Showing papers on "Oxoglutarate dehydrogenase complex published in 1991"
TL;DR: In mitochondria, electrons are transferred from NADH to O2 through a chain of three large enzyme complexes, namely NADH: ubiquinone oxidoreductase (NADH dehydrogenase or complex I), ubiquinol: ferricytochrome c oxidore ductase (cytochrome reductase orcomplex III), and ferrocy tochrome c:O2 oxidoreductionase (Cytochrome oxid enzyme or complex IV).
Abstract: In mitochondria, electrons are transferred from NADH to O2 through a chain of three large enzyme complexes, namely NADH: ubiquinone oxidoreductase (NADH dehydrogenase or complex I), ubiquinol: ferricytochrome c oxidoreductase (cytochrome reductase or complex III), and ferrocytochrome c:O2 oxidoreductase (cytochrome oxidase or complex IV). The function of these enzyme complexes is to link electron transfer with proton translocation out of the mitochondrion. In doing so, they generate a transmembraneous proton motive force which subsequently drives ATP synthesis by the H+-ATPase (complex V, for a review see [1]).
491 citations
TL;DR: The results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors.
Abstract: Relative levels of many individual proteins in Escherichia coli HB101 strains with 0, 37, 56, and 240 plasmids per chromosome were determined by computer image analysis of two-dimensional gel electrophoresis patterns The plasmids investigated had very similar sequences except for small domains encoding the repressor of plasmid replication At the intermediate plasmid copy number of 56, levels of several of the TCA cycle enzymes (oxoglutarate dehydrogenase complex, succinate thiokinase, and succinate dehydrogenase) as well as in aspartate transcarbamoylase increased At a plasmid copy number of 240, higher amounts of PEP carboxylase as well as several of the heat shock proteins were observed Furthermore, at high plasmid levels, significant decreases occurred in growth rate, pyruvate kinase I, pyruvate dehydrogenase complex, unadenylated glutamine synthetase, aspartate transcarbamoylase as well as in several of the proteins involved in translation Decreases in ribosome content as well as in the free 30S and 50S ribosomal subunit pool fractions were also observed in separate analyses These results indicate that recombinant DNA manipulations can cause major alterations in numerous host cell properties which could significantly influence cloned protein production or metabolic engineering endeavors
175 citations
TL;DR: The gene encoding D-lactate dehydrogenase of Lactobacillus plantarum has been sequenced, and expressed in Escherichia coli cells with an inducible expression plasmid, in which the 5'-noncoding region of the gene was replaced with the tac promoter, suggesting that the new family consists of D-isomer-stereospecific enzymes.
170 citations
TL;DR: Modification of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells, and extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruVate.
Abstract: Disruption of the PDX1 gene encoding the protein X component of the mitochondrial pyruvate dehydrogenase (PDH) complex in Saccharomyces cerevisiae did not affect viability of the cells. However, extracts of mitochondria from the mutant, in contrast to extracts of wild-type mitochondria, did not catalyze a CoA- and NAD(+)-linked oxidation of pyruvate. The PDH complex isolated from the mutant cells contained pyruvate dehydrogenase (E1 alpha + E1 beta) and dihydrolipoamide acetyltransferase (E2) but lacked protein X and dihydrolipoamide dehydrogenase (E3). Mutant cells transformed with the gene for protein X on a unit-copy plasmid produced a PDH complex that contained protein X and E3, as well as E1 alpha, E1 beta, and E2, and exhibited overall activity similar to that of the wild-type PDH complex. These observations indicate that protein X is not involved in assembly of the E2 core nor is it an integral part of the E2 core. Rather, protein X apparently plays a structural role in the PDH complex; i.e., it binds and positions E3 to the E2 core, and this specific binding is essential for a functional PDH complex. Additional evidence for this conclusion was obtained with deletion mutations. Deletion of most of the lipoyl domain (residues 6-80) of protein X had little effect on the overall activity of the PDH complex. This observation indicates that the lipoyl domain, and its covalently bound lipoyl moiety, is not essential for protein X function. However, deletion of the putative subunit binding domain (residues approximately 144-180) of protein X resulted in loss of high-affinity binding of E3 and concomitant loss of overall activity of the PDH complex.(ABSTRACT TRUNCATED AT 250 WORDS)
76 citations
TL;DR: In vitro translation of Euglena polyadenylated RNA in a cell-free rabbit reticulocyte lysate system explained the formation of a single polypeptide with a molecular weight of 65,000, suggesting that a putative precursor of 2-oxoglutarate decarboxylase which is about 3000 larger than the subunit of the mature enzyme is synthesized in EuglENA cells.
