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Results for “Pyruvate dehydrogenase”

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PubMed1991

Characteristic changes on brain CT in a case of Leigh encephalopathy with deficiency of pyruvate dehydrogenase.

A girl aged 23 months of Leigh encephalopathy with pyruvate dehydrogenase complex (PDHC) deficiency was reported. Brain CT scan showed atrophy of the frontal and parietal cortex, low density in the midbrain and putamina, and rounded caudatum. CT change was showed from rounded to atrophic caudatum during a three-month period. The rounded caudatum may be an early CT finding in Leigh encephalopathy.

Brain↗

PubMed1984

[Activation of pyruvate dehydrogenase by addition of insulin to a mixture of cell membranes and mitochondria from the brain of normal and alloxan treated rats. (Preliminary note)].

The effect of insulin and ATP on pyruvate dehydrogenase activity has been studied in a mixture of plasma membranes/mitochondria from normal and diabetic rat brain. In both enzymatic preparations ATP inhibits almost completely pyruvate dehydrogenase activity; insulin on the contrary induces a conspicuous activation and removes the inhibiting action of ATP.

Animals↗

PubMed1973

Effect of phenylpyruvate on pyruvate dehydrogenase activity in rat brain mitochondria.

1. The effects of phenylpyruvate, a metabolite produced in phenylketonuria, on the pyruvate dehydrogenase-complex activity were investigated in rat brain mitochondria. 2. Pyruvate dehydrogenase activity was measured by two methods, one measuring the release of (14)CO(2) from [1-(14)C]pyruvate and the other measuring the acetyl-CoA formed by means of the coupling enzyme, pigeon liver arylamine acetyltransferase (EC 2.3.1.5). In neither case was there significant inhibition of the pyruvate dehydrogenase complex by phenylpyruvate at concentrations below 2mm. 3. However, phenylpyruvate acted as a classical competitive inhibitor of the coupling enzyme arylamine acetyltransferase, with a K(i) of 100mum. 4. It was concluded that the inhibition of pyruvate dehydrogenase by phenylpyruvate is unlikely to be a primary enzyme defect in phenylketonuria.

Journal Article↗

PubMed1988

The effects of various anions and cations on the regulation of pyruvate dehydrogenase complex activity from pig kidney cortex.

The activity of pyruvate dehydrogenase complex (PDC) purified from pig kidney cortex was found to be affected by various uni- and bi-valent ions. At a constant strength of 0.13 M at pH 7.8, K+, Na+, Cl-, HCO3- and HPO4(2-) had significant effects on the activity of PDC: Na+, K+ and HPO4(2-) stimulated, but HCO3- and Cl- inhibited. The stimulatory effect of Na+ was mediated by a change in the Vmax. of PDC only, whereas K+ produced an increase in Vmax. and a change in the Hill coefficient (h). The extent of stimulation produced by HPO4(2-)4 on the activity of PDC was dependent on the concentrations of K+ and Na+. Both cations at concentrations higher than 40 mM partially prevented the effect of HPO4(2-)4. Cl- and HCO3- anions decreased the Vmax. of the enzyme and increased the S0.5 for pyruvate. The effects of Na+, K+, Cl-, HPO4(2-) and HCO3- on the activity of PDC were additive. In the presence of 80 mM-K+, 20 mM-Na+, 10 mM-HPO4(2-), 20 mM-Cl- and 20 mM-HCO3- the activity of PDC was increased by 30%, the S0.5 for pyruvate was increased from 75 to 158 microM and h was decreased from 1.3 to 1.1. Under these conditions and at 1.0 mM-pyruvate, the activity of PDC was 80% of the maximal activity achieved in the presence of these ions and 4.5 mM-pyruvate. The present study suggests that PDC may operate under non-saturating concentrations for substrate in vivo.

Animals↗

PubMed1977

Effects of arsenic on pyruvate dehydrogenase activation.

Our studies illuminate a particular site of altered pyruvate utilization by liver mitochondria isolated from arsenic-fed rats. Initially, pyruvate dehydrogenase (PDH) levels were measured before and after in vitro activation. The liver homogenates were prepared from male rats given access to deionized drinking water solutions containing 0, 20, 40, and 85 ppm arsenic as sodium arsenate (As+5) for 3 and 6 weeks. After 3 weeks, the effects of arsenic at the highest dose level were pronounced on the basal activity (before activation), with inhibition up to 48% of the control values. The total PDH (after activation) was inhibited by 14, 15, and 28% of the control values at 20, 40, and 85 ppm As+5, respectively. A similar pattern of inhibition of PDH was observed at 6 weeks, although the inhibition was lower at the highest dose. This effect is probably a reflection of mitochondrial regeneration at this time and dose. The inhibition of PDH both before and after activation suggests a direct arsenic effect on pyruvate utilization which does not involve a lipoic acid moiety. Evidence is also presented which indicates an arsenic effect on the regulating kinase and/or phosphatase. The metabolic effects of impaired mitochondrial utilization by pyruvate are also discussed.

