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PubMed1986

Immunological and biosynthetic studies on the mammalian 2-oxoglutarate dehydrogenase multienzyme complex.

High-titre, monospecific, polyclonal antisera have been raised against purified mitochondrial 2-oxoglutarate dehydrogenase complex (OGDC) from ox heart and two of its three constituent enzymes, 2-oxoglutarate dehydrogenase (E1) and lipoyl succinyltransferase (E2). These specific antisera have been employed to monitor molecular events in the biosynthesis, import and maturation of this multimeric assembly. Lipoamide dehydrogenase (E3) elicits a poor antibody response in comparison to the other polypeptides of the complex. In cultured pig kidney cells (PK-15), incubated with [35S]methionine in the presence of uncouplers of oxidative phosphorylation, appearance of stable higher-Mr forms of the individual enzymes can be detected by specific immunoprecipitation and fluorographic analysis. In the case of 2-oxoglutarate dehydrogenase, E1, the initial cytoplasmic translation product has a subunit Mr value of 1500-3000 greater than in the mature enzyme while the precursor of the lipoyl succinyltransferase, E2, contains an additional sequence of Mr 6000-8000. Competition studies have revealed the immunological similarity of the precursor molecules to the native subunits. On removal of uncouplers, processing of accumulated precursors is rapidly initiated and is complete within 40 min. Interestingly, antiserum to native 2-oxoglutarate dehydrogenase complex fails to recognise E2 precursor molecules (pre-E2), which can be immunoprecipitated, however, by antibodies raised against the denatured E2 subunit. It is concluded that pre-E2 is conformationally dissimilar to native E2, which exists normally as a highly ordered, multimolecular aggregate in the native complex.

Animals↗

PubMed1980

Friedreich's ataxia II. Biochemical studies in cultured cells.

Pyruvate and palmitate oxidations by cultured fibroblast suspensions were measured in optimized conditions and proved to be within normal range in the cells from Friedreich's patients. But when pyruvate oxidation was measured by direct assay of the pyruvate dehydrogenase complex, this enzyme activity proved to be significantly lower in Friedreich's than in controls' cells. These abnormalities were not observed when the cells were sonicated. Moreover, lipoamide dehydrogenase activity Km and Vmax were within the normal range in Friedreich's cells. These data suggest that the low activities of the PDH complex are not a primary defect in Friedreich's ataxia but are more likely to be related to membrane abnormalities in Friedreich's cells.

Adolescent↗

PubMed1980

Friedreich's ataxia in northern Italy. II. Biochemical studies in cultured cells.

Pyruvate and palmitate oxidations by cultured fibroblasts suspensions were measured in optimized conditions and proved to be within normal range in the cells from Friedreich's patients. However, when pyruvate oxidation was measured by direct assay of the pyruvate dehydrogenase complex, this enzyme activity proved to be significantly lower in Friedreich's than in controls' cells. These abnormalities were not observed when the cells were sonicated. Moreover, lipoamide dehydrogenase activity. Km and Vmax were within the normal range in Friedreich's cells. These data suggest that the low activities of the PDH complex are not a primary defect in Friedreich's ataxia, but are more likely related to membrane abnormalities in Friedreich's cells.

Carnitine O-Acetyltransferase↗

PubMed1980

Protein mobility inside pyruvate dehydrogenase complexes as reflected by laser-pulse fluorometry. A new approach to multi-enzyme catalysis.

The fluorescence decay curves of the flavin in all pyruvate dehydrogenase complexes studied here are consistent with a two-exponential fit. One of the lifetimes calculated is very short, as demonstrated by experiments in which a mode-locked argon-ion laser was used for excitation. In three complexes out of the four which were investigated, about equal weights for the amplitudes of the two lifetimes are found. In the three-component complex from Azotobacter vinelandii this is not the case. No effects of the protein concentration on the lifetimes of the fluorophore were found in the concentration range studied. A small but significant difference in lifetime is observed for the A. vinelandii complexes when coenzyme-free complex is compared with complex to which Mg2+ and thiamin diphosphate are added. The correlation time calculated from the polarized decay of the flavin fluorescence at 11 degrees C is around 40 ns and 50 ns for A. vinelandii complexes and Escherichia coli complexes respectively. This correlation time is of the same order as the rotational correlation time of free lipo-amide dehydrogenase itself, but much shorter than would be expected from the molecular weights of the complexes. Models explaining the two lifetimes are discussed. A catalytic mechanism based on the internal mobility of the lipoamide dehydrogenase inside the multi-enzyme complex is proposed.

