Some Characteristics of Central Metabolism in Acinetobacter sp. Grown on Ethanol

Ethanol-grown cells of the mutant Acinetobacter sp. strain 1NG, incapable of producing exopolysaccharides, were analyzed for the activity of enzymes of the tricarboxylic acid (TCA) cycle and some biosynthetic pathways. In spite of the presence of both key enzymes (isocitrate lyase and malate synthase) of the glyoxylate cycle, these cells also contained all enzymes of the TCA cycle, which presumably serves biosynthetic functions. This was evident from the high activity of isocitrate dehydrogenase and glutamate dehydrogenase and the low activity of 2-oxoglutarate dehydrogenase. Pyruvate was formed in the reaction catalyzed by oxaloacetate decarboxylase, whereas phosphoenolpyruvate (PEP) was synthesized by the two key enzymes (PEP carboxykinase and PEP synthase) of gluconeogenesis. The ratio of these enzymes was different in the exponential and the stationary growth phases. The addition of the C₄-dicarboxylic acid fumarate to the ethanol-containing growth medium led to a 1.5- to 2-fold increase in the activity of enzymes of the glyoxylate cycle, as well as of fumarate hydratase, malate dehydrogenase, PEP synthase, and PEP carboxykinase (the activity of the latter enzyme increased by more than 7.5 times). The data obtained can be used to improve the biotechnology of production of microbial exopolysaccharide ethapolan on C₂-substrates.     

Properties of Candida lipolytica mutants with the modified glyoxylate cycle and their ability to produce citric and isocitric acid

Summary Enzyme activities of the tricarboxylic acid (TCA) cycle and the anaplerotic pathways, as well as the cell cytology of two C. lipolytica mutants with the modified glyoxylate cycle and their parent strain were studied during the exponential growth phase on glucose or hexadecane.Among the TCA cycle enzymes, the key enzyme citrate synthase had the highest activity in all three strains grown on both substrates. NAD-dependent isocitrate dehydrogenase had the minimum activity. All strains had well-developed mitochondria.Pyruvate carboxylation was active in the wild strain and mutant 2 grown on glucose, where this reaction is the basic anaplerotic pathway for oxal-acetate synthesis; mutant 1 had actively functioning enzymes for both anaplerotic pathways — pyruvate carboxylase, isocitrate lyase and malate synthase.During hexadecane assimilation, the number of peroxisomes in all strains increased sharply, accompanied by a simultaneous increase in isocitrate lyase activity.The low activities of both isocitrate lyase and pyruvate carboxylase in mutant 2 give reason to believe that this strain has an additional pathway for oxalacetic acid synthesis during the assimilation of n-alkane.     

The tricarboxylic acid cycle in L₃ Teladorsagia circumcincta: metabolism of acetyl CoA to succinyl CoA

Nematodes, like other species, derive much of the energy for cellular processes from mitochondrial pathways including the TCA cycle. Previously, we have shown L₃Teladorsagia circumcincta consume oxygen and so may utilise a full TCA cycle for aerobic energy metabolism. We have assessed the relative activity levels and substrate affinities of citrate synthase, aconitase, isocitrate dehydrogenase (both NAD⁺ and NADP⁺ specific) and α-ketoglutarate dehydrogenase in homogenates of L₃T. circumcincta. All of these enzymes were present in homogenates. Compared with citrate synthase, low levels of enzyme activity and low catalytic efficiency was observed for NAD⁺ isocitrate dehydrogenase and especially α-ketoglutarate dehydrogenase. Therefore, it is likely that the activity of these to enzymes regulate overall metabolite flow through the TCA cycle, especially when [NAD⁺] limits enzyme activity. Of the enzymes tested, only citrate synthase had substrate affinities which were markedly different from values obtained from mammalian species. Overall, the results are consistent with the suggestion that a full TCA cycle exists within L₃T. circumcincta. While there may subtle variations in enzyme properties, particularly for citrate synthase, the control points for the TCA cycle in L₃T. circumcincta are probably similar to those in the tissues of their host species.     

The oxidation of tricarboxylic acid cycle intermediates, with particular reference to isocitrate, by intact mitochondria isolated from the liver of the American eel, Anguilla rostrata leSueur.

