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PubMed1977

Physiological role of yeasts NAD(P)+ and NADP+-linked aldehyde dehydrogenases.

The activity of NAD+ and NADP+-linked aldehyde dehydrogenases has been investigated in yeast cells grown under different conditions. As occurs in other dehydrogenase reactions the NAD(P)+-linked enzyme was strongly repressed in all hypoxic conditions; nervetheless, the NADP+-linked enzyme was active. The results suggest that the NAD(P)+ aldehyde dehydrogenase is involved in the oxidation of ethanol to acetyl-CoA, and that when the pyruvate dehydrogenase complex is repressed the NADP+-linked aldehyde dehydrogenase is operative as an alternative pathway from pyruvate to acetyl-CoA: pyruvate leads to acetaldehyde leads to acetate leads to acetyl-Coa. In these conditions the supply of NADPH is advantageous to the cellular economy for biosynthetic purposes. Short term adaptation experiments suggest that the regulation of the levels of the aldehyde dehydrogenase-NAD(P)+ takes place by the de novo synthesis of the enzyme.

Aldehyde Oxidoreductases↗
PubMed1987

Limited proteolysis and sequence analysis of the 2-oxo acid dehydrogenase complexes from Escherichia coli. Cleavage sites and domains in the dihydrolipoamide acyltransferase components.

The structures of the dihydrolipoamide acyltransferase (E2) components of the 2-oxo acid dehydrogenase complexes from Escherichia coli were investigated by limited proteolysis. Trypsin and Staphylococcus aureus V8 proteinase were used to excise the three lipoyl domains from the E2p component of the pyruvate dehydrogenase complex and the single lipoyl domain from the E2o component of the 2-oxoglutarate dehydrogenase complex. The principal sites of action of these enzymes on each E2 chain were determined by sequence analysis of the isolated lipoyl fragments and of the truncated E2p and E2o chains. Each of the numerous cleavage sites (12 in E2p, six in E2o) fell within similar segments of the E2 chains, namely stretches of polypeptide rich in alanine, proline and/or charged amino acids. These regions are clearly accessible to proteinases of Mr 24,000-28,000 and, on the basis of n.m.r. spectroscopy, some of them have previously been implicated in facilitating domain movements by virtue of their conformational flexibility. The limited proteolysis data suggest that E2p and E2o possess closer architectural similarities than would be predicted from inspection of their amino acid sequences. As a result of this work, an error was detected in the sequence of E2o inferred from the previously published sequence of the encoding gene, sucB. The relevant peptides from E2o were purified and sequenced by direct means; an amended sequence is presented.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1995

Purification and properties of the lipoate protein ligase of Escherichia coli.

Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg(2+)- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38,000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the Km values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.

Amino Acid Sequence↗
PubMed1990

Sjögren's syndrome and primary biliary cirrhosis: presence of autoantibodies to purified mitochondrial 2-OXO acid dehydrogenases.

Sjögren's syndrome is well known for the presence of antibodies directed at specific nuclear antigens. However, the presence of antibodies reacting with a variety of other self antigens, including antimitochondrial antibodies, has often been reported although their significance is unknown. Moreover, patients with Sjögren's syndrome have been occasionally reported to be concordant with primary biliary cirrhosis. To address this issue we studied in a group of 96 patients with Sjögren's syndrome the presence of autoantibodies to the dihydrolipoamide acetyltransferases of both pyruvate dehydrogenase and branch chain ketoacid dehydrogenase and to alpha-ketoglutarate dehydrogenase; these latter enzymes are the mitochondrial target antigens of primary biliary cirrhosis. We report that 7 of the 96 patients reacted with the mitochondrial antigens that are prominent in primary biliary cirrhosis. Moreover, in those patients showing reactivity with mitochondrial antigens, the autoantibodies were directed at the same immunodominant epitopes that have been previously characterized in primary biliary cirrhosis. One of the 7 positive patients was known to have primary biliary cirrhosis. We hypothesize that the remaining 6 patients are at clinical risk for the development of primary biliary cirrhosis and/or that abnormalities would be found on liver biopsy.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed2000

Inherent abnormalities in energy metabolism in Alzheimer disease. Interaction with cerebrovascular compromise.

