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PubMed2001

A novel syndrome affecting multiple mitochondrial functions, located by microcell-mediated transfer to chromosome 2p14-2p13.

We have studied cultured skin fibroblasts from three siblings and one unrelated individual, all of whom had fatal mitochondrial disease manifesting soon after birth. After incubation with 1 mM glucose, these four cell strains exhibited lactate/pyruvate ratios that were six times greater than those of controls. On further analysis, enzymatic activities of the pyruvate dehydrogenase complex, the 2-oxoglutarate dehydrogenase complex, NADH cytochrome c reductase, succinate dehydrogenase, and succinate cytochrome c reductase were severely deficient. In two of the siblings the enzymatic activity of cytochrome oxidase was mildly decreased (by approximately 50%). Metabolite analysis performed on urine samples taken from these patients revealed high levels of glycine, leucine, valine, and isoleucine, indicating abnormalities of both the glycine-cleavage system and branched-chain alpha-ketoacid dehydrogenase. In contrast, the activities of fibroblast pyruvate carboxylase, mitochondrial aconitase, and citrate synthase were normal. Immunoblot analysis of selected complex III subunits (core 1, cyt c(1), and iron-sulfur protein) and of the pyruvate dehydrogenase complex subunits revealed no visible changes in the levels of all examined proteins, decreasing the possibility that an import and/or assembly factor is involved. To elucidate the underlying molecular defect, analysis of microcell-mediated chromosome-fusion was performed between the present study's fibroblasts (recipients) and a panel of A9 mouse:human hybrids (donors) developed by Cuthbert et al. (1995). Complementation was observed between the recipient cells from both families and the mouse:human hybrid clone carrying human chromosome 2. These results indicate that the underlying defect in our patients is under the control of a nuclear gene, the locus of which is on chromosome 2. A 5-cM interval has been identified as potentially containing the critical region for the unknown gene. This interval maps to region 2p14-2p13.

Amino Acids↗
PubMed1993

Molecular mimicry in primary biliary cirrhosis. Evidence for biliary epithelial expression of a molecule cross-reactive with pyruvate dehydrogenase complex-E2.

Sera from patients with primary biliary cirrhosis (PBC) react with enzymes of the 2-oxo dehydrogenase pathways, particularly PDC-E2. These enzymes are present in all nucleated cells, yet autoimmune damage is confined to biliary epithelial cells. Using a panel of eight mouse monoclonal antibodies and a human combinatorial antibody specific for PDC-E2, we examined by indirect immunofluorescence and confocal microscopy sections of liver from patients with PBC, progressive sclerosing cholangitis, and hepatocarcinoma. The monoclonal antibodies gave typical mitochondrial immunofluorescence on biliary epithelium and on hepatocytes from patients with either PBC, progressive sclerosing cholangitis, or hepatocarcinoma. However, one of eight mouse monoclonal antibodies (C355.1) and the human combinatorial antibody reacted with great intensity and specificity with the luminal region of biliary epithelial cells from patients with PBC. Simultaneous examination of these sections with an antiisotype reagent for human IgA revealed high IgA staining in the luminal region of biliary epithelial cells in patients with PBC. IgG and IgA antibodies to PDC-E2 were detected in the bile of patients with PBC but not normal controls. We believe that this data may be interpreted as indicating that a molecule cross-reactive with PDC-E2 is expressed at high levels in the luminal region of biliary epithelial cells in PBC.

Antibodies, Monoclonal↗
PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗
PubMed2002

Pyruvate dehydrogenase E3 binding protein deficiency.

Primary defects of the E3 binding protein component of the pyruvate dehydrogenase complex appear to be a rare cause of pyruvate dehydrogenase deficiency. We describe two new, unrelated patients with mutations in the E3 binding protein gene, in both cases involving the conserved dinucleotides of splice junctions. Both patients presented with delayed development and lactic acidosis, features that are also found in patients with the more common pyruvate dehydrogenase E1 alpha subunit deficiency; however, they both had significant residual enzyme activity in cultured fibroblasts and prolonged survival.

Adolescent↗
PubMed1998

Abnormalities of mitochondrial enzymes in Alzheimer disease.