Animals↗

PubMed2003

Immunoreactivity to pyruvate dehydrogenase complex-E2 in well-defined patients with autoimmune hepatitis: Western blot analysis.

Anti-mitochondrial antibodies (AMA) are frequently detected in sera from patients with primary biliary cirrhosis (PBC). Major autoantigens for AMA have been identified as members of the 2-oxoacid dehydrogenase enzyme complex family, with pyruvate dehydrogenase complex (PDC)-E2 showing strongest reactivity to AMA in PBC patients. Recently, anti-PDC-E2 has been found in patients with other diseases. Since frequency and significance of anti-PDC-E2 in patients with autoimmune hepatitis (AIH) remain obscure, we measured anti-PDC-E2 in sera from well-defined AIH cases by Western blotting using bovine heart mitochondrial protein and recombinant PDC-E2 protein as antigen sources. All 55 enrolled patients fulfilled the international diagnostic criteria for definite or probable AIH. Anti-PDC-E2 positivity showed concordance between native and recombinant antigens. Anti-PDC-E2 was detected in nine of 55 sera from AIH patients (16%). Variables including alkaline phosphatase (ALP) and IgM concentrations, effects of prednisolone, and pathologic findings concerning bile ducts showed no significant differences between anti-PDC-E2-positive and anti-PDC-E2-negative AIH patients. These data indicate that detection of anti-PDC-E2 is not rare in defined AIH, but anti-PDC-E2-positive AIH does not represent an intermediate entity in a clinical spectrum between AIH and PBC.

Journal Article↗

PubMed1988

Insulin modulation of pyruvate dehydrogenase in human circulating lymphocytes.

1. In human circulating lymphocytes pyruvate dehydrogenase (PDH) complex is present in the active (PDHa) and inactive (PDHi) forms. 2. PDHi conversion into PDHa is stimulated when intact lymphocytes are incubated with 5 microU/ml insulin at pH 7.4, for 15 min at 37 degrees C in a medium supplemented with 50 microM Ca2+-Mg2+. 3. The generation of a mediator is strongly suggestive since a cell free preparation from circulating lymphocytes, treated as above described, still stimulates PDHi----PDHa conversion, when combined with either disrupted or intact lymphocytes.

Calcium↗

PubMed1999

Cloning and characterization of a 5.9 kb promoter region of the human pyruvate dehydrogenase alpha subunit gene.

A human genomic clone containing a 5.9 kb promoter region of the human pyruvate dehydrogenase (E1) alpha subunit gene (PDHA1) was isolated from a human X-chromosome library. The nucleotide sequence showed two Alu repeats at the -2880 and -2200 bp regions. Comparison between the -1400 bp E1alpha promoter and the -1241 bp E1beta promoter revealed a 57% homology, with a high degree of homology at the putative protein binding regions in these two promoters. Computer-aided transcription factor binding consensus sequence analysis revealed the presence of PPAR, HOXD, MyoD and other tissue-specific transcription factor binding sites. Promoter function analysis using the chloramphenicol acetyltransferase reporter gene indicated that the -2.2 kb/-1.7 kb and -5.9 kb/-5.2 kb regions of the E1alpha promoter may possess negative regulatory elements which are likely to function in a tissue-specific manner.

Amino Acid Sequence↗

PubMed1996

Tissue-specific expression of the human pyruvate dehydrogenase alpha (Pdha-1)/chloramphenicol acetyltransferase fusion gene in transgenic mice.

A chimeric gene (-763/+33Pdha-1/CAT) containing -763/+33 nucleotides o f the human pyruvate dehydrogenase gene (Pdha-1) and the structural gene of chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. CAT activity was detected predominantly in the brain followed in decreasing order by adipose tissue, spleen, heart, kidney and liver. Developmental expression of CAT activity in the testes was similar to that of the endogenous Pdha-1 subunit expression in the testes. Dietary regulation of the transgene was comparable to the expression of endogenous pyruvate dehydrogenase complex in kidney and adipose tissue. Thus, the -763/+33 bp segment of the human Pdha-1 gene is transcriptionally active in vivo and can direct the expression of CAT in several tissues.

Adipose Tissue↗

PubMed2003

[Cloning and expressing the E2 subunit of pyruvate dehydrogenase complex].