The oxidation of exogenously added substrates has been studied in intact liver mitochondria isolated from the American eel, Anguilla rostrata. These data, coupled to determinations of the activity and localization of critical tricarboxylic acid (TCA) cycle enzymes, have been used to propose a pathway for the eel liver TCA cycle. (1) Isocitric, α-ketoglutaric, succinic, and malic acids are oxidized at essentially equivalent rates by eel mitochondria, with normal ADP:O and respiratory control ratios. No oxidation of citric, oxaloacetic, or pyruvic acids was detected when added alone or with malate, although oxaloacetic acid + pyruvic acid was oxidized but at a much reduced rate. (2) Radioactively labeled isocitrate was incorporated into at least α-ketoglutaric, succinic, and malic acids, indicating the eel liver TCA cycle is normal between isocitrate and malate. (3) No activity of the NAD-linked isocitrate dehydrogenase (IDH) could be detected, but NADP-IDH activities were higher in the mitochondria than cytosolic fractions. An active NADPH:NAD transhydrogenase was localized to the mitochondrial compartment. (4) These data suggest an important role for the NADP-IDH:transhydrogenase enzyme couple in eel liver TCA cycle function, and a pathway incorporating these ideas is proposed.     

Metabolism of [3-13C]pyruvate in TCA cycle mutants of yeast.

The utilization of pyruvate and acetate by Saccharomyces cerevisiae was examined using 13C and 1H NMR methodology in intact wild-type yeast cells and mutant yeast cells lacking Krebs tricarboxylic acid (TCA) cycle enzymes. These mutant cells lacked either mitochondrial (NAD) isocitrate dehydrogenase (NAD-ICDH1),alpha-ketoglutarate dehydrogenase complex (alpha KGDC), or mitochondrial malate dehydrogenase (MDH1). These mutant strains have the common phenotype of being unable to grow on acetate. [3-13C]-Pyruvate was utilized efficiently by wild-type yeast with the major intermediates being [13C]glutamate, [13C]acetate, and [13C]alanine. Deletion of any one of these Krebs TCA cycle enzymes changed the metabolic pattern such that the major synthetic product was [13C]galactose instead of [13C]glutamate, with some formation of [13C]acetate and [13C]alanine. The fact that glutamate formation did not occur readily in these mutants despite the metabolic capacity to synthesize glutamate from pyruvate is difficult to explain. We discuss the possibility that these data support the metabolon hypothesis of Krebs TCA cycle enzyme organization.     

Tricarboxylic acid cycle enzymes of a pseudophyllid cestode Penetrocephalus ganapatii

Studies on the tricarboxylic acid cycle (TCA cycle) enzymes of Penetrocephalus ganapatii reveal that the TCA cycle is only partially operative, as some of the enzymes at the start of the cycle viz. citrate synthase, aconitase and isocitrate dehydrogenase are found to be low in their activities. The high activities of malate dehydrogenase and fumarase, showing affinity towards a reverse direction, indicate that the TCA cycle operates in the reverse direction resulting in the formation of fumarate. The low succinate dehydrogenase/fumarate reductase ratio suggests that ATP generation may occur at site I of the respiratory chain during the reduction of fumarate into succinate.     

细胞质雄性不育水稻幼穗和花药的呼吸酶活性研究

研究珍汕97A和珍汕97B的雌雄蕊原基形成期、花粉母细胞形成期和花粉母细胞减数分裂期的幼穗及单核期、二核期和三核期的花药中呼吸代谢三羧酸循环（TCA）的苹果酸脱氢酶（MDH）和异柠檬酸脱氢酶（IDH）及戊糖途径（PPP）的磷酸葡萄糖脱氢酶（G6PDH）、磷酸葡萄糖酸脱氢酶（6PGDH）和5一磷酸核糖异构酶（RSPI）的活性。结果表明：可育花药的5种酶活性皆高于同期不育花药;而幼穗中,TCA途径中的MDH和IDH在不育系与保持系之间无差异,PPP途径的G6PDH和6PGDH及R5PI则保持系高于不育系。这说明不育系中PPP发生的变化早于TCA途径,PPP途径的改变可能与小孢子败育有着更为直接的关系。     

Enzyme profiles of Thiobacillus versutus after aerobic and denitrifying growth: Regulation of isocitrate lyase