Abundant evidence, including critical information gathered by Prof. Siegfried Hoyer and his colleagues, indicates that abnormalities of cerebral metabolism are common in neurodegenerative diseases, including Alzheimer's Disease (AD). Alterations in mitochondrial enzymes likely underlie these deficits. Replicable reductions in AD brain occur in the pyruvate dehydrogenase complex (the link of glycolysis to the Kreb's cycle), the alpha-ketoglutarate dehydrogenase complex (KGDHC; the link of Kreb's cycle to glutamate metabolism) and cytochrome oxidase (the link of the Kreb's cycle to oxygen utilization). Available evidence suggests that deficiencies in KGDHC may be genetic in some cases, whereas evidence that the other two enzyme systems have a genetic component is lacking. Additional results indicate that the reductions can also be secondary to other causes including oxidative stress. A variety of data suggest that the mitochondrial insufficiencies contribute significantly to the pathophysiology of AD.

Alzheimer Disease↗
PubMed2004

Mitochondrial enzymes in schizophrenia.

The responses of brain metabolism and blood flow to stimulation are diminished in the dorsolateral prefrontal cortexes (DLPFCs) of schizophrenic patients. Reductions in mitochondrial enzymes underlie diminished metabolism in several neurodegenerative diseases. Thus, we tested whether reductions in selected mitochondrial enzymes could underlie the changes in schizophrenia. The activities of the pyruvate dehydrogenase complex (PDHC), aconitase, isocitrate dehydrogenase (ICDH), and the alpha-ketoglutarate dehydrogenase complex (KGDHC) were determined on DLPFCs from patients with schizophrenia (n=26) and normal nonpsychiatric disease controls (n=13). The enzyme activities (mU/mg protein; mean +/- SEM) were similar (values for controls and schizophrenic patients, respectively) for PDHC (11.36 +/-1.5, 10.33 +/- 0.8), aconitase (1.06 +/- 0.1, 1.35 +/- 0.2), ICDH (31.70 +/- 2.7, 32.00 +/- 2.6), and KGDHC (2.62 +/- 0.4, 3.09 +/- 0.3). Separate analyses of the patients matched for age or postmortem interval gave similar conclusions. Cognitive dementia rating scores correlated poorly with activities of PDHC, aconitase, ICDH, and KGDHC. In one schizophrenic patient, activity of aconitase was undetectable, and in two others KGDHC activity was very low. Both had low activities of ICDH. A reduced activity of these enzymes in a subgroup is consistent with other data, suggesting that some patients with schizophrenia have abnormalities in brain mitochondria. However, in schizophrenia, unlike a number of neurodegenerative diseases, reductions in the activities of the key mitochondrial enzymes KGDHC and PDHC are not frequent.

Aconitate Hydratase↗
PubMed1977

[Study of the kinetic mechanism of the pyruvate-2,6-dichlorophenolindophenol reductase activity of muscle pyruvate dehydrogenase].

The mechanism of pyruvate-2,6-dichlorophenol-indophenol (2,6-CPI) reductase reaction catalyzed by the pyruvate dehydrogenase complex from pigeon breast muscle and by its pyruvate dehydrogenase component was studied. The K'm values for 2,6-DCPI in both cases were found equal to 1.3--1.4-10(-5) M. The double reverse values plots obtained at a fixed concentration of the first substrate and a variable concentration of the second one were linear and had a constant K'm/V'max ratio. The substitution of thiamine pyrophosphate and pyruvate by the substrate decarboxylation product, i.e. 2-oxyethyl thiamine pyrophosphate under similar conditions resulted in kinetic plots, typical for the "ping-pong" mechanism of enzymatic reactions. A mechanism of the pyruvate 2,6-DCPI reductase reaction, providing for the interaction of 2-oxyethyl thiamine pyrophosphate after its binding to the apoenzyme with a certain protein group of the pyruvate dehydrogenase active centre, was postulated. The reaction was shown to result in the production of acetyl-substituted reduced form of the enzyme. Regeneration of free enzyme required the presence of 2,6-DCPI as oxidizing agent.

