Activity Of 2-oxoglutarate Dehydrogenase Complex Research Articles

Pentylenetetrazole-Induced Seizures Are Increased after Kindling, Exhibiting Vitamin-Responsive Correlations to the Post-Seizures Behavior, Amino Acids Metabolism and Key Metabolic Regulators in the Rat Brain.

Epilepsy is characterized by recurrent seizures due to a perturbed balance between glutamate and GABA neurotransmission. Our goal is to reveal the molecular mechanisms of the changes upon repeated challenges of this balance, suggesting knowledge-based neuroprotection. To address this goal, a set of metabolic indicators in the post-seizure rat brain cortex is compared before and after pharmacological kindling with pentylenetetrazole (PTZ). Vitamins B1 and B6 supporting energy and neurotransmitter metabolism are studied as neuroprotectors. PTZ kindling increases the seizure severity (1.3 fold, p < 0.01), elevating post-seizure rearings (1.5 fold, p = 0.03) and steps out of the walls (2 fold, p = 0.01). In the kindled vs. non-kindled rats, the post-seizure p53 level is increased 1.3 fold (p = 0.03), reciprocating a 1.4-fold (p = 0.02) decrease in the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) controlling the glutamate degradation. Further, decreased expression of deacylases SIRT3 (1.4 fold, p = 0.01) and SIRT5 (1.5 fold, p = 0.01) reciprocates increased acetylation of 15 kDa proteins 1.5 fold (p < 0.01). Finally, the kindling abrogates the stress response to multiple saline injections in the control animals, manifested in the increased activities of the pyruvate dehydrogenase complex, malic enzyme, glutamine synthetase and decreased malate dehydrogenase activity. Post-seizure animals demonstrate correlations of p53 expression to the levels of glutamate (r = 0.79, p = 0.05). The correlations of the seizure severity and duration to the levels of GABA (r = 0.59, p = 0.05) and glutamate dehydrogenase activity (r = 0.58, p = 0.02), respectively, are substituted by the correlation of the seizure latency with the OGDHC activity (r = 0.69, p < 0.01) after the vitamins administration, testifying to the vitamins-dependent impact of the kindling on glutamate/GABA metabolism. The vitamins also abrogate the correlations of behavioral parameters with seizure duration (r 0.53-0.59, p < 0.03). Thus, increased seizures and modified post-seizure behavior in rats after PTZ kindling are associated with multiple changes in the vitamin-dependent brain metabolism of amino acids, linked to key metabolic regulators: p53, OGDHC, SIRT3 and SIRT5.

Interplay Between Thiamine and p53/p21 Axes Affects Antiproliferative Action of Cisplatin in Lung Adenocarcinoma Cells by Changing Metabolism of 2-Oxoglutarate/Glutamate.

Thiamine (vitamin B1) is often deficient in oncopatients, particularly those undergoing chemotherapy. However, interaction between the thiamine deficiency and anticancer action of drugs has not been characterized. A major natural thiamine derivative, thiamine diphosphate (ThDP), is a coenzyme of central metabolism, also known to affect transcriptional activity of the master metabolic regulator and genome guardian p53. A direct transcriptional target of p53, p21, regulates cell cycle dynamics and DNA damage response. Our work focuses on dependence of the action of the DNA damaging anticancer drug cisplatin on metabolic regulation through p53/p21 axes and cellular thiamine status in human lung adenocarcinoma cells A549. These cells are used as a model of a hardly curable cancer, known to develop chemoresistance to platinum drugs, such as cisplatin. Compared to wild type (A549WT), a stable line with a 60% knockdown of p21 (A549p21–) is less sensitive to antiproliferative action of cisplatin. In contrast, in the thiamine-deficient medium, cisplatin impairs the viability of A549p21– cells more than that of A549WT cells. Analysis of the associated metabolic changes in the cells indicates that (i) p21 knockdown restricts the production of 2-oxoglutarate via glutamate oxidation, stimulating that within the tricarboxylic acid (TCA) cycle; (ii) cellular cisplatin sensitivity is associated with a 4-fold upregulation of glutamic-oxaloacetic transaminase (GOT2) by cisplatin; (iii) cellular cisplatin resistance is associated with a 2-fold upregulation of p53 by cisplatin. Correlation analysis of the p53 expression and enzymatic activities upon variations in cellular thiamine/ThDP levels indicates that p21 knockdown substitutes positive correlation of the p53 expression with the activity of 2-oxoglutarate dehydrogenase complex (OGDHC) for that with the activity of glutamate dehydrogenase (GDH). The knockdown also changes correlations of the levels of OGDHC, GDH and GOT2 with those of the malate and isocitrate dehydrogenases. Thus, a p53/p21-dependent change in partitioning of the glutamate conversion to 2-oxoglutarate through GOT2 or GDH, linked to NAD(P)-dependent metabolism of 2-oxoglutarate in affiliated pathways, adapts A549 cells to thiamine deficiency or cisplatin treatment. Cellular thiamine deficiency may interfere with antiproliferative action of cisplatin due to their common modulation of the p53/p21-dependent metabolic switch between the glutamate oxidation and transamination.

