS), myoclonus epilepsy with ragged red fibers (MERRF), and mitochondrial myopathy-encephalopathy-lactic acidosis-stroke (MELAS). As with other families of inborn errors, both clinical and biochemical heterogeneity occur. Patients with apparently similar clinical syndromes can turn out to have different inborn errors, and patients with abnormalities of the same gene product can have clinically distinguishable syndromes. Secondary disorders are those in which the derangements of energy metabolism are presumably secondary to some other insult but may still be important for the cellular pathophysiology. These include the metabolic encephalopathies and probably a number of well-known neurodegenerative disorders. In the hereditary ataxias, abnormalities of mitochondrial markers are common but do not correlate consistently with the disorders as conventionally classified; a new classification into axonal ataxias, multiple system degenerations, and ataxic encephalopathies may be easier to relate to the pathophysiology.(ABSTRACT TRUNCATED AT 400 WORDS)

Alzheimer Disease↗
PubMed2000

Automated enzymatic mitochondrial antibody assay for the diagnosis of primary biliary cirrhosis.

Primary biliary cirrhosis is a progressive autoimmune disease that affects middle aged women, resulting in liver cirrhosis. We describe here an automated enzymatic mitochondrial antibody assay adapted for performance on laboratory analysers for the serological diagnosis of primary biliary cirrhosis. This assay detects the characteristic autoantibody directed against the 74kDa E2 subunit of the pyruvate dehydrogenase complex. Analysis of receiver operator characteristic curve data indicated that the automated enzymatic mitochondrial assay procedure discriminated clinically identified patients with primary biliary cirrhosis from normal subjects with a sensitivity of 83% and a specificity of 100%. This method compared favourably against a commercial ELISA method which had a sensitivity of 73% and a specificity of 100%. The automated enzymatic mitochondrial antibody assay is a high throughput assay of use for the routine diagnosis of patients with primary biliary cirrhosis with autoantibodies to the E2 subunit of the pyruvate dehydrogenase complex. The method is of potential value for economical and rapid screening to detect asymptomatic primary biliary cirrhosis in the at-risk segment of the population, namely middle aged women.

Autoantibodies↗
PubMed1994

Nucleotide sequence analysis of natural and combinatorial anti-PDC-E2 antibodies in patients with primary biliary cirrhosis. Recapitulating immune selection with molecular biology.

We have analyzed at the nucleotide level the variable region gene sequences of five human mAbs and five recombinant Fab fragments derived from the mesenteric lymph nodes of patients with primary biliary cirrhosis. Both mAbs and Fabs were monospecific for dihydrolipoamide acetyltransferase, the E2 subunit of the pyruvate dehydrogenase complex, which has been shown to be the major autoantigen of primary biliary cirrhosis. We found that although the mAbs, mainly of the IgM isotype, were encoded by a diverse array of VH and VL gene segments either as direct copies of germline genes or somatically mutated, the recombinant IgG Fabs expressed clonally related heavy chains displaying a high number of somatic mutations that very likely occurred in the context of Ag selection. Combinatorial pairing of clonally related heavy chains with highly homologous light chains suggests that the IgG anti-pyruvate dehydrogenase complex repertoire of primary biliary cirrhosis patients is the result of the clonal expansion of a restricted set of B cells.

Amino Acid Sequence↗
PubMed1998

Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase.

The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. The first component (E1) converts pyruvate to bound acetaldehyde using thiamine diphosphate (ThDP) and Mg2+ as cofactors. There is no 3D structure of E1 available but those of other ThDP-dependent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an alpha2beta2 structure and the conserved Glu89 of the beta-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expressed in Escherichia coli and purified. Mutating Glu89 to glutamine, aspartate and alanine markedly reduces catalytic activity and the affinity for ThDP, consistent with a role as the catalytic glutamate.

Amino Acid Sequence↗
PubMed1995

Identification of the tryptophan residue in the thiamin pyrophosphate binding site of mammalian pyruvate dehydrogenase.