OBJECTIVES: To construct the expression vector of the pyruvate dehydrogenase complex E2 subunit gene (PDC-E2). METHODS: The PDC-E2 gene was amplified from human lymphocytes with RT-PCR, and was cloned into pExSecI vector to induce the PDC-E2 expression. The products were identified with western blot and ELISA. RESULTS: The expression vector pExSecI/PDC-E2 was successfully constructed. The products could be identified by the specific self-antibodies in the sera from the primary biliary cirrhosis patients. CONCLUSION: High efficient expression vector of PDC-E2 lays the foundation for serum assay of primary biliary cirrhosis patients with prokaryotic expressing PDC-E2.

Cloning, Molecular↗

PubMed1986

Crossed affinity immunoelectrophoresis of the Escherichia coli pyruvate dehydrogenase complex.

The crossed immunoelectrophoretic pattern obtained with the intact pyruvate dehydrogenase complex of Escherichia coli can be modified when this technique is combined with affinity gel electrophoresis using reactive dyes coupled to agarose as ligands. The patterns that arise have been interpreted with respect to localization of the three component enzymes. This was realized by using antibodies with different specificity, active enzyme staining and E2-E3 subcomplex behaviour. Dissociation of E1 subunits occurs more easily than that of E3 but remains incomplete in this system. The free reactive Procion Blue-MX dyes tested inactivate the complex even at neutral pH. The dyes react with all three components but E3 (80%) and E2 (15-20%) retain part of their catalytic activity. Modification leads to an enhanced dissociation of E1.

Coloring Agents↗

PubMed2002

Synthesis of pyruvate dehydrogenase in Staphylococcus aureus is stimulated by osmotic stress.

The pyruvate dehydrogenase multienzyme complex (PDHC) was found to be upregulated by osmotic stress in the osmotolerant pathogen Staphylococcus aureus. Upregulation was detectable in the levels of both activity and protein and was judged to be about fourfold when sodium chloride was used to adjust the water activity (a(w)) of the growth medium to 0.94. The upregulation of the PDHC was also found to be humectant dependent and was greatest when impermeant, nonmetabolizable humectants were used to adjust a(w). Further experiments provided evidence that in addition to osmotic upregulation, the PDHC complex is also subject to catabolite repression, thus providing a possible explanation for the observation that high concentrations of carbohydrates are generally more inhibitory to the growth of this bacterial pathogen than are high concentrations of salts.

Culture Media↗

PubMed1999

First prenatal diagnosis of defects in the HsPDX1 gene encoding protein X, an additional lipoyl-containing subunit of the human pyruvate dehydrogenase complex.

We have previously reported a genetic study of a neonatal lactic acidosis linked to a pyruvate dehydrogenase complex deficiency due to the absence of the protein X subunit. This rare autosomal recessive disorder is associated with specific deletions in this polypeptide which is encoded by the HsPDX1 gene, located on chromosome 11p1.3. The pathology of the patient was considered to arise from a large homozygous deletion (78del85) found at the 5' end of the HsPDX1 coding sequence. Her heterozygous mother underwent prenatal diagnosis during a subsequent pregnancy. Chorionic villus samples were used for three independent studies: (1) normal levels of the protein X component of the PDH complex were detected by immunoblotting; (2) RT-PCR analysis showed no deletion at the 5' end of the cDNA but the presence of a distinct heterozygous deletion (965del59) at its 3' end inherited from the father; (3) haplotype analysis revealed the presence of the father's mutated allele and the mother's normal allele. It was concluded that the fetus was heterozygous for this separate 3' deletion, so, it was likely to be not affected. This study permitted us to characterize more precisely the genetic abnormalities of the HsPDX1 cDNA occurring in each family's member.

Blotting, Western↗

PubMed1996

Amrinone prevents the inhibition of muscle pyruvate dehydrogenase complex activity during sepsis.

A decreased proportion of active pyruvate dehydrogenase complex (PDH) in skeletal muscle has been implicated as an important factor in elevating plasma lactate concentrations in hypermetabolic sepsis. The mediators of the septic process responsible for the inhibition of PDH complex in muscle are unknown. To assess the role of tumor necrosis factor in mediating the effects of sepsis, the effect of daily injections of amrinone (5 mg/kg/day), which inhibits the release of tumor necrosis factor during sepsis, on the proportion of PDH in the active form (PDHa) was investigated in a model of chronic hypermetabolic sepsis. In skeletal muscle from untreated septic rats, PDHa was decreased 50%. Treatment of septic rats with amrinone for 5 days prevented the sepsis-induced decrease in PDHa. Sepsis caused a 2.5-fold elevation in plasma lactate concentrations. The maintenance of the PDH complex activity at control values following injection of amrinone in septic rats was associated with reduced lactate concentrations in plasma. Thus, amrinone prevented the sepsis-induced abnormalities in skeletal muscle PDH activity and plasma lactate concentrations.

