DT diaphorase. III. Separation of mitochondrial DT diaphorase and respiratory chain.
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Dihydrolipoamide Dehydrogenase↗
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Estimation of pyruvate dehydrogenase (E1) activity in human skeletal muscle; three cases with E1 deficiency.
Pyruvate dehydrogenase (E1) catalyzes the rate-limiting step of the pyruvate dehydrogenase complex. Since E1 activity of human muscle tissue is low, a sensitive method is needed for diagnostic purposes. Measurement of 14CO2 production from [1-14C]pyruvate provides a specific and sensitive assay for measuring E1 activity. We use as artificial electron acceptor dichlorophenolindophenol (DCPIP) instead of the often applied ferricyanide. The method can be applied to small muscle samples obtained by needle or open biopsy. We prefer to use total homogenate because E1 activities in homogenate are higher than in the corresponding 600-g supernatant of skeletal muscle tissue. Control values in homogenate are higher or of the same order as those reported by others.
Adolescent↗
Isolation and characterisation of the mouse pyruvate dehydrogenase E1 alpha genes.
We have characterized two mouse genes that code for the E1 alpha subunit of pyruvate dehydrogenase (PDH), Pdha-1 and Pdha-2. The coding regions show a high degree of homology with each other and with the human PDH genes, PDAH1 and PDHA2. Conserved regions include mitochondrial import sequences, phosphorylation sites and a putative TPP binding site. The PDH genes have an analogous chromosomal arrangement to PGK genes in that two isoforms code for a functionally and structurally similar product. Pdha-1 codes for a somatic isoform and maps to the X-chromosome. Pdha-2 is located on an autosome, is intronless and only expressed in spermatogenic cells. Comparison of human and mouse PDH and PGK gene sequences shows that the somatic sequences are more conserved relative to the testis-specific isoforms, and that the mouse PDH E1 alpha genes have experienced a faster rate of DNA change compared to their human counterparts.
Amino Acid Sequence↗
Characterization of cDNAs encoding the rat testis-specific E1 alpha subunit of the pyruvate dehydrogenase complex: comparison of expression of the corresponding mRNA with that of the somatic E1 alpha subunit.
cDNA clones encoding the testis-specific form of the rat pyruvate dehydrogenase complex E1 alpha subunit have been isolated. Comparison of the predicted amino acid sequence with those of the somatic and testis-specific E1 alpha forms of man and mouse and the somatic E1 alpha form of rat indicates the change of a serine residue, believed to be phosphorylated in vivo by pyruvate dehydrogenase E1 alpha-specific kinase, to an alanine at position 233. The implications of this change are discussed. Northern blot analysis and RNase protection assays indicate that the expression of mRNA encoding testis-specific E1 alpha subunit is restricted to testis whereas mRNA for the somatic form is found in all tissues analyzed, albeit in very small amounts in testis.
Amino Acid Sequence↗
An immunochemical assay model system for the sensitive detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit pyruvate dehydrogenase (E1).
An immunochemical enzyme immunoassay model system was developed and compared for maximum sensitivity with a radioimmunoassay method and the classic enzyme activity method for the detection of pyruvate dehydrogenase complex (PDHc) and its decarboxylating subunit, pyruvate dehydrogenase (E1), isolated from Escherichia coli. Cross-linked large molecular weight antibody-enzyme conjugate systems are compared with heterobifunctional singular antibody conjugates substituted with high levels of horseradish peroxidase. Both polyclonal and monoclonal antibodies generated to the Escherichia coli PDHc and E1 antigens were used to develop a double-antibody sandwich microtiter plate enzyme-linked immunosorbent assay. It is demonstrated that a double sandwich immunochemical assay system can be quantitative for PDHc, can detect PDHc in crude cell lysates and has levels of sensitivity of 2.0.10(-16) mol for the detection of PDHc. This assay model system provides specific antibody selection criteria and coupling methods needed to select specific antisera that cross-react with human PDHc. This rapid and sensitive immunochemical assay method clearly demonstrates that sensitive mass assay systems can be developed for the detection of PDHc. Different from Western blot, this methodology could be used to generate mass assays which could be applied to the rapid detection of mammalian antigens (employing the corresponding antibodies) implicated in a number of pyruvate dehydrogenase deficiencies associated with human disorders.