Azotobacter↗

PubMed1998

Transcription and transcript processing in the sdhCDAB-sucABCD operon of Escherichia coli.

The genes encoding succinate dehydrogenase (sdhCDAB), the specific components of the 2-oxoglutarate dehydrogenase complex (ODH, E1o and E2o; sucAB) and succinyl-CoA synthetase (sucCD) form a cluster containing two promoters at 16.3 min in the chromosome of Escherichia coli: Psdh sdhCDAB-Psuc sucAB-sucCD. The gene encoding the lipoamide dehydrogenase component of both the 2-oxoglutarate and pyruvate dehydrogenase complexes (E3; lpdA) is the distal gene of another cluster containing two promoters located at 2.7 min: Ppdh pdhR-aceEF-Plpd lpdA. The responses of the suc and lpd promoters to different environmental conditions and to regulator defects were investigated with appropriate lacZ fusions, in order to understand how expression of the sucAB genes is co-regulated with other genes in the sdhCDAB-sucABCD cluster and with lpdA expression. Expression from the suc promoter was repressed by IHF and partially activated by sigma 38 but it was not regulated by ArcA, FNR, CRP, FruR or Fis, and not repressed by glucose or anaerobiosis, indicating that the well-established catabolite and anaerobic repression of ODH synthesis is imposed elsewhere. In contrast, the lpd promoter was repressed by both glucose (via a CRP-independent mechanism) and anaerobiosis (mediated by ArcA), and activated by Fis, but it was not regulated by FNR, FruR, IHF or sigma 38. These observations support the view that transcription of the sucABCD genes is primarily initiated and regulated at the upstream sdh promoter, and that the lpd promoter is independently co-regulated with Psdh (primarily by ArcA-mediated repression) rather than with Psuc. Direct evidence for co-transcription of the entire sdhCDAB-sucABCD region from Psdh was obtained by detecting a 10 kb transcript in rnc and rne mutants, but not in the parental strains. Three RNaseIII-specific processing sites, which contribute to the extreme instability of the readthrough transcript, were identified in the sdhCDAB-sucABCD intergenic region. Other sites of endonuclease processing were located by interpreting the patterns of transcript subfragments observed in Northern blotting.

Base Sequence↗

PubMed1990

Three genes for enzymes of the pyruvate dehydrogenase complex map to human chromosomes 3, 7, and X.

The genes for three proteins of the pyruvate dehydrogenase (PDH) complex have been assigned to human chromosomes by Southern analysis of a panel of human-rodent somatic cell hybrid DNAs with cDNA probes for these genes. PDH-E1 alpha has been localized on human chromosome 3p13-q23. The assignments of lipoamide dehydrogenase(E3) and PDH-E1 alpha [corrected] to chromosomes 7 and Xp, respectively, have been confirmed. Restrictive-fragment-length polymorphisms have been identified with E3, which will permit further localization of this gene by genetic linkage analysis.

Animals↗

PubMed1981

Glutathione reductase from yeast. Differential reactivity of the nascent thiols in two-electron reduced enzyme and properties of a monoalkylated derivative.