Amrinone↗

PubMed1999

[Studies on brain pyruvate dehydrogenase (PDH) activity and energy metabolites during ischemia and reperfusion].

Pyruvate dehydrogenase is one of the mitochondrial enzymes considered important in the regulation of oxidative metabolism. To further understand the relationship between its activity and ischemic brain damage we conducted three experiments. We studied the effects of (1) duration of cerebral ischemia, (2) the Ca2+ channel blocker, nicardipine, and (3) the immunosuppressant, FK506, on PDH activity and energy metabolites during ischemia and reperfusion. In the first study we also measured regional cerebral blood flow (rCBF). (1) Increasing the duration of the ischemic insult delayed the deactivation of PDH, slowed the resynthesis of high energy phosphates and the clearance of lactate, and impaired recovery of rCBF. Additionally, (2) nicardipine normalized PDH activities and improved the impaired metabolism after reperfusion, and (3) FK506 did not effect PDH activity, but significantly improved the impaired metabolism during the early phase of reperfusion. From these studies we conclude that PDH plays a role in the recovery of metabolism during reperfusion, and both nicardipine and FK506 improve metabolism during the early phase of reperfusion.

Animals↗

PubMed1993

Characterization of PDH beta 1, the structural gene for the pyruvate dehydrogenase beta subunit from Saccharomyces cerevisiae.

The gene encoding the pyruvate dehydrogenase (PDH) beta subunit (E1 beta) of the PDH complex from Saccharomyces cerevisiae has been cloned, sequenced, disrupted, and expressed. Two overlapping DNA fragments were generated from a yeast genomic DNA library by the polymerase chain reaction with synthetic oligonucleotide primers based on amino acid sequences of the yeast and human E1 beta subunits. The DNA fragments were subcloned and sequenced. The composite sequence has an open reading frame of 1098 nucleotides encoding a putative presequence of 33 amino acid residues and a mature protein of 333 residues with a calculated M(r) = 36,486. Yeast and human E1 beta exhibit 62% sequence identity. The size of the mRNA is approximately 1.5 kilobases. Hybridization analysis showed that the E1 beta gene (PDH beta 1) is localized to chromosome II. Disruption of PDH beta 1 is not lethal under vegetative growth conditions. The null mutant transformed with PDH beta 1 on a unit-copy plasmid produced mature E1 beta and a functional PDH complex.

Amino Acid Sequence↗

PubMed1997

Biochemical and molecular analysis of an X-linked case of Leigh syndrome associated with thiamin-responsive pyruvate dehydrogenase deficiency.

We report molecular analysis of thiamin-responsive pyruvate dehydrogenase complex (PDHC) deficiency in a patient with an X-linked form of Leigh syndrome. PDHC activity in cultured lymphoblastoid cells of this patient and his asymptomatic mother were normal in the presence of a high thiamin pyrophosphate (TPP) concentration (0.4 mmol/L). However, in the presence of a low concentration (1 x 10(-4) mmol/L) of TPP, the activity was significantly decreased, indicating that PDHC deficiency in this patient was due to decreased affinity of PDHC for TPP. The patient's older brother also was diagnosed as PDHC deficiency with Leigh syndrome, suggesting that PDHC deficiency in these two brothers was not a de novo mutation. Sequencing of the X-linked PDHC E1 alpha subunit revealed a C-->G point mutation at nucleotide 787, resulting in a substitution of glycine for arginine 263. Restriction enzyme analysis of the E1 alpha gene revealed that the mother was a heterozygote, indicating that thiamin-responsive PDHC deficiency associated with Leigh syndrome due to this mutation is transmitted by X-linked inheritance.

DNA↗

PubMed2003

Conformational flexibility of pyruvate dehydrogenase complexes: a computational analysis by quantized elastic deformational model.

Pyruvate dehydrogenase complex (PDC) is one of the largest multienzyme complexes known and consists of a dodecahedral E2 core to which other components are attached. We report the results of applying a new computational method, quantized elastic deformational model, to simulating the conformational fluctuations of the truncated E2 core, using low-resolution electron cryomicroscopy density maps. The motional features are well reproduced; especially, the symmetric breathing mode revealed in simulation is nearly identical with what was observed experimentally. Structural details of the motions of the trimeric building blocks, which are critical to facilitating the global expansion and contraction of the complex, were revealed. Using the low-resolution maps from electron cryomicroscopy reconstructions, the simulations showed a picture of the motional mechanism of the PDC core, which is an example without precedent of thermally activated global dynamics. Moreover, the current results support an earlier suggestion that, at low resolution and without the use of amino acid sequence and atomic coordinates, it is possible for computer simulations to provide an accurate description of protein dynamics.

Acetyltransferases↗