Alzheimer disease (AD) is a form of the dementia syndrome. AD appears to have a variety of fundamental etiologies that lead to the neuropathological manifestations which define the disease. Patients who are at high risk to develop AD typically show impairments of cerebral metabolic rate in vivo even before they show any evidence of the clinical disease on neuropsychological, electrophysiological, and neuroimaging examinations. Therefore, impairment in energy metabolism in AD can not be attributed to loss of brain substance or to electrophysiological abnormalities. Among the characteristic abnormalities in the AD brain are deficiencies in several enzyme complexes which participate in the mitochondrial oxidation of substrates to yield energy. There include the pyruvate dehydrogenase complex (PDHC), the alpha-ketoglutarate dehydrogenase complex (KGDHC), and Complex IV of the electron transport chain (COX). The deficiency of KGDHC may be due to a mixture of causes including damage by free radicals and perhaps to genetic variation in the DLST gene encoding the core protein of this complex. Inherent impairment of glucose oxidation by the AD brain may reasonably be expected to interact synergistically with an impaired supply of oxygen and glucose to the AD brain, in causing brain damage. These considerations lead to the hypothesis that cerebrovascular compromise and inherent abnormalities in the brain's ability to oxidize substrates can interact to favor the development of AD, in individuals who are genetically predisposed to develop neuritic plaques.

Alzheimer Disease↗
PubMed1999

alpha-Lipoic acid treatment decreases serum lactate and pyruvate concentrations and improves glucose effectiveness in lean and obese patients with type 2 diabetes.

OBJECTIVE: We examined the effect of lipoic acid (LA), a cofactor of the pyruvate dehydrogenase complex (PDH), on insulin sensitivity (SI) and glucose effectiveness (SG) and on serum lactate and pyruvate levels after oral glucose tolerance tests (OGTTs) and modified frequently sampled intravenous glucose tolerance tests (FSIGTTs) in lean (n = 10) and obese (n = 10) patients with type 2 diabetes. RESEARCH DESIGN AND METHODS: FSIGTT data were analyzed by minimal modeling technique to determine SI and SG before and after oral treatment (600 mg, twice a day, for 4 weeks). Serum lactate and pyruvate levels of diabetic patients after glucose loading were compared with those of lean (n = 10) and obese (n = 10) healthy control subjects in which SI and SG were also determined from FSIGTT data. RESULTS: Fasting lactate and pyruvate levels were significantly increased in patients with type 2 diabetes. These metabolites did not exceed elevated fasting concentrations after glucose loading in lean patients with type 2 diabetes. However, a twofold increase of lactate and pyruvate levels was measured in obese diabetic patients. LA treatment was associated with increased SG in both diabetic groups (lean 1.28 +/- 0.14 to 1.93 +/- 0.13; obese 1.07 +/- 0.11 to 1.53 +/- 0.08 x 10(-2) min-1, P < 0.05). Higher SI and lower fasting glucose were measured in lean diabetic patients only (P < 0.05). Lactate and pyruvate before and after glucose loading were approximately 45% lower in lean and obese diabetic patients after LA treatment. CONCLUSIONS: Treatment of lean and obese diabetic patients with LA prevents hyperglycemia-induced increments of serum lactate and pyruvate levels and increases SG.

Blood Glucose↗
PubMed1988

Reduced activities of thiamine-dependent enzymes in the brains and peripheral tissues of patients with Alzheimer's disease.

A report of cell loss in the nucleus basalis of Meynert in patients with Wernicke-Korsakoff disease prompted the examination of thiamine pyrophosphate (TPP)-dependent enzymes in the brain and peripheral tissues of patients with Alzheimer's disease. In these brains, the activities of the 2-ketoglutarate dehydrogenase complex were reduced more than 75% and those of transketolase more than 45%. Decreases occurred in histologically damaged and in relatively undamaged areas. Small but statistically significant abnormalities of transketolase, but not of 2-ketoglutarate dehydrogenase complex, were identified in red blood cells and cultured fibroblasts. Previous studies have shown deficiencies in the brain and variable effects in peripheral tissues on another TPP-dependent enzyme--the pyruvate dehydrogenase complex. Activities of TPP-dependent enzymes appear to be deficient in the brain and perhaps in some peripheral tissues in patients with Alzheimer's disease.

Aged↗
PubMed1990

Mechanism of pyruvate inhibition of plant pyruvate dehydrogenase kinase and synergism with ADP.