Two-electron reduced glutathione reductase from yeast reacted with iodoacetamide is alkylated almost exclusively in the nascent thiol nearer the amino terminus of the protein. The charge-transfer absorbance, maximal at 530 nm, characteristic of the two-electron reduced enzyme is not lost as the alkylation proceeds, and the product has a spectrum virtually identical with that of the two-electron reduced enzyme. This observation demonstrates that the thiol alkylated is not the charge-transfer-donor thiolate which interacts with the FAD. The spectrum of the monoalkylated derivative is stable in the presence of oxidized glutathione, indicating that the charge-transfer-donor thiol is not involved in interchange with the substrate in the native enzyme. Thus, the nascent thiols produced upon two-electron reduction of glutathione reductase have distinct functions, interchange with the substrate and interaction with the FAD. Treatment of the monoalkylated derivative with the apolar phenylmercuric acetate eliminates the charge-transfer interaction. The spectrum of the resulting species is similar to that of the oxidized enzyme but less resolved and blue shifted by 10 nm. The dependence on pH of the absorbance associated with the thiolate to FAD charge-transfer interaction in native two-electron reduced glutathione reductase is biphasic, with pK values at approximately 4.8 and 7.4. By analogy with glyceraldehyde-3-phosphate dehydrogenase and papain, these data indicate that the thiolate is stabilized by an adjacent basic residue. The pK 7.4 is associated with the titration of the base to give the ion pair, and the pK of 4.8 is associated with the titration of the thiolate. Unlike lipoamide dehydrogenase, glutathione reductase is sufficiently stable to allow titration with dithionite at pH 3.7. The spectrum at this pH is essentially the same as that of the monoalkylated derivative treated with phenylmercuric acetate. The changes with pH are completely reversible.

Alkylation↗

PubMed2005

Mycobacterium tuberculosis appears to lack alpha-ketoglutarate dehydrogenase and encodes pyruvate dehydrogenase in widely separated genes.

Mycobacterium tuberculosis (Mtb) persists for prolonged periods in macrophages, where it must adapt to metabolic limitations and oxidative/nitrosative stress. However, little is known about Mtb's intermediary metabolism or antioxidant defences. We recently identified a peroxynitrite reductase-peroxidase complex in Mtb that included products of the genes sucB and lpd, which are annotated to encode the dihydrolipoamide succinyltransferase (E2) and lipoamide dehydrogenase (E3) components of alpha-ketoglutarate dehydrogenase (KDH). However, we could detect no KDH activity in Mtb lysates, nor could we reconstitute KDH by combining the recombinant proteins SucA (annotated as the E1 component of KDH), SucB and Lpd. We therefore renamed the sucB product dihydrolipoamide acyltransferase (DlaT). Mtb lysates contained pyruvate dehydrogenase (PDH) activity, which was lost when the dlaT gene (formerly, sucB) was disrupted. Purification of PDH from Mtb yielded AceE, annotated as an E1 component of PDH, along with DlaT and Lpd. Moreover, anti-DlaT antibody coimmunoprecipitated AceE. Finally, recombinant AceE, DlaT and Lpd, although encoded by genes that are widely separated on the chromosome, reconstituted PDH in vitro with Km values typical of bacterial PDH complexes. In sum, Mtb appears to lack KDH. Instead, DlaT and Lpd join with AceE to constitute PDH.

Acyltransferases↗

PubMed1979

Polypeptide-chain stoicheiometry and lipoic acid content of the pyruvate dehydrogenase complex of Escherichia coli.

The pyruvate dehydrogenase multienzyme complex was isolated from Escherichia coli grown in the presence of [35S]sulphate. The three component enzymes were separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the molar ratios of the three polypeptide chains were determined by measurement of the radioactivity in each band. The chain ratio of lipoamide dehydrogenase to lipoate acetyltransferase approached unity, but there was a molar excess of chains of the pyruvate decarboxylase component. The 35S-labelled complex was also used in a new determination of the total lipoic acid content. It was found that each polypeptide chain of the lipoate acetyltransferase component appears to bear at least three lipoyl groups.

Chemical Phenomena↗

PubMed1981

Pyruvate dehydrogenase complex from baker's yeast. 2. Molecular structure, dissociation, and implications for the origin of mitochondria.

1. Pyruvate dehydrogenase complex from Saccharomyces cerevisiae is similar in size (s20,w 77 S) and flavin content (1.3--1.4 nmol/mg) to the complexes from mammalian mitochondria. 2. The relative molecular masses of the constituent polypeptide chains, as determined by dodecylsulfate gel electrophoresis at different gel concentrations, were: lipoate acetyltransferase (E2), 58 000; lipoamide dehydrogenase (E3), 56 000; pyruvate dehydrogenase (E1), alpha-subunit, 45 000, and beta-subunit, 35 000. Gel chromatography in the presence of 6 M guanidine . HCl gave a value of 52 000 for E2 indicating anomalous electrophoretic migration as described for the E2 components of other pyruvate dehydrogenase complexes. Thus, the organization and subunit Mr values are similar with the mammalian complexes and virtually identical with the complexes of gram-positive bacteria but differ greatly from the pyruvate dehydrogenase complexes of gram-negative bacteria. 3. The complex was resolved into its component enzymes by the following methods. E1 was obtained by treatment of the complex with elastase followed by gel chromatography on Sepharose CL-2B using a reverse ammonium sulfate gradient for elution. E2 was isolated by gel filtration of the complex in the presence of 2 M KBr, and E3 was obtained by hydroxyapatite chromatography in 8 M urea. The isolated enzymes reassociated spontaneously to give pyruvate dehydrogenase overall activity.

