Characterization of aconitate hydratase from mitochondria and cytoplasm of ascites tumor cells

This paper describes the characterization of aconitate hydratase (EC 4.2.1.3) in cytoplasmic and mitochondrial extracts from Ehrlich ascites tumor cells carried by BALB/C mice. The results show a similar distribution of aconitate hydratase in both extracts, with specific activities much lower than those found in pig and mouse tissues. Mitochondrial aconitate hydratase shows a substrate inhibition by citrate with a Km similar to that found in cytoplasm (Km = 1.0 mM and 0.9 mM, respectively). Oxalacetate produces a mixed type of inhibition in both cytoplasmic and mitochondrial aconitate hydratases with different inhibition constants (Ki = 0.3 mM and 1.0 mM, respectively). Moreover, the specific activities of aconitate hydratase in both cytoplasm and mitochondria decrease when the tumor progresses in the peritoneum of BALB/C mice, as well as the percentage of aconitate hydratase activity in the presence of oxalacetate as the inhibitor. These results indicate that the activity and kinetics of aconitate hydratase are markedly altered by neoplastic transformation as occurs in Ehrlich ascites tumor cells. Since aconitate hydratase is not a key enzyme, these unexpected data are of interest in the study of cancer biochemistry.     

Regulation of aconitate hydratase activity from rat kidney cortex by bicarbonate.

1. The increase in pH value and bicarbonate concentration stimulated citrate synthesis from pyruvate and malate, inhibiting simultaneously conversion of isocitrate to citrate. 2. Bicarbonate inhibited competitively the activity of aconitate hydratase, probably binding with the two active sites of the enzyme. The Ki values for the cytoplasmic and mitochondrial enzyme were, respectively, 27 and 38 mM. The pH optimum for both forms of the enzyme in Tris-HCl buffer was in the range 7.8-8.6, and in bicarbonate buffer varied from 7.2 to 8.0, depending on the form of the enzyme and the substrate used. 3. Only free, completely dissociated citrate anion acts as a substrate for aconitate hydratase. 4. The role of aconitate hydratase as a factor controlling the rate of citrate metabolism in kidney in metabolic alkalosis is discussed.     

Metabolism of Bacillus thuringiensis in Relation to Spore and Crystal Formation

A general pattern of metabolism was determined for Bacillus thuringiensis grown in a glucose-yeast extract-salts medium. The pattern did not differ significantly from that of B. cereus grown in a similar medium. Acetic acid produced from glucose during exponential growth was further catabolized in the early sporulation phase of growth, at which time the specific activity of aconitate hydratase increased markedly. Fluoroacetate and alpha-picolinate prevented the removal of accumulated acid, and the resulting low pH inhibited spore and crystal synthesis. Neither crystal-related antigens nor insect toxicity was shown by cells whose crystal synthesis was inhibited in this way. alpha-Picolinate prevented the normal increase in specific activity of aconitate hydratase without inhibiting exponential growth. It also inhibited aconitate hydratase in vitro, but only if preincubated with the enzyme. alpha-Picolinate did not inhibit the increase in specific activity of aconitate hydratase or spore and crystal synthesis in a medium buffered near neutrality. Chloramphenicol and actinomycin D inhibited crystal enlargement and sporulation when added to cells in which small crystals had already begun to form. Typical messenger ribonucleic acid-dependent protein synthesis, rather than the type associated with peptide antibiotic synthesis, is thus indicated for the synthesis of crystal peptide subunits.     

The inhibition by fluorocitrate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1. The effects of synthetic fluorocitrate were studied on: (a) the oxidation of citrate and cis-aconitate by rat liver mitochondria; (b) the activity of the aconitate hydratase found in the liver cell sap; (c) the activity of the aconitate hydratase solubilized from liver mitochondria. 2. Fluorocitrate was found to be a potent inhibitor of oxidation of citrate but only a weak inhibitor of oxidation of cis-aconitate: 6.7mum-fluorocitrate (containing 4% of the inhibitory isomer) caused 94% inhibition of the oxidation of citrate (2mm) whereas 1.0mm-fluorocitrate was necessary to provoke the same inhibition when cis-aconitate (2mm) was the substrate. The degree of inhibition varied in relation to the respiratory state of mitochondria when fluorocitrate was added. The inhibition could be partially reversed by cis-aconitate. 3. The aconitate hydratase extracted from the mitochondria was much less inhibited by fluorocitrate than was the mitochondria-bound enzyme, and the aconitate hydratase found in the cell sap was even less sensitive. 0.3mm-Fluorocitrate was required to cause 50% inhibition of the reaction citrate-->cis-aconitate, catalysed by the aconitate hydratase extracted from the mitochondria, and 1.2m-fluorocitrate for the extramitochondrial enzyme. For both enzymes the reaction citrate-->cis-aconitate was 2-3 times more sensitive to fluorocitrate than was the reaction isocitrate-->cis-aconitate. The inhibition was of the competitive type for both reactions.     