The effects of various metabolites on pyruvate dehydrogenase (PDH) kinase-catalyzed inactivation of the pyruvate dehydrogenase complex (PDC) were studied in extracts of mitochondria purified from green leaf tissue of Pisum sativum L. Pyruvate was an uncompetitive inhibitor of PDH kinase with respect to ATP whereas ADP was a competitive inhibitor. In the absence of pyruvate a fivefold excess of ADP over ATP was required to inhibit PDH kinase, however, in the presence of pyruvate much lower ADP concentrations were required. Inhibition of PDH kinase by pyruvate and ADP was synergistic and the addition of ADP changed pyruvate from an uncompetitive inhibitor to a noncompetitive inhibitor. This result indicates that pyruvate acts as a "dead-end" inhibitor, binding to the PDH kinase-ADP reaction intermediate. Evidence is also presented that inhibition by pyruvate in the presence of thiamine pyrophosphate is due to the formation of hydroxyethyl thiamine pyrophosphate. The results are discussed in terms of the regulation of PDC activity by pyruvate and ADP during periods of increased demand for carbon skeleton biosynthesis by way of the tricarboxylic acid (TCA) cycle despite constraints imposed on TCA cycle flux by a high ATP/ADP ratio.

Adenosine Diphosphate↗
PubMed2005

Neonatal lactic acidosis, complex I/IV deficiency, and fetal cerebral disruption.

Cerebral developmental abnormalities occur in various inborn errors of metabolism including peroxisomal deficiencies, pyruvate dehydrogenase complex deficiency and others. Associations with abnormalities of the respiratory chain are rare. Here we report male and female siblings with microcephaly, a complex neuromigrational disorder including ependymal cysts, leptomeningeal and subcortical heterotopia, polymicrogyria, multifocal cerebral calcifications, agenesis of the corpus callosum, and spongiform changes in brainstem and cerebellum. Intractable lactic acidosis, causing death on the first day of life, was associated with severely reduced activities of complex I and complex IV. The neuropathological and biochemical findings are closely similar to those reported previously. The findings confirm a distinct genetic syndrome of disrupted brain development with TORCH-like calcifications, and a complex neuronal migration disorder associated with a multicomplex disorder of the respiratory chain.

Acidosis, Lactic↗
PubMed1982

Ammonium ions enhance the flow through the pyruvate dehydrogenase in Ehrlich ascites tumor cells.

The flow through pyruvate dehydrogenase was assayed in glycolysing cells by the evolution of 14CO2 from [1-14C] pyruvate. Parallel incubations were carried out in high bicarbonate buffer (25 mM) and in bicarbonate-free buffer. The activation of the complex by NH+4 was only observed in high bicarbonate buffer, because the dilution of labelled CO2 in the presence of an excess of bicarbonate enables the quantitative determination of labelled CO2 evolved from pyruvate in the decarboxylase step. In the bicarbonate-free buffer the activation of the complex was not observed, because the 14CO2 evolved from pyruvate was consumed by biosynthetic processes inside the cell. On the contrary in isolated hepatocytes the NH+4 activation of the pyruvate dehydrogenase was observed in both buffers. In Ehrlich ascites cells, in common with other mammalian tissues, pyruvate dehydrogenase activity was found to be inversely correlated to the intramitochondrial ATP/ADP ratio.

Adenine Nucleotides↗
PubMed1971

Utilisation de la centrifugation analytique pour la mesure, au sein d'un extrait brut d'E. coli, du coefficient de sédimentation du complexe de décarboxylation oxidative du pyruvate.

We have applied successfully the active enzyme analytical centrifugation method (A.E.C.) to the study of the well known pyruvate dehydrogenase complex (P.D.C.) in crude extracts from E. coli. It is not possible to study the complex in a spectrophotometer (through its dehydrogenase activity) due to NADH oxidase activities. The A.E.C. method, which overcomes this difficulty, can also be viewed as a refined spectrophotometric method.

Journal Article↗
PubMed1995

Diversity of the pyruvate dehydrogenase kinase gene family in humans.