Biological Evolution↗

PubMed1992

Isolation and characterisation of the pyruvate dehydrogenase complex of anaerobically grown Enterococcus faecalis NCTC 775.

In this contribution the isolation and some of the structural and kinetic properties of the pyruvate dehydrogenase complex (PDC) of anaerobically grown Enterococcus faecalis are described. The complex closely resembles the PDC of other Gram-positive bacteria and eukaryotes. It consists of four polypeptide chains with apparent molecular masses on SDS/PAGE of 97, 55, 42 and 36 kDa, and these polypeptides could be assigned to dihydrolipoyl transacetylase (E2), lipoamide dehydrogenase (E3) and the two subunits of pyruvate dehydrogenase (E1 alpha and E1 beta), respectively. The E2 core has an icosahedral symmetry. The apparent molecular mass on SDS/PAGE of 97 kDa of the E2 chain is extremely high in comparison with other Gram-positive organisms (and eukaryotes) and probably due to several lipoyl domains associated with the E2 chain. NADH inhibition is mediated via E3. The mechanism of inhibition is discussed in view of the high PDC activities in vivo that are found in E. faecalis, grown under anaerobic conditions.

Acetyltransferases↗

PubMed1991

Selective proteolysis of the protein X subunit of the bovine heart pyruvate dehydrogenase complex. Effects on dihydrolipoamide dehydrogenase (E3) affinity and enzymic properties of the complex.

Selective proteolysis of the protein X subunit of native bovine heart pyruvate dehydrogenase complex may be accomplished without loss of overall complex activity. Partial loss of function occurs if Mg2+ and thiamin pyrophosphate are not present during proteinase arg C treatment as these cofactors are necessary to prevent cleavage of the E1 alpha subunit. Specific degradation of component X leads to marked alterations in the general enzymic properties of the complex. Lipoamide dehydrogenase (E3) exhibits a decreased affinity for the core assembly and the complex is much more susceptible to inactivation at high ionic strength. The inactive form of the complex is not readily re-activated by removal of salt. It appears that intact protein X and specifically the presence of its cleaved lipoyl domain is not essential for maintenance of an enzymically active pyruvate dehydrogenase complex. However, this protein has an important structural role in promoting the correct association of E3 with the E2 core assembly, an interaction that is required for optimal catalytic efficiency of the complex.

Animals↗

PubMed1995

Reduction and transport of lipoic acid by human erythrocytes.

Reduction of exogenous lipoic acid to dihydrolipoate is known to occur in several mammalian cells and tissues. Dihydrolipoate is a potent radical scavenger, and may provide significant antioxidant protection. Because lipoic acid appears in the bloodstream after oral administration, we have examined the reduction of exogenous lipoate by human erythrocytes. Normal human erythrocytes reduced lipoate to dihydrolipoate only in the presence of glucose; deoxyglucose did not substitute for glucose, indicating that the reduction of lipoate requires glucose metabolism. Furthermore, the reduction was shown to be NADPH dependent. Erythrocytes isolated from a human subject with a genetic deficiency of glucose-6-phosphate dehydrogenase (and, therefore, deficient in the formation of NADPH) did not reduce lipoate. Dehydroepiandrosterone, a specific inhibitor of glucose-6-phosphate dehydrogenase, inhibited lipoate reduction. Our findings imply that some of the reduction of exogenous lipoic acid is catalysed by glutathione reductase, a flavoprotein dehydrogenase; mitomycin C, an inhibitor of FAD-dependent reductases, inhibited lipoate reduction by erythrocytes, and glutathione reductase purified from human erythrocytes was observed to reduce lipoic acid in a cell-free system. We further explored these findings with erythrocyte ghosts and liposomes. Our results indicate that a transport system exists for alpha-lipoic acid and dihydrolipoate; resealed erythrocyte ghosts, containing trapped lipoamide dehydrogenase and pyridine nucleotides, reduced externally added lipoate. By contrast, liposomes prepared with enzyme and pyridine nucleotides did not catalyze reduction of lipoate. This work indicates that uptake of exogenous lipoate and reduction to dihydrolipoate by normal human erythrocytes may contribute to oxidant protection in the human bloodstream.