Control of the citric acid cycle by glyoxylate. The mechanism of inhibition of oxoglutarate dehydrogenase, isocitrate dehydrogenase and aconitate hydratase

1. The effects of glyoxylate on partially purified preparations of aconitate hydratase, isocitrate dehydrogenase and oxoglutarate dehydrogenase were compared with those of oxalomalate and hydroxyoxoglutarate (obtained by condensation of glyoxylate with oxaloacetate and pyruvate respectively). 2. Glyoxylate (1mm) did not affect aconitate hydratase and isocitrate dehydrogenase, whereas oxalomalate (1mm) inhibited the enzyme activities completely. 3. Glyoxylate (0.025mm) inhibited oxoglutarate dehydrogenase irreversibly, whereas the same concentrations of oxalomalate and hydroxyoxoglutarate were ineffective. This inhibitory effect was prevented if oxoglutarate, pyruvate or oxaloacetate was mixed with the enzyme before the glyoxylate. 4. Incubation of oxoglutarate dehydrogenase with radioactive glyoxylate produced radioactive carbon dioxide; radioactivity was also recovered in the portion of the enzyme identified with thiamin pyrophosphate. 5. The behaviour of glyoxylate in producing multiple inhibitions of the citric acid cycle, either by direct interaction with oxoglutarate dehydrogenase, or by means of its condensation compounds which inhibit aconitate hydratase and isocitrate dehydrogenase, is discussed.     

Catalytic Properties of Cytoplasmic and Mitochondrial Aconitate Hydratase from Rat Cardiomyocytes

Enzymatic activity of aconitate hydratase (aconitase, EC 4.2.1.3) from the rat heart is localized in the cytoplasm (65%) and mitochondria (35%). Cytoplasmic and mitochondrial forms of aconitate hydratase were separated by ion-exchange chromatography on CM-Cellulose and CM-Sephadex. The two forms have similar molecular weight, optimal pH range, and spectral properties; however, they have different chromatography properties, K _m for citrate and isocitrate, as well as sensitivity to Fe²⁺ ions.     

Fluorocitrate inhibition of aconitate hydratase and the tricarboxylate carrier of rat liver mitochondria

1. The effect of biologically synthesized and purified fluorocitrate on the metabolism of tricarboxylate anions by isolated rat liver mitochondria was investigated, in relation to the claim by Eanes et al. (1972) that this fluoro compound inhibits the tricarboxylate carrier at concentrations at which it has little effect on the aconitate hydratase activity. 2. That the inhibitory action of fluorocitrate is at the level of the aconitate hydratase and not at the level of the tricarboxylate carrier is indicated by the following findings. Although the oxidation of citrate and cis-aconitate, but not that of isocitrate, was inhibited by fluorocitrate, the exchange of internal citrate for external citrate or l-malate was not. Had the tricarboxylate carrier been affected, these latter exchange reactions would have been inhibited. 3. By using aconitate hydratase solubilized from mitochondria it was found that with citrate as substrate the inhibition by fluorocitrate was partially competitive (K(i)=3.4x10(-8)m), whereas with cis-aconitate as substrate the inhibition was partially non-competitive (K(i)=3.0x10(-8)m).     

Effects of lipoic acid on citrate content, aconitate hydratase activity, and oxidative status during myocardial ischemia in rats

The effects of lipoic acid on intensity of free radical reactions, citrate content, and aconitate hydratase during myocardial ischemia have been investigated. Treatment with lipoic acid normalized biochemiluminescence parameters and citrate level, which were increased in the myocardial pathology. Treatment with lipoic acid also increased specific activity of aconitate hydratase, which was decreased in myocardium and blood of animals with myocardial ischemia. Administration of lipoic acid decreased DNA fragmentation observed during myocardial ischemia. The data suggest that lipoic acid can be effectively used as a cardioprotector preventing the development of free radical oxidation during myocardial ischemia.     