74 citations
TL;DR: The transport mechanism of the reconstituted oxoglutarate carrier, purified from bovine heart mitochondria, was studied kinetically and a sequential type of mechanism became evident, implying that one internal and one external substrate molecule form a ternary complex with the carrier before transport occurs.
Abstract: The transport mechanism of the reconstituted oxoglutarate carrier, purified from bovine heart mitochondria, was studied kinetically. A complete set of half-saturation constants (Km) was established for the two different substrates oxoglutarate and malate on both the external and the internal sides of the membrane. The internal affinities for oxoglutarate (Km 0.17 mM) and malate (Km 0.7 mM) were higher than the corresponding external affinities (Km 0.3 mM and 1.4 mM, respectively). The exclusive presence of a single transport affinity for each substrate on one side of the membrane indicated a unidirectional insertion of the oxoglutarate carrier into the liposomal membrane. The Km values and also the maximum exchange rates (8-11 mumol.min-1.mg protein-1) for oxoglutarate and malate were independent of the nature of the counter substrate on the other side of the membrane. Under these defined conditions we analyzed the antiport mechanism in two-reactant initial velocity studies varying both the internal and external substrate concentrations. From the kinetic patterns obtained, a sequential type of mechanism became evident, implying that one internal and one external substrate molecule form a ternary complex with the carrier before transport occurs. A quantitative analysis of substrate interaction with the unloaded or single-substrate-occupied carrier revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrate. This kinetic mechanism agrees with previous results obtained in intact mitochondria. Considering also the data available for other mitochondrial carriers, a common kinetic mechanism (sequential type) for this carrier family is suggested.
61 citations
TL;DR: Data is presented showing that both polypeptidespecific and cross‐reacting antibodies are present in patients' sera, arguing against a bacterial origin for primary biliary cirrhosis.
61 citations
TL;DR: Characteristics of the reaction suggest this enzyme plays an important role in the response of the colonic mucosa to the mitogenic effect of oxidized fatty acids, and other characteristics suggest the enzyme is neither alcohol dehydrogenase, diol dehydrogen enzyme, nor a prostaglandin dehydrogenases.
50 citations
TL;DR: It is suggested that LPL‐B may be responsible for the octanoylation of lipoyl domains observed previously under lipoate‐deficient conditions.
44 citations
TL;DR: Biochemical screens of a pet mutant collection have led to the identification of a new gene required for the expression of enzymatically active KGDC, most likely by posttranslational modification of one of the subunits.
Abstract: The assembly of alpha-ketoglutarate dehydrogenase complex (KGDC) has been studied in wild-type Saccharomyces cerevisiae and in respiratory-deficient strains (pet) with mutations in KGD1 and KGD2, the structural genes for alpha-ketoglutarate dehydrogenase (KE1) and dihydrolipoyl transsuccinylase (KE2) components, respectively. Mutants unable to express KE1 or KE2 form partial complexes similar to those reported in earlier studies on the resolution and reconstitution of bacterial and mammalian KGDC. Thus mutants lacking KE1 assemble a high-molecular-weight subcomplex consisting of a KE2 core particle with bound dihydrolipoyl dehydrogenase (E3). Similarly, mitochondrial extracts of mutants lacking KE2 contain dimeric KE1 and E3. These components, however, are not associated with each other. The partial complexes detected in the mutants are capable of reconstituting normal KGDC when supplied with the missing subunit. Complete restoration of overall alpha-ketoglutarate dehydrogenase activity is achieved by mixing appropriate ratios of mitochondrial extracts from mutants deficient in KE1 and KE2. The reconstitution of enzymatic activity correlates with binding of KE1 to the KE2-E3 particle to form a complex with the same sedimentation properties as wild-type KGDC. Overexpression of KE2 relative to KE1 results in a preponderance of incompletely assembled complexes with substoichiometric contents of KE1. Formation of a complex with a full complement of KE1 therefore depends on a balanced output of KE1 and KE2 from their respective genes. Biochemical screens of a pet mutant collection have led to the identification of a new gene required for the expression of enzymatically active KGDC. Mitochondria of the mutant have all of the catalytic subunits of KGDC. Sedimentation analysis of these components indicates that while the mutant has a stable KE2-E3 subcomplex, the interaction of KE1 with KE2 core is much weaker in the mutant than in the wild type. The gene product responsible for this phenotype, therefore, appears to function at a late stage of assembly of KGDC, most likely by posttranslational modification of one of the subunits.