Regulation of Thiamine (Vitamin B1)-Dependent Metabolism in Mammals by p53.

Transcriptional factor p53 is a master regulator of energy metabolism. Energy metabolism strongly depends on thiamine (vitamin B1) and/or its natural derivatives. Thiamine diphosphate (ThDP), which is a major thiamine derivative, affects p53 binding to DNA. In order to elucidate the mechanism of regulation of thiamine-dependent metabolism by p53, we assessed putative p53-binding sites near transcription starting points in genes coding for transporters and enzymes, whose function is associated with thiamine and/or its derivatives. The predictions were validated by studying cell metabolic response to the p53 inducer cisplatin. Expression of p53 and its known target, p21, has been evaluated in cisplatin-treated and control human lung adenocarcinoma A549 cells that possess functional p53 pathway. We also investigated the activity of enzymes involved in the thiamine-dependent energy metabolism. Along with upregulating the expression of p53 and p21, cisplatin affected the activities of metabolic enzymes, whose genes were predicted as carrying the p53-binding sites. The activity of glutamate dehydrogenase GDH2 isoenzyme strongly decreased, while the activities of NADP+-dependent isocitrate dehydrogenase (IDH) and malic enzymes, as well as the activity of 2-oxoglutarate dehydrogenase complex at its endogenous ThDP level, were elevated. Simultaneously, the activities of NAD+-dependent IDH, mitochondrial aspartate aminotransferase, and two malate dehydrogenase isoenzymes, whose genes were not predicted to have the p53-binding sequences near the transcription starting points, were upregulated by cisplatin. The p53-dependent regulation of the assayed metabolic enzymes correlated with induction of p21 by p53 rather than induction of p53 itself.

Hypoxic Adaptation of Mitochondrial Metabolism in Rat Cerebellum Decreases in Pregnancy.

Function of brain amino acids as neurotransmitters or their precursors implies changes in the amino acid levels and/or metabolism in response to physiological and environmental challenges. Modelling such challenges by pregnancy and/or hypoxia, we characterize the amino acid pool in the rat cerebellum, quantifying the levels and correlations of 15 amino acids and activity of 2-oxoglutarate dehydrogenase complex (OGDHC). The parameters are systemic indicators of metabolism because OGDHC limits the flux through mitochondrial TCA cycle, where amino acids are degraded and their precursors synthesized. Compared to non-pregnant state, pregnancy increases the cerebellar content of glutamate and tryptophan, decreasing interdependence between the quantified components of amino acid metabolism. In response to hypoxia, the dependence of cerebellar amino acid pool on OGDHC and the average levels of arginine, glutamate, lysine, methionine, serine, phenylalanine, and tryptophan increase in non-pregnant rats only. This is accompanied by a higher hypoxic resistance of the non-pregnant vs. pregnant rats, pointing to adaptive significance of the hypoxia-induced changes in the cerebellar amino acid metabolism. These adaptive mechanisms are not effective in the pregnancy-changed metabolic network. Thus, the cerebellar amino acid levels and OGDHC activity provide sensitive markers of the physiology-dependent organization of metabolic network and its stress adaptations.

Improvement of poly(γ-glutamic acid) biosynthesis and redistribution of metabolic flux with the presence of different additives in Bacillus subtilis CGMCC 0833