Antibodies, Monoclonal↗
LACTATE METABOLISM BY PEPTOSTREPTOCOCCUS ELSDENII: EVIDENCE FOR LACTYL COENZYME A DEHYDRASE.
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Acrylic Resins↗
Antisense and sense poly(A)-RNAs from the Xenopus laevis pyruvate dehydrogenase gene loci are regulated with message production during embryogenesis.
The structure and temporal expression of two Xenopus cDNAs encoding the beta subunit of pyruvate dehydrogenase (XPdhE1 beta) have been determined. XPdhE1 beta was 88% homologous to mature human PdhE1 beta, but the putative N-terminal mitochondrial signal peptide was poorly conserved. Zygotic expression of XPdhE1 beta mRNA was detected at neural tube closure and increased until stage 40. RT-PCR cloning identified a short homology to a protein kinase open reading frame within the 3' non-coding sequence of the XPdhE1 beta cDNAs. This homology, which occurred on the antisense cDNA strand, was shown by strand specific RT-PCR to be transcribed in vivo as part of an antisense RNA. Northern analysis showed that this RNA formed part of an abundant and heterogeneous population of antisense and sense poly(A)-RNAs transcribed from the XPdhE1 beta loci and coordinately regulated with message production.
Amino Acid Sequence↗
Pyruvate dehydrogenase deficiency: clinical and biochemical diagnosis.
A female neonate with pyruvate dehydrogenase (PDH) deficiency is presented with clinical, radiologic, biochemical, neuropathologic, and molecular genetic data. She was dysmorphic, with a high forehead, lowset ears, thin upper lip, upturned nose, and rhizomelic limbs. Cranial MRI revealed severe cortical atrophy, ventricular dilatation, and corpus callosum agenesis. Pyruvate and lactate levels were increased in CSF and blood. Urinary organic acid profile was compatible with PDH deficiency. PDH activity was normal in fibroblasts, lymphocytes, and muscle. The PDH E1-alpha gene was sequenced and a single base mutation was found within the regulatory phosphorylation site in exon 10. It is postulated that this mutation causes a cerebral form of PDH deficiency. Tissue-specific expression of the disease could be explained by differential X chromosome inactivation because the PDH E1-alpha gene is located on this chromosome. Dysmorphism with severe cerebral malformations in female patients merits a metabolic evaluation, including determination of lactate and pyruvate levels in CSF.
Brain↗
Females with PDHA1 gene mutations: a diagnostic challenge.
Biochemical analysis was performed in muscle tissue and in fibroblasts of four unrelated females consecutively diagnosed with a 'de novo' point mutation in the PDHA1 gene. Pyruvate dehydrogenase E1 subunit deficiency was confirmed in the muscle sample of all patients, however, in three out of four cases the activity of the pyruvate dehydrogenase complex in fibroblasts showed a normal activity. A skewed inactivation was confirmed of the maternal X chromosome in fibroblasts in all children. Due to the possibility of a skewed X inactivation pattern enzyme measurements in fibroblasts are not always reliable for the diagnosis of a PDHc defect in females. Based on the overlapping features of PDHc deficiency with those of the disorders of the oxidative phosphorylation we suggest performing a fresh muscle biopsy for detailed biochemical analysis in females with a suspected pyruvate dehydrogenase deficiency, followed by molecular genetic analysis of the PDHA1 gene.
Adolescent↗
First characterization of a large deletion of the PDHA 1 gene.
Pyruvate dehydrogenase complex (PDC) deficiency is one of the major recognized causes of congenital lactic acidosis. The most common form is due to PDHA 1 gene (Xp22.12) defects. Here, we report the case of a Polynesian girl presenting with delayed neurological development, cortical atrophy, and posterior corpus callosum agenesis. Elevated lactate and pyruvate levels in blood and cerebrospinal fluid suggested PDC deficiency. However, PDC activity was within the normal range in lymphocytes and the direct sequencing of the 11 exons and intron-exon junctions of the PDHA 1 gene did not show any changes. Long-range PCR amplification of the whole gene (16 kb) from blood DNA revealed a heterozygous deletion of approximately 4.2kb. Fine mapping of the deletion breakpoint was achieved using purified long-range PCR products for restriction enzyme analysis and direct sequencing. The deletion removed a 4,227 bp region covering part of intron 5 to part of intron 9 [g.10,145_14,371 del 4,227]. The deletion breakpoint contained a short direct repeat (GTAG), which may be derived either from the upstream or the downstream homologous sequence. The presence of a GAG triplet and inverted repeats in the vicinity of the deletion suggest replication slippage at a polymerase alpha arrest site. This is the first time that a large intragenic deletion of the PDHA 1 gene has been characterized.