Enzymatic Activity in Mitochondria from Orobanche

Mitochondria from Orobanche were analysed for the activities of aconitate hydratase, isocitrate dehydrogenase, succinate dehydro-genase, fumarate hydratase, malate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases, glutamate dehydrogenase, aminotransferases, ATPase and “malic” enzyme. The specific activities of isocitrate dehydrogenase, NADH oxidase, substrate-cytochrome c oxidoreductases and glutamate dehydrogenase in the mitochondria) fraction from parasite tissue compared favourably with those reported for most of the mitochondria from growing and storage tissues. Succinate dehydrogenase, fumarate hydratase and aspartate aminotransferase were of intermediate activity, while aconitate hydratase and malate dehydrogenase had rather low activity, and “malic” enzyme had very low activity in comparison with other preparations. The relevance of these findings in relation to mitochondrial metabolism in the parasite is discussed. No evidence was obtained to suggest any basic abnormality in the biochemical properties of the mitochondria from Orobanche centua which may be correlated with its obligatorily parasitic existence.     

Oxalate accumulation from citrate by Aspergillus niger. II. Involvement of the tricarboxylic acid cyclase.

Carbon-14 was incorporated into oxalate and CO2 from either citrate-1,5-14C, succinate-1,4-14C, or fumarate-1,4-14C by cultures of Aspergillus niger pregrown on a medium which contained glucose as the sole carbon source and which did not allow citrate accumulation. In cell-free extracts of mycelium forming oxalate and CO2 from added citrate the following enzymes of the tricarboxylic acid (TCA) cycle were identified: citrate synthase CE 4.1.3.7), aconitate hydratase (EC4.2.1.3), NAD and NADP-dependent isocitrate dehydrogenase (EC 1.1.1.41, 1.1.1.42), (alpha-oxoglutarate dehydrogenase (EC 1.2.4.2), succinate dehydrogenase (EC 1.3.99.1), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37). The in vitro activity of aconitate hydratase and of NADP-dependent isocitrate dehydrogenase was shown to be almost identical to the rate of in vivo degradation of citrate or to exceed this rate. The degradation of citrate to oxalate was inhibited completely by 9 mM fluoroacetate. It is concluded that the TCA cycle is involved in the formation of oxalate from citrate.     

Citrate Cycle and Related Metabolism of Listeria monocytogenes

The growth response of Listeria monocytogenes strains A4413 and 9037-7 to carbohydrates was determined in a defined medium. Neither pyruvate, acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, nor malate supported growth. Furthermore, inclusion of any of these carbohydrates in the growth medium with glucose did not increase the growth of Listeria over that observed on glucose alone. Resting cell suspensions of strain A4413 oxidized pyruvate but not acetate, citrate, isocitrate, alpha-ketoglutarate, succinate, fumarate, or malate. Cell-free extracts of strain A4413 contained active citrate synthase, aconitate hydratase, isocitrate dehydrogenase, malate dehydrogenase, fumarate hydratase, fumarate reductase, pyruvate dehydrogenase system, and oxidases for reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. The alpha-ketoglutarate oxidation system, succinate dehydrogenase, isocitrate lyase, and malate synthase were not detected. Cytochromes were not detected. The data suggest that strain A4413, under these conditions, utilizes a split noncyclic citrate pathway which has an oxidative portion (citrate synthase, aconitate hydratase, and isocitrate dehydrogenase) and a reductive portion (malate dehydrogenase, fumarate hydratase, and fumarate reductase). This pathway is probably important in biosynthesis but not for a net gain in energy.     

Relationship between Aconitate Hydratase Activity and Citric Acid Productivity in Fluoroacetate-sensitive Mutant Strain of Candida lipolytica

Fluoroacetate-sensitive mutant strains, K–20 and S–22, of Candida lipolytica could not grow or could only slightly grow on agar media containing di- or tricarboxylic acid involved in the TCA-cycle as the sole source of carbon. Relative activities of aconitate hydratase in the cells of the mutant strains, K-20 and S-22, were approximately 1/10 and 1/100, against that of the parent strain, respectively. This facts support the statement that the mutant strains were extremely sensitive to monofiuoroacetate.

The aconitate hydratase activities of these mutant strains and the parent strain corresponded well to the citric to (+)-isocitric acid ratio in the final fermented broths.     

