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PubMed2000

Symbiotic induction of pyruvate dehydrogenase genes from Sinorhizobium meliloti.

Genes coding for components of the pyruvate dehydrogenase (PDH) multienzyme complex (PDHc) from Sinorhizobium meliloti, the alfalfa symbiont, have been isolated on the basis of their high expression in symbiotic bacteria. The Elp component, PDH, is encoded by two genes, pdhAalpha (1,047 bp) and pdhAbeta (1,383 bp), a situation encountered in the alpha-proteobacteria Rickettsia prowazekii and Zymomonas mobilis as well as in some gram-positive bacteria and in mitochondria. pdhAalpha and pdhAbeta precede pdhB (1,344 bp), which encodes the E2p component, dihydrolipoamide acetyltransferase, of the PDHc. No gene encoding the E3 component, lipoamide dehydrogenase, was found in the immediate vicinity of pdhA and pdhB genes. pdhAalpha, pdhAbeta and pdhB likely constitute an operon. Here, we provide evidence that pdhA expression is induced in the symbiotic stage, compared with free-living conditions. We demonstrate that symbiotic expression of pdhA genes does not depend on the fix LJ regulatory cascade that regulates nitrogen fixation and respiration gene expression in symbiotic S. meliloti cells. Induction of pdhA expression could be obtained under free-living conditions upon the addition of pyruvate to the culture medium. Induction by pyruvate and symbiotic activation of pdh gene expression take place at the same promoter.

Amino Acid Sequence↗

PubMed1991

Mitochondrial pyruvate dehydrogenase complex subunits as autoantigens in human primary biliary cirrhosis.

Primary biliary cirrhosis is an evolutive and chronic human liver disease characterized by presence of antimitochondrial autoantibodies in the serum. We present the biochemical definition of these autoantigens and show that purified pyruvate dehydrogenase complex contains three of the major mitochondrial antigens of M2 type i.e. the E2 subunit (dihydrolipoamide acetyl transferase), the X subunit and the E1 alpha subunit (pyruvate dehydrogenase), by immunoblotting experiments and inhibition of enzyme activity with several types of serums.

Autoantigens↗

PubMed2001

Azorhizobium caulinodans pyruvate dehydrogenase activity is dispensable for aerobic but required for microaerobic growth.

Azorhizobium caulinodans mutant 62004 carries a null allele of pdhB, encoding the E1beta subunit of pyruvate dehydrogenase, which converts pyruvate to acetyl-CoA. This pdhB mutant completely lacks pyruvate oxidation activities yet grows aerobically on C(4) dicarboxylates (succinate, L-malate) as sole energy source, albeit slowly, and displays pleiotropic growth defects consistent with physiological acetyl-CoA limitation. Temperature-sensitive (ts), conditional-lethal derivatives of the pdhB mutant lack (methyl)malonate semialdehyde dehydrogenase activity, which thus also allows L-malate conversion to acetyl-CoA. The pdhB mutant remains able to fix N(2) in aerobic culture, but is unable to fix N(2) in symbiosis with host Sesbania rostrata plants and cannot grow microaerobically. In culture, A. caulinodans wild-type can use acetate, beta-D-hydroxybutyrate and nicotinate--all direct precursors of acetyl-CoA--as sole C and energy source for aerobic, but not microaerobic growth. Paradoxically, acetyl-CoA is thus a required intermediate for microaerobic oxidative energy transduction while not itself oxidized. Accordingly, A. caulinodans energy transduction under aerobic and microaerobic conditions is qualitatively different.

Acetyl Coenzyme A↗

PubMed1995

Skeletal muscle pyruvate dehydrogenase activity during maximal exercise in humans.