The pyruvate dehydrogenase (E1) component of the mammalian pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate with the formation of an acetyl residue and reducing equivalents, which are transferred sequentially to the dihydrolipoyl acetyltransferase and dihydrolipoamide dehydrogenase components. To examine the role of tryptophanyl residue(s) in the active site of E1, the enzyme was modified with the tryptophan-specific reagent N-bromosuccinimide. Modification of 2 tryptophan residues/mol of bovine E1 (out of 12 in a tetramer alpha 2 beta 2) resulted in complete inactivation of the enzyme. The inactivation was prevented by preincubation with thiamin pyrophosphate (TPP), indicating that the modified tryptophan residue(s) is part of the active site of this enzyme. Fluorescence studies showed that thiamin pyrophosphate interacts with tryptophan residue(s) of E1. The magnetic circular dichroism (MCD) spectral intensity at approximately 292 nm was decreased by approximately 15% for E1 + TPP relative to the intensity for E1 alone. Because this MCD band is uniquely sensitive to and quantitative for tryptophan, the simplest interpretation is that 1 out of 6 tryptophan residues present in E1 (alpha beta dimer) interacts with TPP. The natural circular dichroism (CD) spectrum of E1 is dramatically altered upon binding TPP, with concomitant induction of optical activity at approximately 263 nm for the nonchiral TPP macrocycle. From CD studies, it is also inferred that loss of activity following N-bromosuccinimide treatment occurred without significant changes in the overall secondary structure of the protein. A single peptide was isolated by differential peptide mapping in the presence and absence of thiamin pyrophosphate following modification with N-bromosuccinimide. This peptide generated from human E1 was found to correspond to amino acid residues 116-143 in the deduced sequence of human E1 beta, suggesting that the tryptophan residue 135 in the beta subunit of human E1 functions in the active site of E1. The amino acid sequence surrounding this tryptophan residue are conserved in E1 beta from several species, suggesting that this region may constitute a structurally and/or functionally essential part of the enzyme.

Amino Acid Sequence↗
PubMed1995

Mitochondrial antigens, molecular mimicry and autoimmune disease.

The immune system is normally tolerant to mitochondrial self-antigens, but responsive against bacteria. Low-titre anti-mitochondrial antibodies (AMA) might be involved in this discrimination. Tolerance is broken in diseases characterised by high titre AMA. Some of these AMA, against cardiolipin, cross-react with DNA. The best studied AMA are those characterising primary biliary cirrhosis (PBC). These are directed against E2 subunits of the oxo-acid dehydrogenase complexes, and also against subunits E1 alpha, E1 beta and X of the pyruvate dehydrogenase complex. AMA of PBC patients also react with bacterial E2s. Reactivities are primarily peptide-specific but with cross-reactivity between mitochondrial and microbial antigens and between E2s of respective complexes. Immunodominant epitopes, for anti E2 AMA, include the conserved sequence flanking the site of lipoyl attachment. It is proposed that the initial stimulus for antibody production is chronic urinary tract infection. AMA themselves are not pathogenic, but CD4+ T-cells would be primed, recognising the lipoyl domain epitope in association with class II HLA. Inappropriate expression of class II antigens on bile duct epithelia, (as found in PBC), might lead to presentation of a particular fragment of HLA-DR alpha, known to be a major MHC presented self-peptide in the mouse. That sequence strongly mimics the lipoyl domain and might be recognised by primed T-cells, initiating the autoimmune cascade. In the mouse, a peptide of ND1 of Complex I is presented in association with class I MHC. Cells exhibiting somatic mutation of such a peptide might thus be subject to attack by CD8+ T-cells. If such peptides were presented by class II HLA, autoimmune diseases might arise, related to mimicry between such peptides and microbial sequences and/or self-antigens. These considerations might apply in Leber's disease and in age-related pathology.

Animals↗
PubMed1989

2-ketoacid dehydrogenase complexes of Escherichia coli: stereospecificities of the three components for (R)-lipoate.

Stereospecificities of component enzymes in the pyruvate dehydrogenase complex and 2-ketoglutarate dehydrogenase complex from Escherichia coli for lipoate and dihydrolipoate are determined. Assays of the component enzymes using R,S-, R-, or S-lipoate or the enantiomers of dihydrolipoate show that only the R-enantiomers are substrates for these enzymes. Nonenzymatic reactions involving acetyl group transfer and coupled electron and acetyl group transfer between enantiomeric molecules of lipoate or/and dihydrolipoate proceed at significant rates. Coupled acetyl group and electron transfer from enzyme-bound acetyldihydrolipoyl moieties to free lipoate is also observed. The S-enantiomers are neither substrates nor inhibitors; however, products of S-enantiomers are slowly generated in enzymatic reactions owing to nonenzymatic reactions between enzyme-bound acetyldihydrolipoyl-groups and free S-lipoate or S-dihydrolipoate.

Acetylation↗
PubMed1992

Purification and cellular localization of wild type and mutated dihydrolipoyltransacetylases from Azotobacter vinelandii and Escherichia coli expressed in E. coli.