42 citations
TL;DR: Acetate-grown GS-15 whole-cell suspensions were disrupted with detergent and assayed for enzymes associated with acetate catabolism, andCatabolic levels of acetokinase and phosphotransacetylase were observed.
Abstract: Acetate-grown GS-15 whole-cell suspensions were disrupted with detergent and assayed for enzymes associated with acetate catabolism. Carbon monoxide dehydrogenase and formate dehydrogenase were not observed in GS-15. Catabolic levels of acetokinase and phosphotransacetylase were observed. Enzyme activities of the citric acid cycle, i.e., isocitrate dehydrogenase, 2-oxoglutarate sythase, succinate dehydrogenase, fumarase, and malate dehydrogenase, were observed.
TL;DR: The analysis of three patients with pyruvate dehydrogenase E1α deficiency is reported, one female has a three base pair deletion which affects dephosphorylation of the subunit, and two males analysed, one has a two base pair delete causing a shift in the reading frame.
Abstract: The human pyruvate dehydrogenase complex catalyses the oxidative decarboxylation of pyruvate to acetyl-CoA Defects in several of the seven subunits have been reported, but the majority of mutations affect the E1 component and especially the E1α subunit However, the clinical presentation of patients with pyruvate dehydrogenase E1α deficiency is extremely variable Dependency of the brain on pyruvate dehydrogenase activity and localization of the gene for the somatic form of the pyruvate dehydrogenase E1α subunit to the X chromosome provide the basis for a better understanding of the variation in the clinical manifestations Further understanding of the function and interaction of subunits and the pathophysiology of pyruvate dehydrogenase deficiency necessitates the characterization of mutations in the pyruvate dehydrogenase complex We report the analysis of three patients with pyruvate dehydrogenase E1α deficiency One female has a three base pair deletion which affects dephosphorylation of the subunit Of two males analysed, one has a two base pair deletion causing a shift in the reading frame The other has a base change, resulting in an Arg to His substitution All three mutations are located near the carboxyl terminus of the subunit
TL;DR: 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.
Abstract: 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.
TL;DR: It is suggested that the α and β subunits of the pyruvate and 2-oxoisovalerate dehydrogenases have been derived by the cleavage of the α-ketoglutarate dehydrogenase.
TL;DR: Results show that the rat succinyltransferase does not have the sequence motif of the putative E3- and/or E1-binding site in the dihydrolipoamide acyltransferases of three alpha-ketoacid dehydrogenase complexes.
TL;DR: The ethanol route has significant advantages (minimal acidosis, avoidance of osmotic problems, and endproduct inhibition of the glycolytic chain) but also has disadvantages (relatively low energy yield, loss of carbobydrate carbon).
Abstract: 1. Ethanol is an end product of anaerobic metabolism in a surprisingly large variety of multicellular organisms. These include Angiosperms, Platyhelminthes, Aschelminthes, Acanthocephala, Arthropoda, and Vertebrata. 2. Ethanol formation proceeds in two steps (via pyruvate decarboxylase and alcohol dehydrogenase). 3. Pyruvate decarboxylase of plants is located in the cytosol, whereas that of animals is intramitochondrial and probably part of the pyruvate dehydrogenase complex. 4. Alcohol dehydrogenases of facultative anaerobes are NAD-rather than NADP-dependent, with the enzyme in the parasitic worm Moliniformis dubius as the only known exception. 5. Regulation at the pyruvate branch point in plants seems to involve three different mechanisms: (i) increases of pyruvate decarboxylase and alcohol dehydrogenase activity due to increased gene expression, (ii) control of pyruvate decarboxylase by the intracellular pH and the cytoplasmic NADH/NAD⁺ ratio, and (iii) inhibition of lactate dehydrogenase by ATP at lo...
TL;DR: In situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition, which is indicated by the inhibition by malonate and the apparent lack of coupling.