2,6-Dichloroindophenol↗
PubMed1986

[Dehydrogenases of alpha-keto acids from pigeon breast muscle].

The structure, function and regulation of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase complexes from pigeon breast muscle are reviewed. The nature of essential groups involved in the formation of active centers of the first components of the complexes, i.e., pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase, is described. The catalytic mechanism of the pyruvate dehydrogenase reaction and the peculiarities of cooperative interactions of the active centers of the above enzymes are discussed.

Animals↗
PubMed1991

Evidence for two protein-lipoylation activities in Escherichia coli.

The lipoate acyltransferase subunits of the 2-oxo acid dehydrogenase complexes are post-translationally modified with one or more covalently-bound lipoyl cofactors. Two distinct lipoate-protein ligase activities, LPL-A and LPL-B, have been detected in E. coli by their ability to modify purified lipoyl apo-domains of the bacterial pyruvate dehydrogenase complex. Both enzymes require ATP and Mg2+, use L-lipoate, 8-methyllipoate, lipoyl adenylate and octanoyl adenylate as substrates, and both activate lipoyl-deficient pyruvate dehydrogenase complexes. In contrast, only LPL-B uses D-lipoate and octanoate and there are differences in the metal-ion and phosphate requirements. It is suggested that LPL-B may be responsible for the octanoylation of lipoyl domains observed previously under lipoate-deficient conditions.

Chromatography, Affinity↗
PubMed2006

An acute increase in skeletal muscle carnitine content alters fuel metabolism in resting human skeletal muscle.

CONTEXT: Carnitine plays an essential role in the integration of fat and carbohydrate oxidation in skeletal muscle, which is impaired in obesity and type 2 diabetes. OBJECTIVE: The aim of the present study was to investigate the effect of an increase in skeletal muscle total carnitine (TC) content on muscle fuel metabolism. DESIGN: A 5-h iv infusion of saline (control) or l-carnitine was administered while serum insulin was maintained at a physiologically high concentration during two randomized visits. PARTICIPANTS: Seven healthy, nonvegetarian young men (body mass index, 26.1 +/- 1.6 kg/m2) participated in the present study at the University of Nottingham. MAIN OUTCOME MEASURES: Skeletal muscle pyruvate dehydrogenase complex (PDC) activity and associated muscle metabolites were measured. RESULTS: The combination of hypercarnitinemia (600 micromol/liter) and hyperinsulinemia (160 mU/liter) increased muscle TC content by 15% (P < 0.01) and was associated with decreased pyruvate dehydrogenase complex activity (P < 0.05) and muscle lactate content (P < 0.05) by 30 and 40%, respectively, and an overnight increase in muscle glycogen (P < 0.01) and long-chain acyl-coenzyme A content (P < 0.05) by 30 and 40%, respectively, compared with control. CONCLUSIONS: These results suggest that an acute increase in human skeletal muscle TC content results in an inhibition of carbohydrate oxidation in conditions of high carbohydrate availability, possibly due to a carnitine-mediated increase in fat oxidation. These novel findings may have important implications for our understanding of the regulation of muscle fat oxidation, particularly during exercise, when carnitine availability may limit fat oxidation, and in obesity and type 2 diabetes where it is known to be impaired.

Acyl Coenzyme A↗
PubMed2004

Metabolic inertia in contracting skeletal muscle: a novel approach for pharmacological intervention in peripheral vascular disease.