Biological Transport↗

PubMed1981

Limited proteolysis and proton n.m.r. spectroscopy of the 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli.

The 2-oxoglutarate dehydrogenase multienzyme complex of Escherichia coli was treated with trypsin at pH 7.0 at 0 degrees C. Loss of the overall catalytic activity was accompanied by rapid cleavage of the lipoate succinyltransferase polypeptide chains, this apparent Mr falling from 50 000 to 36 000 as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. A slower shortening of the 2-oxoglutarate decarboxylase chains was also observed, whereas the lipoamide dehydrogenase chains were unaffected. The inactive trypsin-treated enzyme had lost the lipoic acid-containing regions of the lipoate succinyltransferase polypeptide chains, yet remained a highly assembled structure, as judged by gel filtration and electron microscopy. The lipoic acid-containing regions are therefore likely to be physically exposed in the complex, protruding from the structural core formed by the lipoate succinyltransferase component between the subunits of the other component enzymes. Proton nuclear magnetic resonance spectroscopy of the 2-oxoglutarate dehydrogenase complex revealed the existence of substantial regions of polypeptide chain with remarkable intramolecular mobility, most of which were retained after removal of the lipoic acid-containing regions by treatment of the complex with trypsin. By analogy with the comparably mobile regions of the pyruvate dehydrogenase complex of E. coli, it is likely that the highly mobile regions of polypeptide chain in the 2-oxoglutarate complex are in the lipoate succinyltransferase component and encompass the lipoyl-lysine residues. It is clear, however, that the mobility of this polypeptide chain is not restricted to the immediate vicinity of these residues.

Electrophoresis, Polyacrylamide Gel↗

PubMed1997

Identification of mitochondrial antigens recognized by antibodies in sera of patients with idiopathic dilated cardiomyopathy by two-dimensional gel electrophoresis and protein sequencing.

Antimitochondrial antibodies in sera of patients with idiopathic dilated cardiomyopathy (IDC) have been described previously, but the corresponding antigens have not been analyzed systematically. We therefore used both 1-dimensional and high-resolution 2-dimensional gel electrophoresis followed by immunoblotting and N-terminal amino acid sequencing to identify the relevant mitochondrial antigens, which are recognized by serum antibodies. Sera were obtained from patients with IDC (n = 75) and healthy controls (n = 182). For detection of antimitochondrial antibodies the mitochondrial antigen fraction, consisting of submitochondrial particles isolated from a bovine heart, was separated on SDS-PAGE and all sera were examined by immunoblot analysis. For further characterization of the mitochondrial epitopes the antigen fraction was separated in the first dimension according to isoelectric points using isoelectric focusing followed by gel electrophoresis. Proteins recognized by serum antibodies in 2-dimensional immunoblots were analyzed by N-terminal amino acid sequencing. In 1-dimensional immunoblot analysis, 51% of patients with IDC and 34% of controls contained serum antibodies reacting with mitochondrial protein bands with molecular weights of about 30, 43, 60, and preferentially 50 to 55 and 70 to 75 kD (p <0.01). We identified a 75-kD subunit of nicotinamide-adenine dinucleotide dehydrogenase (17% in IDC patients vs 5% in controls, p <0.05) and 2 core proteins of ubiquinol-cytochrome-c reductase (core P1: 39% in IDC patients vs 15% in controls, p <0.05; core P2: 20% in IDC patients vs 10% in controls, p <0.1), both enzymes of the respiratory chain, as are most relevant mitochondrial antigens. Furthermore, serum antibodies of patients with IDC were directed against lipoamide-dehydrogenase (15% vs 10% in controls) and a subunit of pyruvate-dehydrogenase (9% vs 3% in controls). Because these antigens play an important role in energy metabolism, the respective antibodies can be more than merely diagnostic markers of cell damage. To attribute them also to pathogenetic relevance appears to be a most attractive but still speculative hypothesis.