The specific activities of the tricarboxylic acid (TCA) cycle enzymes in Thiobacillus versutus were invariably lower after aerobic growth as compared to denitrifying growth in acetate- or succinate-limited chemostat cultures. Of the glyoxylate cycle enzymes, isocitrate lyase (ICL) activity was nil during aerobic and 76 nmol·min^-1·mg^-1 protein during denitrifying growth on acetate whereas malate synthase (MS) did not change. In succinate-grown cells ICL was always near nil. The change in ICL and MS was followed after pulse additions of acetate and nitrate to an aerobic acetate-limited chemostat culture made anaerobic prior to the first pulse. ICL remained nil during denitrifying growth after the first pulse but increased to 47 and 81 nmol ·min^-1·mg^-1 protein after the second and third pulse, respectively. MS remained unaltered. The appearance of ICL was dependent upon de novo protein synthesis. During transition in a steady state culture on acetate from oxygen to nitrate as terminal electron acceptor, denitrifying growth started after 0.6 volume replacements. The resumption of growth was concomitant with an increase in TCA cycle enzyme activities. ICL was observed only after two volume replacements. During the reverse transition, ICL disappeared at a rate twice the dilution rate. SDS polyacrylamide gelectrophoresis of cell-free extracts containing ICL showed a major protein band with a R_f value identical to purified ICL and a mol·wt. of 60,000. ICL from T. versutus was inhibited by 1.5 mM itaconate but not by 10 mM phosphoenolpyruvate. Its activity was dependent upon the presence of Mg²⁺ and cysteine.Abbreviations TCA Tricarboxylic acid - ICL isocitrate lyase - MS malate synthase - FPLC fast protein liquid chromatography - maximum specific oxygen consumption rate     

Enzymes of the tricarboxylic acid cycle in Ancylostoma ceylanicum and Nippostrongylus brasiliensis.

Enzymes of the tricarboxylic acid (TCA) cycle and glyoxylate pathway were investigated in adults and infective larvae of Ancylostoma ceylanicum and Nippostrongylus brasiliensis, and their activities were compared with those obtained in rat liver. A complete sequence of enzymes of the TCA cycle, with most of them showing activities quite similar to those in the rat liver homogenate, was detected in adults of both species. All the enzymes except fumarase and malate dehydrogenase were located predominantly in mitochondria where they showed a variable distribution of activities between the soluble and the membranes fractions. Malate dehydrogenase and fumarase were found in both the mitochondria and the 9,000-g supernatant fraction. Succinyl CoA synthetase, which was present in minimum activity, appeared rate limiting. Enzymes of the glyoxylate pathway, particularly isocitrate lyase, seemed to aid the functioning of the Krebs cycle by allowing the formation of succinate from isocitrate. The infective larvae of both species also were found equipped with all the enzymes of the Krebs cycle. Nonetheless, only isocitrate lyase of the glyoxylate pathway could be detected in these parasites.     

Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae.

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.     

Physiology and metabolism of two alkane oxidizing and citric acid producing strains of Candida parapsilosis

Summary The activity of enzymes of the tricarboxylic acid (TAC) and glyoxylate (GC) cycles in Candida parapsilosis (wild type KSh 21 and mutant 337) were studied under different physiological and metabolic conditions. C. parapsilosis differed in most of its enzyme activities from other non-citric acid producing yeasts. Furthermore, pH-value, temperature and age of culture proved to act differently on both strains of the tested organism.The addition of trans-aconitate increased not only the growth but also the activities of citrate synthase and some other enzymes while that of aconitase decreased enormously.The high citrate synthase activity might be connected with the role of citrate in the transport of acetyl groups.Abbreviations CS citrate synthase - AC aconitase - ICDH isocitrate dehydrogenase - GDH glutamate dehydrogenase - Fum fumarase - MDH malate dehydrogenase - ICL isocitrate lyase - MS malate synthase     

Effect of thyroidectomy on testicular enzymes of the pyruvate/malate cycle involved in lipogenesis

The specific activities of testicular enzymes of the pyruvate/malate cycle involved in lipogenesis after thyroidectomy and thyroxine replacement were studied in prepubertal, pubertal and adult rats. Thyroidectomy induced testicular ATP citrate-lyase, malate dehydrogenase and malic enzyme activities and inhibited isocitrate dehydrogenase (NADP⁺) activity. Thyroxine treatment on thyroidectomized animals reverted all enzyme activities to normal. The result suggests that thyroid hormones have a differential effects on testicular enzymes of the pyruvate/malate cycle involved in lipogenesis.     

Chemopreventive efficacy of selenium against N-nitrosodiethylamine-induced hepatoma in albino rats.

The chemopreventive/chemotherapeutic effect of sodium selenite on tricarboxylic acid cycle key enzymes was investigated against hepatoma induced by environmental carcinogen N-nitrosodiethylamine. Decreased activities of TCA cycle key enzymes such as isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) and alpha-ketoglutarate dehydrogenase (alpha-KGDH) in hepatoma and surrounding tissues of hepatoma-bearing rats were observed. Upon selenium supplementation the above biochemical changes were reverted in a dose- and duration-dependent manner. This study further confirms the chemopreventive/chemotherapeutic effect of sodium selenite which is found to be more effective in the initiation phase of carcinogenesis.     