Base Sequence↗
Effects of bezafibrate on beta-cell function of rat pancreatic islets.
Bezafibrate is an activator of peroxisome proliferator-activated receptors (PPAR) alpha. The present study was performed to investigate the effects of bezafibrate and the PPAR alpha activator, 4-Cholro-6-(2.3-xylidino)-2-pyrimidin-ylthio acetic acid (WY14643), on the beta-cell function of rat pancreatic islets in vitro. In islets cultured with 300 microM bezafibrate or WY14643 for 8 h, a low glucose concentration induced insulin release and increased the levels of mRNA for PPAR alpha, acyl CoA oxidase, carnitine palmitoyl transferase-1, pyruvate dehydrogenase E1 alpha or pyruvate carboxylase. In contrast, after a 48-h culture period, a high glucose concentration induced insulin release and islet insulin content, but decreased the levels of mRNA for glucose transporter-2 (GLUT-2), preproinsulin or pancreatic/duodenal homeobox-1. Diazoxide, the KATP channel opener, restored these responses. We conclude that bezafibrate enhances insulin release through the activation of PPAR alpha gene expression during a short culture period, whereas it may contribute to beta-cell dysfunction through the mechanism of "excessive stimulation" during longer culture periods.
Animals↗
Identification of the catalytic glutamate in the E1 component of human pyruvate dehydrogenase.
The pyruvate dehydrogenase complex catalyzes the conversion of pyruvate to acetyl-CoA. The first component (E1) converts pyruvate to bound acetaldehyde using thiamine diphosphate (ThDP) and Mg2+ as cofactors. There is no 3D structure of E1 available but those of other ThDP-dependent enzymes show some similarities including a glutamate residue that assists in ThDP activation. Eukaryotic E1 has an alpha2beta2 structure and the conserved Glu89 of the beta-subunit was identified as a possible catalytic residue by sequence alignment. Human E1 was expressed in Escherichia coli and purified. Mutating Glu89 to glutamine, aspartate and alanine markedly reduces catalytic activity and the affinity for ThDP, consistent with a role as the catalytic glutamate.
Amino Acid Sequence↗
Defect in the lipoyl-bearing protein X subunit of the pyruvate dehydrogenase complex in two patients with encephalomyelopathy.
Among the many metabolic encephalomyelopathies caused by deficiencies in the pyruvate dehydrogenase complex (PDHC), nearly all involve its E1 subunit. We describe two new familial cases of PDHC deficiency with encephalomyelopathy, chronic lactic acidemia, and a normal E1 subunit of PDHC but deficiency in another component. Activity of PDHC was measured in cultured skin fibroblasts and skeletal muscle, and immunoblot studies were performed on mitochondrial extracts from skin fibroblasts. Spectra of muscle tissue, obtained in vivo with phosphorus 31 nuclear magnetic resonance, were recorded both at rest and with exercise. The PDHC activity was markedly reduced to 10% to 20% of normal values in both cultured skin fibroblasts and skeletal muscle. Immunoblotting of skin fibroblast mitochondrial extracts showed a specific deficiency in the protein X component of PDHC but normal E1, E2, and E3 components. Spectra obtained with 31P nuclear magnetic resonance showed alterations compatible with those found in mitochondrial myopathies. This is the second description of an encephalomyelopathy associated with a specific absence of the lipoyl-containing protein X component, which has a structural role in the formation of a functional PDHC.
Child, Preschool↗
Diagnosis and molecular analysis of three male patients with thiamine-responsive pyruvate dehydrogenase complex deficiency.