Adult↗

PubMed2001

Electrocatalytic reduction of lipoic acid and electroenzymatic reduction of NAD(P)(+) for integrated dehydrogenase biosensors.

Biosensors for the oxidized substrates of NAD(P)(+)-specific dehydrogenases demand the reductive recycling of the coenzymes. So far, suitable catalysts for the corresponding two-electron transfer are not available. In the present paper, this transport has been realized by a combined electrocatalytical and electroenzymatic process. Lipoic acid has been reduced on graphite electrodes functionalized with Fe(II)-phthalocyanine in 95% yield at-1200 mV in phosphate buffer pH 7.0. With the electrocatalytically reduced product, dihydrolipoic acid, lipoamide dehydrogenase could reduce NAD(+) in 20% yield and thioredoxin reductase NADP(+) in 18.4% yield. So far, the combined electrocatalytic/electroenzymatic system has not yet been realized, mainly because at the potential needed for the lipoic acid reduction, a parallel one-electron reduction of NAD(P)(+) was observed, implying the dimerization of the coenzyme.

Biosensing Techniques↗

PubMed1998

Glyceraldehyde 3-phosphate dehydrogenase abnormality in metabolically stressed Huntington disease fibroblasts.

Huntington disease (HD) fibroblasts subjected to stress exhibit an enzyme profile that is different from that exhibited by escapee (unaffected members of families with HD) or control fibroblasts. The specific activity of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in normally cultured HD fibroblasts was not different from that in control and escapee fibroblasts. However, in escapee and control fibroblasts subjected to stress by withholding fresh medium, the specific activity of GAPDH in cells harvested by trypsinization increased greatly 3 weeks after withholding medium ( approximately 8-fold), but the increase was significantly less pronounced ( approximately 3-fold) in the HD fibroblasts. In contrast, only small changes occurred in the specific activity of lipoamide dehydrogenase (LADH) over the same time period, and the values were not significantly different among the three groups at any time point. The specific activity of hexokinase (HK) was significantly higher in the HD fibroblasts at 1-3 weeks after withholding fresh medium than in the escapee/control fibroblasts. Finally, the total yield of fibroblasts per culture flask (as judged by protein content) was significantly greater for the stressed HD fibroblasts than for the escapee and control fibroblasts at 2 and 3 weeks after withholding medium. The present results are in accord with the hypothesis that HD is a disease associated with latent, generalized metabolic abnormalities.

Cells, Cultured↗

PubMed1989

Purification and partial characterization of the glycine decarboxylase multienzyme complex from Eubacterium acidaminophilum.

The proteins P1, P2, and P4 of the glycine cleavage system have been purified from the anaerobic, glycine-utilizing bacterium Eubacterium acidaminophilum. By gel filtration, these proteins were determined to have Mrs of 225,000, 15,500, and 49,000, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein P1 was determined to have two subunits with Mrs of 59,500 and 54,100, indicating an alpha 2 beta 2 tetramer, whereas the proteins P2 and P4 showed only single bands with estimated Mrs of 15,500 and 42,000, respectively. In reconstitution assays, proteins P1, P2, P4 and the previously reported lipoamide dehydrogenase (P3) had to be present to achieve glycine decarboxylase or synthase activity. All four glycine decarboxylase proteins exhibited highest activities when NADP+ was used as the electron acceptor or when NADPH was used as the electron donor in the glycine synthase reaction. The oxidation of glycine depended on the presence of tetrahydrofolate, dithioerythreitol, NAD(P)+, and pyridoxal phosphate. The latter was loosely bound to the purified protein P1, which was able to catalyze the glycine-bicarbonate exchange reaction only in combination with protein P2. Protein P2 could not be replaced by lipoic acid or lipoamide, although lipoic acid was determined to be a constituent (0.66 mol/mol of protein) of protein P2. Glycine synthase activity of the four isolated proteins and in crude extracts was low and reached only 12% of glycine decarboxylase activity. Antibodies raised against P1 and P2 showed cross-reactivity with crude extracts of Clostridium cylindrosporum.

Amino Acid Oxidoreductases↗