Tween-80, dimethyl sulfoxide (DMSO), and glycerol could be used as novel materials to regulate the central carbon metabolic pathway and improve gamma-PGA biosynthesis by Bacillus subtilis CGMCC 0833. With glycerol in the medium, the activity of 2-oxoglutarate dehydrogenase complex at the key node of 2-oxoglutarate was depressed, more carbon flux distribution was directed to synthesize glutamate, the substrate of gamma-PGA, which led to overproducing of gamma-PGA, reached 31.7 g/l, compared to the original value of 26.7 g/l. When Tween-80 or DMSO was in the medium, the activity of isocitrate dehydrogenase was stimulated, the branch flux from 2-oxoglutarate to glutamate was also enhanced due to the increasing of total flux from iso-citrate to 2-oxoglutarate, then a large amount of glutamate was produced, and formation of gamma-PGA was also improved, which was a different process compared with that of glycerol. Moreover, with the addition of Tween-80 or DMSO, cell membrane permeability was increased, which facilitated the uptake of extracellular substrates and the secretion of gamma-PGA by this strain; therefore, gamma-PGA production was further stimulated, and 34.4 and 32.7 g/l gamma-PGA were obtained, respectively. This work firstly employed additives to improve the biosynthesis of gamma-PGA and would be helpful in understanding the biosynthesis mechanism of gamma-PGA by Bacillus species deeply.

Glutamate Overproduction inCorynebacterium glutamicumTriggered by a Decrease in the Level of a Complex Comprising DtsR and a Biotin-containing Subunit

Glutamate overproduction in Corynebacterium glutamicum is induced by Tween 40, biotin-limitation, or sublethal amounts of penicillin. Disruption of the dtsR gene, which encodes a putative component of a biotin-containing enzyme complex involved in fatty acid synthesis, causes constitutive overproduction of glutamate. We report here that overexpression of dtsR inhibits the induction of glutamate overproduction. In contrast, the level of DtsR in the wild type strain was found to decrease in the presence of Tween 40 or limited amounts of biotin. Tween 40, biotin-limitation, or dtsR disruption also reduced the activity of 2-oxoglutarate dehydrogenase complex (ODHC), which is involved in the synthesis of succinate from 2-oxoglutarate. These results indicate that decrease in the level of DtsR or a complex containing DtsR triggers the increased synthesis of glutamate from 2-oxoglutarate by lowering the ODHC activity.

An enzyme and13C-NMR study of carbon metabolism in heliobacteria

Heliobacteria are a group of anoxygenic phototrophs that can grow photoheterotrophically in defined minimal media on only a limited range of organic substrates as carbon sources. In this study the mechanisms which operate to assimilate carbon and the routes employed for the biosynthesis of cellular intermediates were investigated in a newHeliobacterium strain, HY-3. This was achieved using two approaches (1) by measuring the activities of key enzymes in cell-free extracts and (2) by the use of(13)C nuclear magnetic resonance (NMR) spectroscopy to analyze in detail the labelling pattern of amino-acids of cells grown on [(13)C] pyruvate and [(13)C] acetate.Heliobacterium strain HY-3 was unable to grow autotrophically on CO2/H2 and neither (ATP)-citrate lyase nor ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBPcase) were detectable in cell-free extracts. The enzyme profile of pyruvate grown cells indicated the presence of a pyruvate:acceptor oxidoreductase at high specific activity which could convert pyruvate to acetyl-Coenzyme A. No pyridine nucleotide dependent pyruvate dehydrogenase complex activity was detected. Of the citric-acid cycle enzymes, malate dehydrogenase, fumarase, fumarate reductase and an NADP-specific isocitrate dehydrogenase were readily detectable but no aconitase or citrate synthase activity was found. However, the labelling pattern of glutamate in long-term 2-[(13)C] acetate incorporation experiments indicated that a mechanism exists for the conversion of carbon from acetyl-CoA into 2-oxoglutarate. A 2-oxoglutarate:acceptor oxidoreductase activity was present which was also assayable by isotope exchange, but no 2-oxoglutarate dehydrogenase complex activity could be detected. Heliobacteria appear to use a type of incomplete reductive carboxylic acid pathway for the conversion of pyruvate to 2-oxoglutarate but are unable to grow autotrophically using this metabolic route due to the absence of ATP-citrate lyase.

Effect of ionic strength on the regulatory properties of 2-oxoglutarate dehydrogenase complex