Wild type dihydrolipoyltransacetylase(E2p)-components from the pyruvate dehydrogenase complex of A. vinelandii or E. coli, and mutants of A. vinelandii E2p with stepwise deletions of the lipoyl domains or the alanine- and proline-rich region between the binding and the catalytic domain have been overexpressed in E. coli TG2. The high expression of A. vinelandii wild type E2p (20% of cellular protein) and of a mutant enzyme with two lipoyl domains changed the properties of the inner bacterial membrane. This resulted in a solubilization of A. vinelandii E2p after degradation of the outer membrane by lysozyme without any contamination by E. coli pyruvate dehydrogenase complex (PDC) or other high-molecular-weight contaminants. The same effect could be detected for A. vinelandii E2o, an E2 which contains only one lipoyl domain, whereas almost no solubilization of A. vinelandii E2p with one lipoyl domain or of E2p consisting only of the binding and catalytic domain was found. Partial or complete deletion of the alanine- and proline-rich sequence between the binding and the catalytic domain did also decrease the solubilization of the E2p-mutants after lysozyme treatment. Immunocytochemical experiments on E. coli TG2 cells expressing A. vinelandii wild type E2p indicated that the enzyme was present as a soluble protein in the cytoplasm. In contrast, overexpressed A. vinelandii E2p with deletion of all three lipoyl domains and E. coli wild type E2p aggregated intracellularly. The solubilization by lysozyme is therefore ascribed to excluded volume effects leading to changes in the properties of the inner bacterial membrane.

Acetyltransferases↗
PubMed1996

Increased acetyl group availability enhances contractile function of canine skeletal muscle during ischemia.

Skeletal muscle contractile function is impaired during acute ischemia such as that experienced by peripheral vascular disease patients. We therefore, examined the effects of dichloroacetate, which can alter resting metabolism, on canine gracilis muscle contractile function during constant flow ischemia. Pretreatment with dichloroacetate increased resting pyruvate dehydrogenase complex activity and resting acetylcarnitine concentration by approximately 4- and approximately 10-fold, respectively. After 20-min contraction the control group had demonstrated an approximately 40% reduction in isomeric tension whereas the dichloroacetate group had fatigued by approximately 25% (P < 0.05). Dichloroacetate resulted in less lactate accumulation (10.3 +/- 3.0 vs 58.9 +/- 10.5 mmol.kg-1 dry muscle [dm], P < 0.05) and phosphocreatine hydrolysis (15.6 +/- 6.3 vs 33.8 +/- 9.0 mmol.kg-1 dm, P < 0.05) during contraction. Acetylcarnitine concentration fell during contraction by 5.4 +/- 1.8 mmol.kg-1 dm in the dichloroacetate group but increased by 10.0 +/- 1.9 mmol.kg-1 dm in the control group. In conclusion, dichloroacetate enhanced contractile function during ischemia, independently of blood flow, such that it appears oxidative ATP regeneration is limited by pyruvate dehydrogenase complex activity and acetyl group availability.

Acetylcarnitine↗
PubMed1993

Sera from patients with tuberculosis recognize the M2a-epitope (E2-subunit of pyruvate dehydrogenase) specific for primary biliary cirrhosis.

Anti-M2 antibodies in primary biliary cirrhosis (PBC) have been shown to react with the alpha-ketoacid dehydrogenase complex of the inner mitochondrial membrane consisting of six epitopes (E2 subunit of the pyruvate dehydrogenase complex (PDC), 70 kD; protein X of the PDC, 56 kD; alpha-ketoglutarate dehydrogenase complex, 52 kD; branched-chain alpha-ketoacid dehydrogenase, 52 kD; E1 alpha subunit of PDC, 45 kD; and E1 beta-subunit of PDC, 36 kD). These epitopes are also present in the M2 fraction which is a chloroform extract from beef heart mitochondria. The E2 subunit of the PDC at 70 kD (M2a), especially, is a major target epitope which is recognized by about 85% of all PBC sera. However, analysing sera from 28 patients with active pulmonary tuberculosis it became evident that 12 (43%) also recognized the PDC-E2 subunit (M2a), as shown by Western blotting using the M2 fraction, the purified PDC, and the recombinant PDC-E2. In contrast, only two of 82 patients with other bacterial and viral infections including 25 patients with Escherichia coli infections reacted with the PBC-specific epitope at 70 kD. Naturally occurring mitochondrial antibodies (NOMA) were present in 54% of the patients with tuberculosis and in 50% of patients with other infectious disorders. They recognized either a determinant at 65 kD (epsilon) or at 60/55 kD (zeta/eta). None of the sera from 100 blood donors had anti-M2 but 14 had NOMA. Testing anti-M2 and NOMA-positive marker sera by Western blotting against membrane fractions derived from mycobacteria and E. coli it could be shown that--like mammalian mitochondria--they contain both the PBC-specific M2 antigen as well as the non-PBC-specific naturally occurring mitochondrial antigen system (NOMAg). The observation that PBC-specific antibodies were preferentially induced in patients suffering from a mycobacterial infection may provide some new clues to the still unknown etiology of PBC.

Adolescent↗