Recent evidence from this laboratory indicates that at least two isoenzymic forms of pyruvate dehydrogenase kinase (PDK1 and PDK2) may be involved in the regulation of enzymatic activity of mammalian pyruvate dehydrogenase complex by phosphorylation (Popov, K.M., Kedishvili, N.Y., Zhao, Y., Gudi, R., and Harris, R.A. (1994) J. Biol. Chem. 269, 29720-29724). The present study was undertaken to further explore the diversity of the pyruvate dehydrogenase kinase gene family. Here we report the deduced amino acid sequences of three isoenzymic forms of PDK found in humans. In terms of their primary structures, two isoenzymes identified in humans correspond to rat PDK1 and PDK2, whereas a third gene (PDK3) encodes for a new isoenzyme that shares 68% and 67% of amino acid identities with PDK1 and PDK2, respectively. PDK3 cDNA expressed in Eschierichia coli directs the synthesis of a polypeptide with a molecular mass of approximately 45,000 Da that possesses catalytic activity toward kinase-depleted pyruvate dehydrogenase. PDK3 appears to have the highest specific activity among the three isoenzymes tested as recombinant proteins. Tissue distribution of all three isoenzymes of human PDK was characterized by Northern blot analysis. The highest amount of PDK2 mRNA was found in heart and skeletal muscle, the lowest amount in placenta and lung. Brain, kidney, pancreas, and liver expressed an intermediate amount of PDK2 (brain > kidney = pancreas > liver). The tissue distribution of PDK1 mRNA differs markedly from PDK2. The message for PDK1 was expressed predominantly in heart with only modest levels of expression in other tissues (skeletal muscle > liver > pancreas > brain > placenta = lung > kidney). In contrast to PDk1 and PDK2, which are expressed in all tissues tested, the message for PDK3 was found almost exclusively in heart and skeletal muscle, indicating that PDK3 may serve specialized functions characteristic of muscle tissues. In all tissues tested thus far, the level of expression of PDK2 mRNA was essentially higher than that of PDK1 and PDK3, consistent with the idea that PDK2 is a major isoenzyme responsible for regulation of pyruvate dehydrogenase in human tissues.

Amino Acid Sequence↗
PubMed2005

Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor.

The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression.

Animals↗
PubMed2005

Apparent presence of Ser133-phosphorylated cyclic AMP response element binding protein (pCREB) in brain mitochondria is due to cross-reactivity of pCREB antibodies with pyruvate dehydrogenase.

Cyclic AMP response element binding protein (CREB) is a constitutive transcription factor that activates transcription following stimulus-dependent phosphorylation at Ser133, implicated in synaptic plasticity and neuronal survival pathways. The prevailing view that CREB is exclusively nuclear has been questioned by several studies, and, for example, mitochondrial localization has been reported. Using subcellular fractionation of rat brain cortex coupled with western immunoblotting with Ser133-phospho-CREB (pCREB) antibodies, we found a robust pCREB immunoreactivity (IR) in mitochondria-enriched fractions. The pCREB antibodies also stained the mitochondria, in addition to nuclei, of glial cells in primary cortical cultures. However, two CREB antibodies against different epitopes and gel shift assay detected the CREB protein mainly in the nuclear fraction. The two-dimensional electrophoretic mobility of mitochondrial pCREB IR differed markedly from the nuclear CREB/pCREB IR, indicating that the pCREB antibody cross-reacts with another mitochondrial protein. Immunoprecipitation of the mitochondrial pCREB IR produced three bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry as E2, E1 alpha-subunit, and E1 beta-subunit of pyruvate dehydrogenase complex. The cross-reacting epitope was identified as phospho-Ser300 of the alpha-subunit. In conclusion, this study confirms the presence of pCREB-like IR in brain mitochondria that, after careful scrutiny, turned out to be pyruvate dehydrogenase rather than authentic CREB.

Adenosine Triphosphate↗
PubMed2004

Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria--case studies.

Oxidative phosphorylation analysis, performed on freshly-isolated mitochondria, assesses the integrated function of the electron transport chain (ETC) coupled to ATP synthesis, membrane transport, dehydrogenase activities, and the structural integrity of the mitochondria. In this review, a case study approach is employed to highlight detection of defects in the adenine nucleotide translocator, the pyruvate dehydrogenase complex, fumarase, coenzyme Q function, fatty acid metabolism, and mitochondrial membrane integrity. Our approach uses the substrates glutamate, pyruvate, 2-ketoglutarate (coupled with malonate), malate, and fatty acid substrates (palmitoylcarnitine, octanoylcarnitine, palmitoyl-CoA (with carnitine), octanoyl-CoA (with carnitine), octanoate and acetylcarnitine) in addition to succinate, durohydroquinone and TMPD/ascorbate to uncover metabolic defects that would not be apparent from ETC assays performed on detergent-solubilized mitochondria.

Journal Article↗
PubMed2003

Starvation and diabetes reduce the amount of pyruvate dehydrogenase phosphatase in rat heart and kidney.