The regulation of the active form of pyruvate dehydrogenase (PDHa) and related metabolic events were examined in human skeletal muscle during repeated bouts of maximum exercise. Seven subjects completed three consecutive 30-s bouts of maximum isokinetic cycling, separated by 4 min of recovery. Biopsies of the vastus lateralis were taken before and immediately after each bout. PDHa increased from 0.45 +/- 0.15 to 2.96 +/- 0.38, 1.10 +/- 0.11 to 2.91 +/- 0.11, and 1.28 +/- 0.18 to 2.82 +/- 0.32 mmol.min-1.kg wet wt-1 during bouts 1, 2, and 3, respectively. Glycolytic flux was 13-fold greater than PDHa in bouts 1 and 2 and 4-fold greater during bout 3. This discrepancy between the rate of pyruvate production and oxidation resulted in substantial lactate accumulation to 89.5 +/- 11.6 in bout 1, 130.8 +/- 13.8 in bout 2, and 106.6 +/- 10.1 mmol/kg dry wt in bout 3. These events coincided with an increase in the mitochondrial oxidation state, as reflected by a fall in mitochondrial NADH/NAD, indicating that muscle lactate production during exercise was not an O2-dependent process in our subjects. During exercise the primary factor regulating PDHa transformation was probably intracellular Ca2+. In contrast, the primary regulatory factors causing greater PDHa during recovery were lower ATP/ADP and NADH/NAD and increased concentrations of pyruvate and H+. Greater PDHa during recovery facilitated continued oxidation of the lactate load between exercise bouts.

Adenine Nucleotides↗

PubMed1996

Protein-protein interactions in the pyruvate dehydrogenase multienzyme complex: dihydrolipoamide dehydrogenase complexed with the binding domain of dihydrolipoamide acetyltransferase.

BACKGROUND: The ubiquitous pyruvate dehydrogenase multienzyme complex is built around an octahedral or icosahedral core of dihydrolipoamide acetyltransferase (E2) chains, to which multiple copies of pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) bind tightly but non-covalently. E2 is a flexible multidomain protein that mediates interactions with E1 and E3 through a remarkably small binding domain (E2BD). RESULTS: In the Bacillus stearothermophilus complex, the E2 core is an icosahedral assembly of 60 E2 chains. The crystal structure of the E3 dimer (101 kDa) complexed with E2BD (4 kDa) has been solved to 2.6 A resolution. Interactions between E3 and E2BD are dominated by an electrostatic zipper formed by Arg135 and Arg139 in the N-terminal helix of E2BD and Asp344 and Glu431 of one of the monomers of E3. E2BD interacts with both E3 monomers, but the binding site is located close to the twofold axis. Thus, in agreement with earlier biochemical results, it is impossible for two molecules of E2BD to bind simultaneously to one E3 dimer. CONCLUSIONS: Combining this new structure for the E3-E2BD complex with previously determined structures of the E2 catalytic domain and the E2 lipoyl domain creates a model of the E2 core showing how the lipoyl domain can move between the active sites of E2 and E3 in the multienzyme complex.

Acetyltransferases↗

PubMed1987

Accelerated loss of lean body mass in fasting rats due to activation of pyruvate dehydrogenase by dichloroacetate.

In order to test the hypothesis that increased pyruvate dehydrogenase (PDH) activity during fasting will result in accelerated loss of lean body mass, we administered sodium dichloroacetate (DCA) intraperitoneally to eight rats during the last three days of a six-day fast, while fasting control rats were given normal saline. DCA treatment resulted in an increased proportion of PDH in the active form in liver (26.5 +/- 4.3% v 13.4 +/- 0.5%, P less than 0.01). During the three-day period of administration, DCA treated rats lost more weight than control animals (42 +/- 2 v 25 +/- 1 g, P less than 0.001) and excreted more nitrogen in the urine (18.1 +/- 1.0 v 6.8 +/- 0.6 mmol/d, P less than 0.001). Calculations from nitrogen balance data suggest that 85% of the increase in weight loss of DCA treated rats over that of control animals was attributable to loss of lean body mass. We conclude that increased flux of pyruvate through the PDH reaction in the DCA-treated animals resulted in increased protein catabolism.

Acetates↗

PubMed1989

Effect of starvation and insulin in vivo on the activity of the pyruvate dehydrogenase complex in rat skeletal muscles.

The in vivo responses of pyruvate dehydrogenase (PDH) complex to starvation and insulin was assessed in heart, diaphragm and red quadriceps muscle. PDH complex activity was decreased by starvation (3.4-10.2-fold), the magnitude of change depending on muscle type. Insulin increased PDH activity in all muscle types. In fed rats, this effect was relatively small (1.25-1.29-fold). In starved rats there were effects in heart (4.3-fold) and red quadriceps (1.7-fold) but no effect in diaphragm. These results demonstrate that PDH complex in different groups of muscle has different insulin sensitivity (particularly in tissues from starved animals).

Adipose Tissue, Brown↗

PubMed1990

Longer-term regulation of pyruvate dehydrogenase kinase in cultured rat cardiac myocytes.