Peripheral vascular disease (PVD) is generally accepted to result in the failure of skeletal muscle blood flow to increase adequately at the onset of muscular work. There are currently no routine pharmacological interventions towards the treatment of PVD, however, recent Phase III trials in the USA have demonstrated the clinical potential of the phosphodiesterase III inhibitor Cilostazol for pain-free and maximal walking distances in patients with intermittent claudication. PVD is characterized by a marked reliance on oxygen-independent routes of ATP regeneration (phosphocreatine hydrolysis and glycolysis) in skeletal muscle during contraction and the rapid onset of muscular pain and fatigue. The accumulation of metabolic by-products of oxygen-independent ATP production (hydrogen and lactate ions and inorganic phosphate) has long been associated with an inhibition in contractile function in both healthy volunteers and PVD patients. Therefore, any strategy that could reduce the reliance upon ATP re-synthesis from oxygen-independent routes, and increase the contribution of oxygen-dependent (mitochondrial) ATP re-synthesis, particularly at the onset of exercise, might be expected to improve functional capacity and be of considerable therapeutic value. Historically, the increased contribution of oxygen-independent ATP re-synthesis to total ATP generation at the onset of exercise has been attributed to a lag in muscle blood flow limiting oxygen delivery during this period. However, recent evidence suggests that limited inertia is present at the level of oxygen delivery, whilst considerable inertia exists at the level of mitochondrial enzyme activation and substrate supply. In support of this latter hypothesis, we have reported on a number of occasions that activation of the pyruvate dehydrogenase complex, using pharmacological interventions, can markedly reduce the dependence on ATP re-synthesis from oxygen-independent routes at the onset of muscle contraction. This review will focus on these findings and will highlight the pyruvate dehydrogenase complex as a novel therapeutic target towards the treatment of peripheral vascular disease, or any other disease state where premature muscular fatigue is prevalent due to metabolite accumulation.

Acetyl Coenzyme A↗
PubMed2000

Starvation increases the amount of pyruvate dehydrogenase kinase in several mammalian tissues.

Covalent modification of the pyruvate dehydrogenase complex provides an important regulatory mechanism for controlling the disposal of glucose and other compounds metabolized to pyruvate. Regulation of the complex by this mechanism is achieved in part by tissue-specific expression of the genes encoding isoenzymes of pyruvate dehydrogenase kinase (PDK). Starvation is known from our previous work to increase PDK activity of heart and skeletal muscle by increasing the amount of PDK isoenzyme 4 (PDK4) present in these tissues. This study demonstrates that increased expression of both PDK4 and PDK2 occurs in rat liver, kidney, and lactating mammary gland in response to starvation. PDK4 and PDK2 message levels were also increased by starvation in the two tissues examined (liver and kidney), suggesting enhancement of gene transcription. Changes in PDK2 message and protein were of similar magnitude, but changes in PDK4 message were greater than those in PDK4 protein, suggesting regulation at the level of translation. In contrast to these tissues, starvation had little or no effect on PDK2 and PDK4 protein in brain, white adipose tissue, and brown adipose tissue. Nevertheless, PDK4 message levels were significantly increased in brain and white adipose tissue by starvation. The findings of this study indicate that increased expression of PDK isoenzymes is an important mechanism for bringing about inactivation of the pyruvate dehydrogenase complex during starvation in many but not all tissues of the body. The absence of this mechanism preserves the capacity of neuronal tissue to utilize glucose for energy during starvation.

Animals↗
PubMed2005

Electron cryotomography of the E. coli pyruvate and 2-oxoglutarate dehydrogenase complexes.

The E. coli pyruvate and 2-oxoglutarate dehydrogenases are two closely related, large complexes that exemplify a growing number of multiprotein "machines" whose domains have been studied extensively and modeled in atomic detail, but whose quaternary structures have remained unclear for lack of an effective imaging technology. Here, electron cryotomography was used to show that the E1 and E3 subunits of these complexes are flexibly tethered approximately 11 nm away from the E2 core. This result demonstrates unambiguously that electron cryotomography can reveal the relative positions of features as small as 80 kDa in individual complexes, elucidating quaternary structure and conformational flexibility.

Cryoelectron Microscopy↗
PubMed1984

Pyruvate dehydrogenase subcomplex with lipoamide dehydrogenase deficiency in a patient with lactic acidosis and branched chain ketoaciduria.

The overall and three component activities of pyruvate dehydrogenase complex were measured in the liver and muscle from a patient who died at 1.9 years with increased concentrations of serum lactate, alpha-ketoglutarate and branched chain amino acids. The component activities of both lipoate acetyltransferase and pyruvate dehydrogenase were similar to those of normal controls, but the overall pyruvate dehydrogenase complex activity was 11 to 30% of controls and lipoamide dehydrogenase activity was not detected. The overall activity was significantly increased by the addition of lipoamide dehydrogenase purified from human liver. Immunochemical studies carried out with antibody prepared against lipoamide dehydrogenase from rat liver, could detect no immunoreactive material in liver and muscle homogenates from the patient, suggesting that the deficiency of lipoamide dehydrogenase activity was due to the lack of enzyme protein.