Kinetic model of functioning and regulation of <Emphasis Type="Italic">Escherichia coli</Emphasis> isocitrate dehydrogenase

To describe published experimental data on the functioning of E. coli isocitrate dehydrogenase (IDH), a Rapid Equilibrium Random Bi Ter mechanism involving the formation of two dead-end enzyme complexes is proposed and a kinetic model of enzyme functioning is constructed. The enzyme is shown to be regulated through reversible phosphorylation by IDH kinase/phosphatase; the latter, in its turn, is controlled by IDH substrates and also by a number of central metabolites—pyruvate, 3-phosphoglycerate, and AMP—reflecting the energy demand of the cell. Using the model, it is shown that an increase in the concentration of the above effectors raises the fraction of active IDH and thus enhances the Krebs cycle flux. The ratio between the free and the phosphorylated forms of IDH is more sensitive to AMP, NADP, and isocitrate than to pyruvate and 3-phosphoglycerate. The model also predicts changes in the ratio between phosphorylated and active forms of IDH in the Krebs cycle that occur with a change in the energy and biosynthetic loads on E. coli cells.     

Enzyme activities in mitochondria isolated from ripening tomato fruit

Mitochondria were isolated from tomato (Lycopersicon esculentum L.) fruit at the mature green, orange-green and red stages and from fruit artificially suspended in their ripening stage. The specific activities of citrate synthase (EC 4.1.3.7), malate dehydrogenase (EC 1.1.1.37), NAD-linked isocitrate dehydrogenase (EC 1.1.1.41) and NAD-linked malic enzyme (EC 1.1.1.38) were determined. The specific activities of all these enzymes fell during ipening, although the mitochondria were fully functional as demonstrated by the uptake of oxygen. The fall in activity of mitochondrial malate dehydrogenase was accompanied by a similar fall in the activity of the cytosolic isoenzyme. Percoll-purified mitochondria isolated from mature green fruit remained intact for more than one week and at least one enzyme, citrate synthase, did not exhibit the fall in specific activity found in normal ripening fruit.     

Global metabolic regulation analysis for<Emphasis Type="Italic"> Escherichia coli</Emphasis> K12 based on protein expression by 2-dimensional electrophoresis and enzyme activity measurement

Regulation of the main metabolic pathways of Escherichia coli K12 was investigated based on 2-dimensional electrophoresis (2DE) and the measurement of enzyme activities. The cells were grown aerobically in different carbon sources, such as glucose, acetate, gluconate or glycerol. Microaerobic cultivation was also conducted with glucose as a carbon source. Fifty-two proteins could be identified based on 2DE, and 26 enzyme activities from the main metabolic pathways-including glycolysis, pentose phosphate pathway, TCA cycle, Entner-Doudoroff pathway and fermentative pathway-were assayed. These enzyme activities, together with global and quantitative protein expression, gave us a clear picture of metabolic regulation. The results show that, compared with the control experiment with glucose as a carbon source under aerobic conditions, glycolytic enzymes were slightly up-regulated (<2-fold), TCA cycle enzymes were significantly down-regulated (2- to 10-fold), and fermentative enzymes such as pfl and adhE were highly up-regulated (>10-fold) under microaerobic conditions in glucose medium. When acetate was used as a carbon source, pfkA, pykF, ppc and zwf were down-regulated, while fbp, pckA, ppsA and mez were significantly up-regulated. Glyoxylate enzymes such as aceA and aceB were strongly up-regulated (>10-fold) and TCA-cycle-related enzymes were also up-regulated to some extent. With gluconate as a carbon source, edd, eda, fbp and TCA cycle enzymes were up-regulated. With glycerol as a carbon source, fbp and TCA cycle enzymes were up-regulated, while ackA was significantly down-regulated. Protein abundance obtained by 2DE correlated well with enzyme activity, with a few exceptions (e.g., isocitrate dehydrogenase), during aerobic growth on acetate.     