Abstract: In vitro, the pyruvate dehydrogenase complex is sensitive to product inhibition by NADH and acetyl-coenzyme A (CoA). Based upon Km and Ki relationships, it was suggested that NADH can play a primary role in control of pyruvate dehydrogenase complex activity in vivo (JA Miernyk, DD Randall [1987] Plant Physiol 83:306-310). We have now extended the in vitro studies of product inhibition by assaying pyruvate dehydrogenase complex activity in situ, using purified intact mitochondria from green pea (Pisum sativum) seedlings. In situ activity of the pyruvate dehydrogenase complex is inhibited when mitochondria are incubated with malonate. In some instances, isolated mitochondria show an apparent lack of coupling during pyruvate oxidation. The inhibition by malonate, and the apparent lack of coupling, can both be explained by an accumulation of acetyl-CoA. Inhibition could be alleviated by addition of oxalacetate, high levels of malate, or l-carnitine. The CoA pool in nonrespiring mitochondria was approximately 150 micromolar, but doubled during pyruvate oxidation, when 60 to 95% of the total was in the form of acetyl-CoA. Our results indicate that in situ activity of the mitochondrial pyruvate dehydrogenase complex can be controlled in part by acetyl-CoA product inhibition.
TL;DR: The unicellular cyanobacterium Synechocystis sp.
TL;DR: The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified and it is concluded that KAP is not a distinct protein, but is kinase which has dissociated from the complex.
Abstract: The kinase-activator protein (KAP) of pyruvate dehydrogenase complex (PDC) has been purified approx. 2250-fold from high-speed supernatants of mitochondrial extracts from the liver of 48 h-starved rats. Purified KAP demonstrates kinase activity towards both the E1 component of PDC and towards a synthetic peptide corresponding to the major phosphorylation site on E1. Furthermore, the activities of KAP and PDC kinase co-fractionate through several stages of purification and have the same apparent mass. We conclude that KAP is not a distinct protein, but is kinase which has dissociated from the complex.
TL;DR: The nucleotide sequence of the last gene of the S. aureus pyruvate dehydrogenase operon, pdhD, is reported, which encodes lipoamide dehydrogen enzyme (LPD), which is closely related to other lipoamia dehydrogenases from bacteria and eukaryotes.
TL;DR: Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.
Abstract: The physiology and biochemistry of Sarcina ventriculi was studied in order to determine adaptations made by the organism to changes in environmental pH. The organism altered carbon and electron flow from acetate, formate and ethanol production at neutral pH, to predominantly ethanol production at pH 3.0. Increased levels of pyruvate dehydrogenase (relative to pyruvate decarboxylase) and acetaldehyde dehydrogenase occurred when the organism was grown at neutral pH, indicating the predominance of carbon flux through the oxidative branch of the pathway for pyruvate metabolism. When the organism was grown at acid pH, there was a significant increase in pyruvate decarboxylase levels and a decrease in acetaldehyde dehydrogenase, causing flux through the non-oxidative branch of the pathway. CO2 reductase and formate dehydrogenase were not regulated as a function of growth pH. Pyruvate dehydrogenase possessed Michaelis-Menten kinetics for pyruvate with an apparent Km of 2.5 mM, whereas pyruvate decarboxylase exhibited sigmoidal kinetics, with a S0.5 of 12.0 mM. Differences in total protein banding patterns from cells grown at pH extremes suggested that synthesis of pyruvate decarboxylase and other enzymes was in part responsible for metabolic regulation of the fermentation products formed.
TL;DR: Enterocytes and colonocytes isolated from hypothyroid rats showed decreased rates of utilization and metabolism of glucose and glutamine, and the maximal activities of hexokinase, 6-phosphofructokinase and phosphate-dependent glutaminase were decreased in intestinal mucosal scrapings from hyp Timothyroid rats.
Abstract: 1. The metabolism of glucose and glutamine was studied in the small intestine and the colon of rats after 4-5 weeks of hypothyroidism. 2. Hypothyroidism resulted in increases in the plasma concentrations of ketone bodies (P less than 0.05), cholesterol (P less than 0.001) and urea (P less than 0.001), but decreases in the plasma concentrations of free fatty acids (P less than 0.05) and triacylglycerol (P less than 0.001). These changes were associated with decreases in the plasma concentrations of total tri-iodothyronine, free tri-iodothyronine, total thyroxine and free thyroxine. 3. Hypothyroidism decreased both the DNA content (by 30.5%) and the protein content (by 23.6%) of intestinal mucosa, with the protein/DNA ratio remaining unchanged. The villi in the jejunum were shorter (P less than 0.05) and the crypt depth was decreased by about 26.5% in hypothyroid rats. 4. Portal-drained visceral blood flow showed no marked change in response to hypothyroidism, but was accompanied by decreased rates of extraction of glucose, lactate and glutamine and release of glutamate, alanine and ammonia. 5. Enterocytes and colonocytes isolated from hypothyroid rats showed decreased rates of utilization and metabolism of glucose and glutamine. 6. The maximal activities of hexokinase (EC 2.7.1.1), 6-phosphofructokinase (EC 2.7.1.11), pyruvate kinase (EC 2.7.1.40), citrate synthase (EC 4.1.3.28), oxoglutarate dehydrogenase (EC 1.2.4.2) and phosphate-dependent glutaminase (EC 3.5.1.2) were decreased in intestinal mucosal scrapings from hypothyroid rats. Similar decreases were obtained in colonic mucosal scrapings (except for citrate synthase and oxoglutarate dehydrogenase) from hypothyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: The maximum activities of some key enzymes of metabolism were studied in lungs of fed and 48-h-starved rats, suggesting the importance of the pentose phosphate pathway in the lung and the activities of both oxoglutarate dehydrogenase and citrate synthase were decreased by starvation.