Acetyltransferases↗
PubMed1986

Pigeon liver phosphoprotein phosphatase: an effective activator of pyruvate dehydrogenase in tissue homogenates.

A fluoride-insensitive, non-metal-requiring pyruvate dehydrogenase phosphatase has been purified 730-fold from pigeon liver acetone powder and proven to be a convenient reagent for studies of pyruvate dehydrogenase complex and its activation (phosphorylation) state in brain and other tissues. This phosphatase is a cytoplasmic enzyme (Mr = 80,000), and fits the functional definition of a type 1 phosphoprotein phosphatase. The pigeon liver phosphatase can be used to activate pyruvate dehydrogenase complex in vitro in brain and other crude tissue homogenates. Addition of the cytoplasmic pigeon liver phosphatase to a homogenate from rat or mouse brain frozen in situ activated pyruvate dehydrogenase to levels comparable to that found in ischemic brain. The fluoride insensitivity of this phosphatase was used to develop a convenient technique for stopping the pyruvate dehydrogenase activation state in situ in cultured skin fibroblasts and then fully activating the complex in vitro in 5 min. The use of this phosphatase as a reagent can facilitate the study of pyruvate dehydrogenase activation defects in mammalian tissues including cultured cells in normal and disease states.

Animals↗
PubMed1983

Energy utilization and pyruvate as determinants of pyruvate dehydrogenase in norepinephrine-stimulated heart.

The quantitative effects of norepinephrine (NE) on the active form of the cardiac pyruvate dehydrogenase complex (PDCa) and the rate of oxidative decarboxylation of pyruvate (MVPyr) were compared with those of pyruvate (Pyr). Isolated working guinea pig hearts metabolized pyruvate alone or in combination with alternative energy-providing substrates (ketone bodies, octanoate, glucose). NE produced proportional increases in PDCa and myocardial oxygen uptake (MVO2). Total PDC activity (PDC1) remained constant. The PDCa/PDC1 ratios in NE depleted hearts (reserpine pretreatment) compared well with those in hearts containing endogenous NE, provided myocardial substrate supply and MVO2 were also comparable. No evidence was obtained indicating that NE or perfusate Ca2+ can dissociate PDCa or MVPyr from MVO2, even in presence of the alternative cosubstrates. In contrast, 0.2-10 mM Pyr produced stepwise but only submaximum increases in PDCa and MVPyr, with MVO2 remaining constant. Thus, at all Pyr concentrations tested, NE stimulations of myocaridal energy utilization and MVO2 were followed by further increases in PDCa and MVPyr. Evidently, pyruvate, and particularly cellular respiration are important determinants in the regulation of cardiac PDC, also during adrenergic stimulation of the heart.

Animals↗
PubMed1984

Defects in citric acid cycle and the electron transport chain in progressive poliodystrophy.

We will present 8 children with progressive infantile or juvenile poliodystrophy (Alpers' disease), associated with a defect in pyruvate metabolism. Laboratory studies showed elevated levels of lactate in CSF and, in 4 children, elevated levels in serum. Histopathologic studies revealed lipid storage in liver and/or muscle tissue, sometimes myopathy with abnormal mitochondria and slight axonal degeneration in the peripheral nerve. Autopsy showed the characteristics of progressive poliodystrophy with degeneration and loss of neurons. Electron microscopy of cerebral cortex showed no mitochondrial abnormalities in neurons or astroglia. Biochemical studies in muscle and/or liver and/or cerebral tissue showed different deficiencies in pyruvate metabolism: in the pyruvate dehydrogenase complex, in the second part of the citric acid cycle (after the oxoglutarate dehydrogenase complex), in the NADH oxidation, in cytochrome aa3 and in pyruvate carboxylase.

Adolescent↗