The effects of changing ionic strength on the activity of the 2-oxoglutarate dehydrogenase complex from pig kidney cortex were explored. This enzyme complex is found to be influenced in many ways by the ionic strength of the reaction medium. The enzyme shows an optimum activity at 0.1 M ionic strength. Increase in ionic strength from 0.1 M to 0.2 M resulted in a decrease of S 0.5 for 2-oxoglutarate, and in an increase of S 0.5 for NAD. Changes in ionic strength over the range of 0.05–0.2 M have little, if any, effect on S 0.5 for CoA. The Hill coeffient for 2-oxoglutarate and NAD at 0.2 M ionic strength was 1.0, whereas at 0.05 M ionic strength it was 0.85 and 1.2 for 2-oxoglutarate and NAD, respectively At 0.05 M ionic strength the pH optimum of the enzyme ranges between 7.4–7.6, but at 0.15 M ionic strength the pH optimum shifts to 7.8. The magnitude of inhibition of enzyme activity by ATP is not influenced by changes in ionic strength in the absence of calcium. However, in the presence of Ca 2+, increases in ionic strength lower the inhibitory effects of ATP. The S 0.5 i for ATP in both presence and absence of Ca 2+ was not affected by changes in ionic strength in the range of 0.1–0.2 M. In contrast, the a 0.5 for ADP in the absence of Ca 2+ decreases as ionic strength increases. In the presence of calcium and 0.2 M ionic strength ADP has no effect on 2-oxoglutarate dehydrogenase complex activity.

S-Ethyl dipropylthiocarbamate (EPTC) and 2,2-dichloro- N,N-di-2-propenylacetamide (Dichlormid) inhibitions of synthesis of acetyl-coenzyme A derivatives

[2- 14C]Acetate incorporation into acetyl-coenzyme A (acetyl-CoA) in wheat ( Triticum aestivum L. cv Stacy) chloroplasts was inhibited by S-ethyl dipropylthiocarbamate (EPTC, 0.01 μ M) and the inhibition was reversed by 90 μ M acetate. [2- 14C]Pyruvate was converted to acetyl-CoA by the pyruvate dehydrogenase complex (PDC) in wheat chloroplasts and the conversion was inhibited by EPTC (0.01 μ M). The inhibition was reversed by 90 μ M pyruvate. Production of NADH by wheat (cv. Holly) mitochondrial PDC was inhibited 10% by 3.3 10 −14 M EPTC and 90% by 0.33 μ M EPTC. Mitochondrial 2-oxoglutarate dehydrogenase complex activity was inhibited 65% by 0.033 μ M EPTC. Dichlormid (2,2-dichloro- N,N-di-2-propenylacetamide, 0.01 μ M) inhibited the incorporation of [2- 14C]acetate into wheat chloroplast acetyl-CoA and the inhibition was reversed by 90 μ M acetate. Dichlormid (0.01 μ M) also inhibited the chloroplastic conversion of [2- 14C]pyruvate into acetyl-CoA and partially reversed the inhibition by EPTC (0.1 μ M) of conversion of [2- 14C]pyruvate to acetyl-CoA.

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.

Substrate-level phosphorylation in isolated yeast mitochondria.

The activity and the control of substrate-level phosphorylations in isolated yeast mitochondria were investigated. The oligomycin-insensitive ATP synthesis rate linked to 2-oxoglutarate oxidation is of similar order of magnitude to that observed in oxidative phosphorylation. The flux control coefficients of respiratory chain activity, translocase and phosphate carrier activities were close to zero. Kinetic control was confined to 2-oxoglutarate supply and 2-oxoglutarate dehydrogenase complex activity. The study of endogenous nucleotide phosphorylation showed that ADP is the first phosphate acceptor during this process. Moreover, the comparison between the whole intramitochondrial content of nucleotides and phosphate to the fraction involved in substrate-level phosphorylation indicated a metabolic compartmentation of nucleotides and phosphate. A linear relationship was observed between the 2-oxoglutarate-linked ATP synthesis rate and the internal phosphate potential: delta G'p = delta G'o + RT 1n ([ATP]/[ADP] [Pi]). The fact that the substrate-level phosphorylation process alone was able to maintain a high internal phosphate potential has an important bioenergetic consequence, particularly for yeast grown on fermentable substrates.

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of neurospora crassa and compared localization with the pyruvate dehydrogenase complex

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.

The effects of calcium ions and adenine nucleotides on the activity of pig heart 2-oxoglutarate dehydrogenase complex.