The increased activity of pyruvate dehydrogenase (PDH) kinase induced in hearts of rats by starvation for 48 h was maintained following preparation of cardiac myocytes, and it was also maintained, though at a decreased level, after 25 h of culture in medium 199. This loss of PDH kinase activity was not prevented by n-octanoate, dibutyryl cyclic AMP or glucagon. The PDH kinase activity of myocytes from fed rats was increased to that of starved rats after 25 h of culture with n-octanoate, dibutyryl cyclic AMP or both agents together.

Animals↗

PubMed1992

Incorporation of the enantiomers of lipoic acid into the pyruvate dehydrogenase complex from Escherichia coli in vivo.

The uptake of 35S-labelled enantiomers of lipoic acid into cells from Escherichia coli was studied. The R-enantiomer was taken up by a factor of two more efficiently than the S-form. Autoradiography of polyacrylamide gels of partially purified pyruvate dehydrogenase complex from these cells showed that only the R-lipoic acid was covalently incorporated as a cofactor into the dihydrolipoamide acetyltransferase component of the pyruvate dehydrogenase complex.

Autoradiography↗

PubMed1977

The development of pyruvate dehydrogenase in the subhuman primate Macaca mulatta.

The development of total pyruvate dehydrogenase activity in the subhuman primate, Macaca mulatta, was investigated in brain, liver, kidney cortex, and skeletal and cardiac muscle. Newborn primates delivered prematurely at 135 days of gestation had only 32% of this enzyme activity present in the brain compared to the full-term animal. Full-term animals were similar to adults. All tissues showed high activities of enzyme at 1 year of age compared either with neonates or adults. Kidney cortex showed an increase in activity from 807 +/- 74 mumol/g/min in prematurely delivered neonates to a maximum of 3,769 +/- 275 mumol/g/min in 1-year-old animals. The results are discussed in relation to the developing energy requirements of the tissues concerned.

Age Factors↗

PubMed1985

An immunochemical study of the pyruvate dehydrogenase deficit in Alzheimer's disease brain.

The activity of the pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) was reduced to about 30% of control values in histologically unaffected occipital cortex of the brains of patients with Alzheimer's disease, as well as in histologically affected frontal cortex. In contrast, activity of another mitochondrial enzyme, glutamate dehydrogenase, was normal. Neither age nor time until postmortem study correlated significantly with PDHC activity in either Alzheimer or control samples, and PDHC was not inactivated significantly on incubation with homogenates of either Alzheimer or control brain. Antibodies against the highly purified bovine PDHC inhibited Alzheimer and control PDHC equally per unit of enzyme activity. Immunoblots also indicated that the PDHC antigens were not different in normal and Alzheimer brains. This antibody, however, inhibited Alzheimer PDHC more effectively than it did control PDHC, based on milligrams of protein, suggesting a reduced amount of normal PDHC protein. Other data suggest that the PDHC deficiency is related to mitochondrial damage and to impaired calcium homeostasis in Alzheimer nerve cells, which may then mediate a variety of other cellular impairments.

Aged↗

PubMed1993

The Regulation of Pyruvate Dehydrogenase Activity in Pea Leaf Mitochondria (The Effect of Respiration and Oxidative Phosphorylation).

The regulation of the pea (Pisum sativum) leaf mitochondrial pyruvate dehydrogenase complex by respiratory rate and oxidative phosphorylation has been investigated by measuring the respiratory activity, the redox poise of the quinone pool (Q-pool), and mitochondrial pyruvate dehydrogenase (mtPDC) activity under various metabolic conditions. It was found that, under state 4 conditions, mtPDC activity was unaffected by either the addition of succinate, 2-oxoglutarate, or glycine or the overall respiratory rate and redox poise of the Q-pool but was partially inhibited by NADH due to product inhibition. In the presence of ADP significant inactivation of PDC, which was sensitive to oligomycin, was observed with all substrates, apart from pyruvate, suggesting that inactivation was due to ATP formation. Inactivation of PDC by ADP addition was observed even in the presence of carboxyatractyloside, an inhibitor of the ATP/ADP translocator, suggesting that other mechanisms to facilitate the entry of adenylates, in addition to the adenylate carrier, must exist in plant mitochondria.

Journal Article↗

PubMed1993

Three-dimensional structure of the lipoyl domain from Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.