2-Oxoglutarate: Oxidation and Role as a Potential Precursor of Cytosolic Acetyl-CoA for the Synthesis of Acetylcholine in Rat Brain Synaptosomes

The possibility that 2-oxoglutarate may supply acetyl units for the cytosolic synthesis of acetylcholine in rat brain synaptosomes was investigated. The contribution of [14C]2-oxoglutarate to the synaptosomal synthesis of [14C]acetylcholine was found to be negligible despite evidence for its uptake and oxidation. The activity of the enzymes NADP-isocitrate dehydrogenase (EC 1.1.1.42), aconitate hydratase (EC 4.2.1.3), and ATP citrate-lyase (EC 4.1.3.8) were measured in the synaptosol. NADP-isocitrate dehydrogenase and aconitate hydratase are present at three- to 1.5-fold higher activities than ATP citrate-lyase. It seems likely that these enzymes contribute to the metabolism of citrate and prevent detectable formation of cytosolic acetyl-CoA from exogenously added 2-oxoglutarate (or citrate). The data further suggest that ATP citrate-lyase may in part be associated with the mitochondrial fraction.     

Control of the citric acid cycle by glyoxylate: Mechanism of the inhibition by oxalomalate and γ-hydroxy-α-oxoglutarate

1. Hydroxyoxoglutarate was obtained by three methods: decarboxylation of oxalomalic acid, and synthesis from glyoxylate and pyruvate by using either Mg²⁺ or an enzyme from rat liver as catalysts. 2. The inhibitory effects of oxalomalate and hydroxyoxoglutarate upon aconitate hydratase, isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase were investigated. 3. Oxalomalate at low concentrations (1mm) inhibited almost completely both aconitate hydratase and isocitrate dehydrogenase. Hydroxyoxoglutarate also inhibited these enzymes, but at concentrations approximately tenfold that of oxalomalate. 4. Oxalomalate and hydroxyoxoglutarate, at the higher concentrations, inhibited oxoglutarate dehydrogenase to approximately the same extent. 5. It is suggested that the ability of glyoxylate to control reaction rates in the tricarboxylic acid cycle must in some degree be due to its condensation with oxaloacetate and pyruvate to form enzyme inhibitors.     

Inhibition by oxalomalate of rat liver mitochondrial and extramitochondrial aconitate hydratase

1. The effect of oxalomalate on the oxidation of citrate and cis-aconitate in rat liver mitochondria, and on the activity of mitochondrial and cytoplasmic aconitate hydratase, has been investigated. 2. Oxalomalate that was added to intact rat liver mitochondria at high concentrations (2mm) produced complete inhibition of citrate and cis-aconitate oxidation, but lower concentrations (0.1-0.25mm) inhibited oxidation of citrate more than that of cis-aconitate. 3. Aconitate hydratase that was either extracted from mitochondria or soluble in the cytoplasm, was strongly inhibited by low concentrations of oxalomalate (0.01-0.2mm), the mitochondrial enzyme being more sensitive than the soluble one. 4. Oxalomalate, when added together with citrate, produced competitive inhibition; the K(i) values calculated were 1x10(-6)m for the mitochondrial and 2.5x10(-6)m for the cytoplasmic enzyme. 5. With both the enzymic preparations oxalomalate added together with the substrates inhibited the initial rate of the reaction citrate-->cis-aconitate more than that of the reaction isocitrate-->cis-aconitate. 6. After 2min of preincubation of the inhibitor with either of the enzymic preparations the inhibition increased tenfold and became irreversible; under these conditions both the reactions were inhibited to the same extent. 7. The inhibition by oxalomalate of aconitate hydratase appeared to be similar in many respects to that produced by fluorocitrate on the same enzyme.     

Speciation of coagulase negative staphylococci, isolated from indoor air, using SDS PAGE gel bands of expressed proteins followed by MALDI TOF MS and MALDI TOF-TOF MS-MS analysis of tryptic peptides

A group of coagulase negative staphylococcal strains isolated from indoor air of occupied school rooms were the subject of this study. Conventional MALDI TOF MS profiling of cellular extracts and physiological tests (including API STAPH) provided incomplete identification of the set of strains. After separation of a 100 kDa band using 1D gel electrophoresis, profiling of peptides (released with tryptic digestion) using MALDI TOF MS allowed improved bacterial speciation in addition to determination of the identity of the protein of origin (aconitate hydratase). This was performed by Mascot search, empirical observation and computer-generated cross-correlation analysis of environmental isolates versus reference strains. The species studied included some with sequenced genomes and others with un-sequenced genomes. Peptide sequences were confirmed to originate from aconitate hydratase using MALDI TOF-TOF MS-MS analysis of a diverse set of m/z values representing variable and conserved sequences. The methodological approach described here might have widespread application in speciation of environmental isolates of diverse origin and in identification of their expressed proteins.     