1. The effects of Ca2+ (mainly by using EGTA buffers), pH, ATP and ADP on the activity of the 2-oxoglutarate dehydrogenase complex from pig heart were explored. 2. Ca2+ (about 30 micrometer) resulted in a decrease in the apparent Km for 2-oxoglutarate from 2.1 to 0.16 mM (at pH 7) without altering the maximal velocity. At 0.1 mM-oxoglutarate there was a 4--5-fold activation by Ca2+, with an apparent Km for Ca2+ of 1.2 micrometer. A similar activation was also observed with Sr2+ (Km 15.1 micrometer), but not wised markedly from pH 7.4 TO 6.6. The effects of Ca2+ remained evident over this pH range. 4. In the presence of Mg2+, ATP resulted in a marked increase in the apparent Km for oxoglutarate, whereas ADP greatly decreased thisp arameter. The concentrations of adenine nucleotide required for half-maximal effects were about 10 micrometer in each case. 5. The effects of the adenine nucleotides and Ca2+ on the apparent Km for oxoglutarate appeared to be essentially independent of each other, reversible, and demonstrable in the presence of end product inhibition by NADH and obtained. 6. Effects similar to those described above were also observed on the activity of 2-oxoglutarate dehydrogenase from rat heart and brown adipose tissue. 7. We discuss the mechanisms controlling this enzyme's activity and compare these regulatory features with those of NAD-isocitrate dehydrogenase and the pyruvate dehydrogenase system, which are also sensitive to Ca2+ and adenine nucleotides.

Suppression of the Succinate Requirement of Lipoamide Dehydrogenase Mutants of Escherichia coli by Mutations Affecting Succinate Dehydrogenase Activity

Reversion studies with a variety of different lipoamide dehydrogenase mutants of Escherichia coli k12 led to the discovery of a class of partial revertant which no longer required succinate for aerobic growth on glucose but retained all the other nutritional characteristics of the Lpd− phenotype. These revertants were given the phenotypic designation Lpd−Sin+ to denote their succinate independence, and mutations in the gene(s) designated sin were considered responsible for generating the succinate-independent phenotype of lpd mutants. Enzymological and genetic studies confirmed that the revertants retained their lpd mutations. Moreover, all of the 24 independent revertants tested possessed sin mutations located in the gal region of the chromosome, presumably suppressing by an intergenic and indirect mechanism. The sin mutations exhibited no allele specificity for suppression and they were recessive to the wild-type sin + allele in merodiploid strains. A common feature of the Lpd−Sin+ revertants (succinate-independent derivatives of lpd mutants) was a deficiency in succinate dehydrogenase. On their own, the sin mutations produced a phenotype analogous to that of sdh mutants. Furthermore, the probable identity of sin and sdh mutations was confirmed by reconstruction studies in which an lpd sdh double mutant was shown to exhibit an Lpd−Sin+ phenotype. It was concluded that the requirement for exogenous succinate by mutants lacking lipoamide dehydrogenase (and hence overall 2-oxoglutarate dehydrogenase complex activity) can be eliminated by inactivating succinate dehydrogenase because the latter enzyme depletes the intracellular succinate and succinylCoA pools by removing succinate faster than it can be supplied by other endogenous mechanisms. In fact, it would appear that the presence of active succinate dehydrogenase is responsible for the nutritional requirement for succinate of mutants lacking overall 2-oxoglutarate dehydrogenase complex activity during aerobic growth on glucose. A gene-dosage effect on the activities of the 2-oxoglutarate dehydrogenase complex was also demonstrated with corresponding merodiploid strains.

Inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in developing rat and human brain.

1. The effect of the branched-chain amino acids, namely leucine, isoleucine and valine and their corresponding 2-oxo acids on the metabolism of 2-oxoglutarate by developing rat and human brain preparations was investigated. 2. The decarboxylation of 2-oxo[1-(14)C]glutarate to (14)CO(2) by mitochondria from adult rat brain was inhibited by the branched-chain 2-oxo acids whereas the branched-chain amino acids had no inhibitory effect on this process. 3. The activity of 2-oxoglutarate dehydrogenase complex was about 0.2unit/g of brain from 2-day-old rats and increased by about fourfold reaching an adult value by the end of the third postnatal week. 4. The K(m) value for 2-oxoglutarate of the 2-oxoglutarate dehydrogenase complex in rat and human brain was 100 and 83mum respectively. 5. The branched-chain 2-oxo acids competitively inhibited this enzyme from suckling and adult rats brains as well as from foetal and adult human brains, whereas the branched-chain amino acids had no effect on this enzyme. 6. Approximate K(i) values for the branched-chain 2-oxo acids found for this enzyme were in the range found for these 2-oxo acids in plasma from patients with maple-syrup-urine disease. 7. The possible significance of the inhibition by the branched-chain 2-oxo acids of the 2-oxoglutarate dehydrogenase complex in brains of untreated patients with maple-syrup-urine disease is discussed in relation to the energy metabolism and the biosynthesis of lipids from ketone bodies.