The structure of the lipoyl domain from the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been determined by means of nuclear magnetic resonance spectroscopy. A total of 452 nuclear Overhauser effect distance constraints and 76 dihedral angle restraints were employed as the input for the structure calculations, which were performed using a hybrid distance geometry-simulated annealing strategy and the programs DISGEO and X-PLOR. The overall structure of the lipoyl domain (residues 1 to 79 of the dihydrolipoamide acetyltransferase polypeptide chain) is that of a flattened eight-stranded beta-barrel folded around a core of well-defined hydrophobic residues. The lipoylation site, lysine 42, is located in the middle of a beta-turn, and the N and C-terminal residues of the domain are close together in adjacent beta-strands at the opposite end of the molecule. The polypeptide backbone exhibits a 2-fold axis of quasi-symmetry, with the C alpha atoms of residues 15 to 39 and 52 to 76 being almost superimposable on those of residues 52 to 76 and 15 to 39, respectively (root-mean-square deviation = 1.48 A). The amino acid residues at key positions in the structure are conserved among all the reported primary structures of lipoyl domains, suggesting that the domains all fold in a similar way.

Amino Acid Sequence↗

PubMed1995

Changes in the structure of pyruvate dehydrogenase complex induced by mono- and divalent ions.

The activity of pyruvate dehydrogenase complex purified from pig kidney cortex was affected by various mono- and di-valent ions and changes in ionic strength. The fluorescence emission spectrum of PDC exposed to 0.04 M ionic strength and excited at 280 nm exhibited a maximum at 334 nm; the fluorescence intensity of PDC appeared to depend upon the ionic strength and the K+ and Na+ content of the incubation buffer. Alteration of ionic strength to which the enzyme complex was exposed produced a change in the absorption of the complex at 230 nm. The presence of HPO4(2-) ions prevented changes in the UV absorption spectrum of PDC induced by the variation in ionic strength. The K+ and Na+ ions alone had no effect on the UV spectrum of PDC. Upon increasing the ionic strength to which the enzyme complex was exposed, dramatic changes in the circular dichroism (CD) pattern were observed. At 0.04 M ionic strength PDC exhibited a CD spectrum with minima at 216, 218 and 222 nm and a cross-over point at 215 nm. At 0.15 M ionic strength the CD spectrum of PDC exhibited minima at 223, 226, 228 nm and a cross-over point at 221 nm. The presence of HPO4(2-) ions prevented alterations in the CD spectrum of PDC induced by variations in ionic strength. The K+ and Na+ ions had no effect on the CD spectrum of PDC.

Animals↗

PubMedDate not supplied

Derangements of pyruvate dehydrogenase in circulating lymphocytes of NIDDM patients and their healthy offspring.

Pyruvate dehydrogenase (PDH) is poorly active in circulating lymphocytes of NIDDM patients; in vitro, it is unresponsive to insulin at 5 microU/ml and activated at 50 microU/ml, instead of activated and inhibited as in healthy controls. This study examines whether healthy offspring of NIDDM patients with a family history for this disease have these alterations. Twenty seven healthy offspring (23+/-10 yr, median 18 yr) and their parents (13 diabetic with a family history for NIDDM and 11 healthy without this history) were enrolled. Twenty healthy individuals without the history and matched for age and gender with the offspring served as controls. Minimum levels for enzyme activity before and after cell stimulation with insulin at 5 microU/ml were computed for a 95% CI with no more than 5% of the controls excluded. Increased or unvaried enzyme activity in response to insulin at 50 microU/ml was defined as abnormal. All NIDDM parents and 11/27 offspring had below normal enzyme activity and defective and reversed enzyme response to insulin at 5 and 50 microU/ml; three offspring had altered enzyme response to insulin at both concentrations, four to insulin at 5 microU/ml, three to insulin at 50 microU/ml and six, together with the healthy parents, had no alterations. We conclude that in healthy individuals a family history for NIDDM is frequently signaled, irrespective of age, by molecular derangements, with an apparent genetic background, in their circulating lymphocytes.

Adolescent↗

PubMed1993

Pyruvate dehydrogenase activity and acetyl group accumulation during exercise after different diets.