Pyruvate dehydrogenase complex (PDHC) deficiency is a major cause of congenital lactic acidemia in children. PDHC catalyzes the thiamine-dependent decarboxylation of pyruvate. Thiamine treatment was effective for some patients with PDHC deficiency. We reexamined 30 patients with congenital lactic acidemia of unknown origin who had normal PDHC activity in their cultured fibroblasts using a routine assay with a high (0.4 mM) thiamine pyrophosphate (TPP) concentration. We measured the activity of PDHC in the presence of a low (1x10(-4) mM) TPP concentration, and analyzed for mutations in the E1alpha subunit gene. Three males had low PDHC activity in the presence of 1x10(-4) mM TPP. The DNA sequence of these three patients' X-linked E1alpha subunit revealed a substitution of alanine for valine at position 71 (V71A) in exon 3, phenylalanine for cysteine at position 101 (C101F) in exon 4, and glycine for arginine at position 263 (R263G) in exon 8, respectively. Thiamine treatment was effective in these three patients. Therefore, they had a thiamine-responsive PDHC deficiency due to a point mutation in the E1alpha subunit gene. PDHC activity should be measured at a low TPP concentration to detect thiamine-responsive PDHC deficiency so that thiamine treatment can be initiated as soon as possible.
Acidosis, Lactic↗
Concomitant administration of sodium dichloroacetate and thiamine in west syndrome caused by thiamine-responsive pyruvate dehydrogenase complex deficiency.
We treated a female patient with West syndrome caused by thiamine-responsive pyruvate dehydrogenase complex (PDHC) deficiency. Infantile spasms occurred in association with elevated blood and CSF lactate concentrations; these symptoms disappeared when lactate concentrations had been lowered by treatment with concomitant sodium dichloroacetate (DCA) and high dose thiamine. Sequencing the patient's PDHC E(1)alpha subunit revealed a substitution of serine for glycine at position 89 in exon 3 (G89S). This mutation must be a de novo mutation because it was not found in either parents' genome DNA. To our knowledge, five previously described patients with PDHC deficiency have displayed the West syndrome. All six known patients, including our own, were female, even though an approximately equal number of males and females have been identified with PDHC deficiency and overall West syndrome occurs somewhat more frequently in males. These results indicated that West syndrome occurred more frequently in female patients with PDHC deficiency. It is suggested that lactate concentration should be measured in patients with West syndrome for potential PDHC deficiency, especially in females.
Amino Acid Substitution↗
Altered phosphorylation/inactivation of a novel pyruvate dehydrogenase in adult Ascaris suum muscle.
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Animals↗
Characterization of the regulatory region of the human testis-specific form of the pyruvate dehydrogenase alpha-subunit (PDHA-2) gene.
The alpha-subunit of human pyruvate dehydrogenase (E(1)) is encoded by two separate genes. The gene located on chromosome X (PDHA-1) is expressed in somatic tissues, whereas the second gene (PDHA-2), located on chromosome 4, is expressed only in post-meiotic spermatogenic cells. A genomic fragment harboring the human gene encoding PDHA-2 has been isolated and approximately 800 nucleotides of the promoter region have been characterized. Functional studies of the promoter indicate the presence of both enhancer and repressor elements that are common to other genes that are only expressed in mature sperm.
Base Sequence↗
The pyruvate dehydrogenase multi-enzyme complex from Gram-negative bacteria.
Pyruvate dehydrogenase multi-enzyme complexes from Gram-negative bacteria consists of three enzymes, pyruvate dehydrogenase/decarboxylase (E1p), dihydrolipoyl acetyltransferase (E2p) and dihydrolipoyl dehydrogenase (E3). The acetyltransferase harbors all properties required for multi-enzyme catalysis: it forms a large core of 24 subunits, it contains multiple binding sites for the E1p and E3 components, the acetyltransferase catalytic site and mobile substrate carrying lipoyl domains that visit the active sites. Today, the Azotobacter vinelandii complex is the best understood oxo acid dehydrogenase complex with respect to structural details. A description of multi-enzyme catalysis starts with the structural and catalytic properties of the individual components of the complex. Integration of the individual properties is obtained by a description of how the many copies of the individual enzymes are arranged in the complex and how the lipoyl domains couple the activities of the respective active sites by way of flexible linkers. These latter aspects are the most difficult to study and future research need to be aimed at these properties.
Acetyltransferases↗