The pyruvate dehydrogenase complex (PDC) is inactivated in many tissues during starvation and diabetes to conserve three-carbon compounds for gluconeogenesis. This is achieved by an increase in the extent of PDC phosphorylation caused in part by increased pyruvate dehydrogenase kinase (PDK) activity due to increased PDK expression. This study examined whether altered pyruvate dehydrogenase phosphatase (PDP) expression also contributes to changes in the phosphorylation state of PDC during starvation and diabetes. Of the two PDP isoforms expressed in mammalian tissues, the Ca(2+)-sensitive isoform (PDP1) is highly expressed in rat heart, brain, and testis and is detectable but less abundant in rat muscle, lung, kidney, liver, and spleen. The Ca(2+)-insensitive isoform (PDP2) is abundant in rat kidney, liver, heart, and brain and is detectable in spleen and lung. Starvation and streptozotocin-induced diabetes cause decreases in PDP2 mRNA abundance, PDP2 protein amount, and PDP activity in rat heart and kidney. Refeeding and insulin treatment effectively reversed these effects of starvation and diabetes, respectively. These findings indicate that opposite changes in expression of specific PDK and PDP isoenzymes contribute to hyperphosphorylation and therefore inactivation of the PDC in heart and kidney during starvation and diabetes.

Animals↗
PubMed1978

Purification and characterization of branched chain alpha-keto acid dehydrogenase complex of bovine kidney.

A branched chain alpha-keto acid dehydrogenase-dihydrolipoyl transacylase complex was purified to apparent homogeneity from bovine kidney mitochondria. As usually isolated, the complex (s(20,w) = 40 S) contained little, if any, dihydrolipoyl dehydrogenase. When saturated with the latter enzyme the complex had a specific activity of about 12 mumol of alpha-ketoisovalerate oxidized per min per mg of protein at 30 degrees with NAD(+) as electron acceptor. In addition to alpha-ketoisovalerate, the complex also oxidized alpha-ketoisocaproate, alpha-keto-beta-methylvalerate, alpha-ketobutyrate, and pyruvate. The ratios of the specific activities were 2.0:1.5:1.0:1.0:0.4, and the apparent K(m) values were 40, 50, 37, 56, and 1000 muM. The complex was separated into its component enzymes. The branched chain alpha-keto acid dehydrogenase (6 S) consists of two different subunits with estimated molecular weights of 46,000 and 35,000. The dihydrolipoyl transacylase (20 S) contains apparently identical subunits of molecular weight about 52,000. In the electron microscope, the transacylase has the appearance of a cube, and the molecules of branched chain alpha-keto acid dehydrogenase appear to be distributed on the surface of the cube. In contrast to the pyruvate dehydrogenase complex of bovine kidney, the branched chain alpha-keto acid dehydrogenase complex apparently is not regulated by phosphorylation-dephosphorylation. Its activity, however, is subject to modulation by end-product inhibition.

Electrophoresis, Polyacrylamide Gel↗
PubMed2006

Decreased activities of mitochondrial respiratory chain complexes in non-mitochondrial respiratory chain diseases.

The aim of this study was to illustrate the difficulties in establishing a diagnosis of mitochondrial respiratory chain (MRC) disorders based on clinical grounds in combination with intermediate activities of the MRC enzyme complexes. We reviewed retrospectively all medical and laboratory records of patients initially considered likely to have MRC disorders on clinical grounds, and subsequently diagnosed with other disorders (n = 20; 11 males, 9 females). Data were retrieved from hospital records, referral letters, and results of enzymatic analysis at a reference laboratory. Clinical symptoms included developmental delay, epilepsy, hypotonia, movement disorder, spastic quadriplegia, tetany, microcephaly, visual problems, carpopedal spasms, dysmorphism, hearing loss, muscle weakness and rhabdomyolysis, and fulminant hepatitis. Blood and cerebrospinal fluid lactate levels were elevated in 13/20 and 9/20 respectively. One or more MRC complex activities (expressed as ratios relative to citrate synthase and/or complex II activity) were less than 50% of control mean activity in 11/20 patients (including patients with deficiencies of pyruvate dehydrogenase complex, pantothenate kinase, holocarboxylase synthetase, long-chain hydroxy acyl-CoA dehydrogenase, molybdenum co-factor, and neonatal haemochromatosis). One patient had a pattern suggestive of mitochondrial proliferation. We conclude that intermediate results of MRC enzymes should be interpreted with caution and clinicians should be actively looking for other underlying diagnoses.

Child↗