TL;DR: Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruVate dehydrogenases from Escherichia coli and Bacillus stearothermophilus and gives a simple way of making 1-aminoalkylph phosphonic acids.
Abstract: Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps R-CO-C1 R-CO- PO(OMe)2 R-CO-PO(OMe)-O R-CO-PO(OH)-O- of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus slearolhermophilus. The inhibition was competitive with pyruvate, with K1 of 6 μM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4mM fo the B. slearolhermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15mM and 10mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.
TL;DR: The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogensase, isolated from E. coli, on denaturing, nondenaturing and isoelectric focusing polyacrylamide gels and by sequence analysis.
Abstract: Glutamate dehydrogenase fromEscherichia coli is phosphorylated in vitro in an ATP-dependent enzymatic reaction. The phosphorylated protein, when exposed to acid conditions, releases the phosphate; this implies that the phosphorylation site is not on a serine, tyrosine, or threonine residue(s). Treatment of glutamate dehydrogenase with diethyl pyrocarbonate, a highly specific histidine-modifying reagent, blocks incorporation of32P-phosphate from [γ-32P]ATP into the enzyme, suggestive that the phosphorylation site is a histidine residue(s). The phosphorylated glutamate dehydrogenase was identified on the basis of its comigration with highly purified glutamate dehydrogenase, isolated fromE. coli, on denaturing, nondenaturing, and isoelectric focusing polyacrylamide gels and by sequence analysis.
TL;DR: The different responses of OGDH in two mitochondrial populations to HE and ammonium ions in vitro would appear to be due to intrinsic differences between the properties of the enzyme in the synaptic and nonsynaptic brain compartments.
TL;DR: In this paper, pH-dependent kinetics implicate two ionizable groups in the dehydrogenase and esterase reactions catalysed by high-Km aldehyde deacetylase from rat liver mitochondria.
Abstract: Studies of pH-dependent kinetics implicate two ionizable groups in the dehydrogenase and esterase reactions catalysed by high-Km aldehyde dehydrogenase from rat liver mitochondria. Sensitized photo...
TL;DR: It was shown that dehydrogenase is reversible and strongly inhibited by 4-MA and that thus the irreversible step in the transformation of DHEA to 4-androstenedione is due to the isomerase activity.
TL;DR: In examining the PDH-E1 c~ cDNA from a male neonate who died of severe lactic acidosis, an insertion of 21 base pairs interfering with one of the serine phosphorylation sites is found.
Abstract: Pyruvate dehydrogenase (PDH) is a multienzyme complex of five subunits and is a key enzyme in aerobic glucose metabolism. Functional deficiency of PDH is a major cause of congenital lactic acidosis. Over the last few years, information on the metabolic and genetic basis of PDH deficiency has emerged. An abnormal E1 c~ subunit of the PDH complex is considered to be the cause of the deficiency in some cases where a biochemical E1 deficiency has been found. This subunit contains several serine phosphorylation sites of regulatory importance for the whole enzyme complex. The normal E1 c~ cDNA (Dahl et al., 1987; De Meirleir et al., 1988; Koike et al., 1988; Ho et al., 1989) and corresponding genomic DNA (Maragos et al., 1989) have been cloned and sequenced recently; the gene was mapped unexpectedly to the X chromosome at Xp 22.1-22.2 (Brown et al., 1989). This work constitutes a basis for the study of female and mate patients with lactic acidosis and PDH deficiency, In examining the PDH-E1 c~ cDNA from a male neonate who died of severe lactic acidosis, we found an insertion of 21 base pairs interfering with one of the serine phosphorylation sites.
TL;DR: The results suggest that covalent adduct formation is not necessary for the observed inhibition of E. coli pyruvate dehydrogenase in absence of its cofactor, NAD+.