Biochemical lesions of respiratory enzymes and configurational changes of mitochondria in vivo

Summary Correlative biochemical and electron microscopic alterations were observed in chick embryo myoblasts in vitro after treatment with fluoroacetate. Fluoroacetate poisoning caused an increase of citrate and a decrease of ATP in the cultures. Cell respiration was only slightly impaired by fluoroacetate in the first 10 min but was inhibited to 30% one hour after exposure to the poison. Fluoroacetate did not affect oxidative phosphorylation. The evidence suggests that fluoroacetate was transformed in myoblasts into fluorocitrate which inhibited the mitochondrial-bound aconitate hydratase as in adult tissues. Ultrastructural changes in the majority of the fluoroacetate-treated cells were observed. Very few myoblasts appeared unaffected by the poison. Mitochondria were specifically altered. The early changes occurred in the mitochondrial matrix where the inhibited enzyme is known to be located and were followed by modifications in the configuration and structure of cristae. Exogenous fluorocitrate caused ultrastructural changes in the mitochondria similar to that provoked by fluoroacetate. The localization of the early change in the mitochondrial matrix and the evaluation of the structural modifications suggest a correlation between the biochemical lesion, i.e. the inhibition of aconitate hydratase, and the change revealed in the mitochondrial structure containing the inhibited enzyme.This work was supported by grants of the Consiglio Nazionale delle Ricerche to both InstitutesThe present study is dedicated to Prof. Otto Bucher on occasion of his 65th birthday     

刺芹侧耳不同发育时期的转录组差异分析

为了探讨刺芹侧耳子实体生长发育时期的基因表达变化,本文利用高通量测序技术对刺芹侧耳不同发育时期（菌丝期、原基期、子实体时期）进行RNA-Seq分析,在转录水平上解析差异表达基因在刺芹侧耳生长发育过程中的作用和功能。KEGG功能富集显示,菌丝期差异表达基因主要富集在碳代谢和氨基酸代谢中,其中三羧酸循环中编码柠檬酸合酶、乌头酸水合酶、异柠檬酸脱氢酶、琥珀酰辅酶A合成酶、琥珀酸脱氢酶、苹果酸脱氢酶的基因表达量均上调,说明碳代谢和氨基酸代谢是菌丝时期的主要能量来源;原基期上调的差异表达基因主要富集在脂肪酸代谢,其中RT-PCR定量结果显示原基期编码脂肪酸合酶的基因和编码脂酰辅酶A合成酶的基因下调,编码超氧化物酶的基因和编码过氧化氢酶的基因上调,表明脂肪酸代谢和抗氧化酶对刺芹侧耳原基期维持机体的稳定和生物应激方面起着重要作用。子实体时期上调的差异表达基因主要富集在剪接体、类固醇的生物合成以及AMPK信号通路中,说明环境因子对子实体时期有一定的影响。     

Regulation of biosynthesis of secondary metabolites. I. Biosynthesis of chlortetracycline and tricarboxylic acid cycle activity

In the course of submerged cultivation of low-production and industrial production strains of Streptomyces aureofaciens, the activity of enzymes of the tricurboxylic acid cycle was studied. The activities of citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2), and malate dehydrogenase (EC 1.1.1.37) were estimated spectrophotometrically in cell-free preparations. In the growth phase, mainly the initial reactions of the cycle were active with both strains. In production-phase, the activities of enzymes in the low-production strain were 2–5 × higher than in the production strain. Benzylthioeyanate, at a concentration of 5 × l0^?5M, stimulated chlortetracycline production of both strains with accompanying decrease in activity of the enzymes of the tricarboxylic acid cycle. The role of the tricarboxylic acid cycle in control of chlortetracycline biosynthesis is discussed.     

Organic acid metabolism in Sclerotinia sclerotiorum

Summary Citrate synthase (EC 4.1.3.7), aconitate hydratase (EC 4.2.1.3), NADP specific isocitrate dehydrogenase (EC 1.1.1.42), fumarate hydratase (EC 4.2.1.2) and malate dehydrogenase (EC 1.1.1.37) were detected in cell-free preparations of Sclerotinia sclerotiorum (Lib.) D By. grown on liquid glucose-salts medium in stationary culture. Isocitrate lyase (EC 4.1.3.1) was present when the fungus grew on a carbohydrate-free medium but was not detected when the cultures grew on the glucose-salts medium. The amount of oxalate in the culture filtrate declined as the specific activity of citrate synthase and malate dehydrogenase in the mycelium declined. Increasing the initial pH of the medium resulted in an increase of the dicarboxylic acids in the culture filtrate and the specific activity of malate dehydrogenase in the mycelium. The specific reaction(s) leading to oxalic acid formation were not identified.