Metabolic characteristics of recombinant Chinese hamster ovary cells expressing glutamine synthetase in presence and absence of glutamine

To elucidate the metabolic characteristics of recombinant CHO cells expressing glutamine synthetase (GS) in the medium with or without glutamine, the concentrations of extra- and intracellular metabolites and the activities of key metabolic enzymes involved in glutamine metabolism pathway were determined. In the absence of glutamine, glutamate was utilized for glutamine synthesis, while the production of ammonia was greatly decreased. In addition, the expression of recombinant protein was increased by 18%. Interestingly, the intracellular glutamine maintained almost constant, independent of the presence of glutamine or not. Activities of glutamate-oxaloacetate aminotransferase (GOT), glutamate-pyruvate aminotransferase (GPT), and glutamate dehydrogenase (GDH) increased in the absence of glutamine. On the other hand, intracellular isocitrate and the activities of its downstream isocitrate dehydrogenase in the TCA cycle increased also. In combination with these two factors, a 8-fold increase in the intracellular α-ketoglutarate was observed in the culture of CHO-GS cells in the medium without glutamine.     

Dependence of Malate Dehydrogenase Structure on the Type of Metabolism in Freshwater Filamentous Colorless Sulfur Bacteria of the Genus Beggiatoa

Major pathways of carbon metabolism were studied in strains D-402 and D-405 of freshwater colorless sulfur bacteria of the genus Beggiatoa grown organotrophically and mixotrophically. The bacteria were found to possess all the enzymes of the tricarboxylic acid (TCA) and glyoxylate cycles. When organotrophic growth changed to mixotrophic growth, the activity of the TCA cycle enzymes decreased 2- to 3-fold, but the activity of enzymes of the glyoxylate cycle increased threefold. It follows that, in the oxidation of thiosulfate, organic compounds no longer play the leading part in the energy metabolism, and most of electrons that enter the electron transport chain (ETC) derive from inorganic sulfur compounds. A connection was established between the structure and kinetic characteristics of malate dehydrogenase—an enzyme of the TCA and glyoxylate cycles—and the type of carbon metabolism in the strains studied. Malate dehydrogenase in organotrophically grown cells of strains D-402 and D-405 is dimeric, whereas in strain D-402 grown mixotrophically it is tetrameric.     

Metabolism of organic acids, nitrogen and amino acids in chlorotic leaves of ‘Honeycrisp’ apple (Malus domestica Borkh) with excessive accumulation of carbohydrates

Metabolite profiles and activities of key enzymes in the metabolism of organic acids, nitrogen and amino acids were compared between chlorotic leaves and normal leaves of ‘Honeycrisp’ apple to understand how accumulation of non-structural carbohydrates affects the metabolism of organic acids, nitrogen and amino acids. Excessive accumulation of non-structural carbohydrates and much lower CO₂ assimilation were found in chlorotic leaves than in normal leaves, confirming feedback inhibition of photosynthesis in chlorotic leaves. Dark respiration and activities of several key enzymes in glycolysis and tricarboxylic acid (TCA) cycle, ATP-phosphofructokinase, pyruvate kinase, citrate synthase, aconitase and isocitrate dehydrogenase were significantly higher in chlorotic leaves than in normal leaves. However, concentrations of most organic acids including phosphoenolpyruvate (PEP), pyruvate, oxaloacetate, 2-oxoglutarate, malate and fumarate, and activities of key enzymes involved in the anapleurotic pathway including PEP carboxylase, NAD-malate dehydrogenase and NAD-malic enzyme were significantly lower in chlorotic leaves than in normal leaves. Concentrations of soluble proteins and most free amino acids were significantly lower in chlorotic leaves than in normal leaves. Activities of key enzymes in nitrogen assimilation and amino acid synthesis, including nitrate reductase, glutamine synthetase, ferredoxin and NADH-dependent glutamate synthase, and glutamate pyruvate transaminase were significantly lower in chlorotic leaves than in normal leaves. It was concluded that, in response to excessive accumulation of non-structural carbohydrates, glycolysis and TCA cycle were up-regulated to “consume” the excess carbon available, whereas the anapleurotic pathway, nitrogen assimilation and amino acid synthesis were down-regulated to reduce the overall rate of amino acid and protein synthesis.     

The RpoS-Mediated Regulation of Isocitrate Dehydrogenase Gene Expression in Escherichia coli

The Escherichia coli NADP⁺-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42), encoded by an icd gene, is a tricarboxylic acid (TCA) cycle enzyme responsible for the oxidative decarboxylation of isocitrate to α-ketoglutarate. In order to examine how the icd gene expression is regulated, an icd-lacZ reporter fusion was constructed. While the icd gene was induced in exponential growth phase, it was repressed in stationary growth phase. Genetic inactivation of an rpoS gene, whose product is an alternative sigma factor, induced the icd gene expression approximately 4.8 times more in the stationary phase and the IDH enzyme activity in the rpoS mutant was 3.2 times higher than that in the wild type, indicating that the RpoS factor acts as a negative regulator of the icd gene expression in the stationary phase.