Pyruvate dehydrogenase activity (PDHa) and acetyl group accumulation were examined in human skeletal muscle at rest and during exercise after different diets. Five males cycled at 75% of maximal O2 uptake (VO2 max) to exhaustion after consuming a low-carbohydrate diet (LCD) for 3 days and again 1-2 wk later for the same duration after consuming a high-carbohydrate diet (HCD) for 3 days. Resting PDHa was lower after a LCD (0.20 +/- 0.04 vs. 0.69 +/- 0.05 mmol.min-1.kg wet wt-1; P < 0.05) and coincided with a greater intramuscular acetyl-CoA-to-CoASH ratio, acetyl-CoA content, and acetylcarnitine content. PDHa increased during exercise in both conditions but at a lower rate in the LCD condition compared with the HCD condition (1.46 +/- 0.25 vs. 2.65 +/- 0.23 mmol.min-1.kg wet wt-1 at 16 min and 1.88 +/- 0.20 vs. 3.11 +/- 0.14 at the end of exercise; P < 0.05). During exercise muscle acetyl-CoA and acetylcarnitine content and the acetyl-CoA-to-CoASH ratio decreased in the LCD condition but increased in the HCD condition. Under resting conditions PDHa was influenced by the availability of fat or carbohydrate fuels acting through changes in the acetyl-CoA-to-CoASH ratio. However, during exercise the activation of PDHa occurred independent of changes in the acetyl-CoA-to-CoASH ratio, suggesting that other factors are more important.

Acetyl Coenzyme A↗

PubMed1999

Dichloroacetate stabilizes the mutant E1alpha subunit in pyruvate dehydrogenase deficiency.

OBJECTIVE: To determine whether dichloroacetate (DCA) treatment can increase pyruvate dehydrogenase (PDH) activity in PDH-deficient cell lines harboring pathogenic mutations in the PDH E1alpha gene. BACKGROUND: PDH deficiency is a nuclear-encoded mitochondrial disorder and a major recognized cause of neonatal encephalomyopathies associated with primary lactic acidosis. Over the last decade, DCA has been used therapeutically, but it has not been clear which patients might benefit. Recent studies suggest that chronic DCA treatment may act by increasing the stability of mutant E1alpha polypeptide. The relative effects of DCA treatment on PDH-deficient cell lines with E1alpha mutations primarily affecting polypeptide stability or catalytic activity were determined and the mechanism of enhancement of residual PDH activity explored. METHODS: The effect of chronic 5-day DCA treatment on PDH activity was assessed in PDH-deficient cell lines containing the R378H, R141Q, K387(FS), and R302C E1alpha mutations. PDH subunit turnover and steady-state E1alpha levels before and after DCA treatment were measured in the R378H mutant line. RESULTS: Chronic DCA treatment resulted in 25% (p = 0.0434), 31% (p = 0.0014) increases in PDH activity in the K387(FS) and R378H cell lines, both of which are associated with decreased mutant polypeptide stability. In the R378H mutant cell line, chronic DCA treatment increased steady-state E1alpha levels and slowed the rate of E1alpha turnover twofold. In contrast, PDH activity did not change in the chronically DCA-treated R302C mutant line, in which the mutant polypeptide has normal stability and reduced catalytic activity. CONCLUSIONS: Chronic DCA treatment can increase PDH activity in PDH-deficient cell lines harboring mutations that affect E1alpha stability, suggesting a biochemical criterion by which DCA-responsive patients might be selected.

Cell Line↗

PubMed1990

Deficiency of the alpha and beta subunits of pyruvate dehydrogenase in a patient with lactic acidosis and unexpected sudden death.

An infant with moderate muscular hypotonia and congenital lactic acidosis died suddenly at the age of 3 months. Autopsy revealed no abnormalities responsible for this unexpected death. Measurement of mitochondrial enzymes involved in energy production indicated a severely decreased total pyruvate dehydrogenase complex (PDHC) activity in muscle tissue (0.23 nmoles x min-1 x mg protein-1, control range 2.8-8.7) and moderately decreased PDHC activity in fibroblasts (0.27 nmoles x min-1 x mg protein-1, control range 0.37-2.32). The activity of the first component E1 (pyruvate dehydrogenase) in muscle tissue was 10 times lower than that of controls (0.008 nmoles x min-1 x mg protein-1, control range 0.10-0.25). The activities of dihydrolipoyl dehydrogenase (E3) and various other mitochondrial enzymes were normal. Immunochemical analysis in skeletal muscle tissue and fibroblasts demonstrated a decrease in the amount of the alpha and beta subunits of E1. The features of this patient are compared with those of other patients reported in the literature with immunochemically confirmed combined E1 alpha and beta deficiency.

Acidosis, Lactic↗