Tricarboxylic Acid Cycle Enzymes and Morphogenesis in Blastocladiella emersonii

During exponential growth, ordinary colorless (OC) plants of Blastocladiella emersonii consumed little glucose and produced no lactic acid. Similarly, resistant sporangial (RS) plants did not utilize glucose or produce lactic acid during the first 24 hr of exponential growth. During the next 24 hr of RS development, glucose was consumed with the concomitant production of lactic acid which was then reutilized. Lactic acid gradually accumulated again at maturity. Enzyme studies on cell-free extracts indicated the presence of all tricarboxylic cycle enzymes except α-ketoglutarate dehydrogenase at all stages of development of both RS and OC plants. Included among the enzymes detected were an adenosine monophosphate-stimulated, nicotinamide adenine dinucleotide-isocitric dehydrogenase, and citrate-condensing enzyme. When measured on a per plant basis, tricarboxylic cycle enzyme levels increased during the exponential growth of both kinds of plants. Only after the bicarbonate ceased to have effect on RS plant morphogenesis was there a decrease in the levels of the tricarboxylic cycle enzymes when measured on a per plant basis. Specific activity measurements indicated some differences in the differential rates of synthesis among the enzymes studied previous to 36 hr. Preliminary studies utilizing short periods of ¹⁴C-bicarbonate fixation in young RS plants indicated that during the first 4 min most of the label was located in aspartic acid. These results are discussed in terms of previous results and particularly Cantino's hypothesis concerning the relationship between bicarbonate induction and tricarboxylic-cycle enzymes in the morphogenesis of B. emersonii.     

Activities of Tricarboxylic Acid Cycle Enzymes, Glyoxylate Cycle Enzymes, and Fructose Diphosphatase in Bakers' Yeast During Adaptation to Acetate Oxidation

Bakers' yeast oxidizes acetate at a high rate only after an adaptation period during which the capacity of the glyoxylate cycle is found to increase. There was apparently no necessity for the activity of acetyl-coenzyme A synthetase, the capacity of the tricarboxylic acid cycle, or the concentrations of the cytochromes to increase for this adaptation to occur. Elevation of fructose 1,6 diphosphatase occurred only when acetate oxidation was nearly maximal. Cycloheximide almost completely inhibited adaptation as well as increases in the activities of isocitrate lyase and aconitate hydratase, the only enzymes assayed. p-Fluorophenylalanine was partially effective and chloramphenicol did not inhibit at all. The presence of ammonium, which considerably delayed adaptation of the yeast to acetate oxidation, inhibited the increases in the activities of the glyoxylate cycle enzymes to different degrees, demonstrating noncoordinate control of these enzymes. Under the various conditions, the only enzyme activity increase consistently related to the rising oxygen uptake rate was that of isocitrate lyase which apparently limited the activity of the cycle.     

Photosynthesis in Rhodospirillum rubrum. III. Metabolic Control of Reductive Pentose Phosphate and Tricarboxylic Acid Cycle Enzymes

Enzymes of the reductive pentose phosphate cycle including ribulose-diphosphate carboxylase, ribulose-5-phosphate kinase, ribose-5-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase and alkaline fructose-1,6-diphos-phatase were shown to be present in autotrophically grown Rhodospirillum rubrum. Enzyme levels were measured in this organism grown photo- and dark heterotrophically as well. Several, but not all, of these enzymes appeared to be under metabolic control, mediated by exogenous carbon and nitrogen compounds. Light had no effect on the presence or levels of any of these enzymes in this photosynthetic bacterium.

The enzymes of the tricarboxylic acid cycle and enolase were shown to be present in R. rubrum cultured aerobically, autotrophically, or photoheterotrophically, both in cultures evolving hydrogen and under conditions where hydrogen evolution is not observed. Light had no clearly demonstrable effect on the presence or levels of any of these enzymes.

     

The Deficient Carbohydrate Metabolic Pathways and the Incomplete Tricarboxylic Acid Cycle in an Obligately Autotrophic Hydrogen-oxidizing Bacterium

The activities of enzymes of carbohydrate metabolism, enzymes of the tricarboxylic acid cycle and some related enzymes were measured in cell-free extracts of strain TK-6, an extremely thermophilic, obligately autotrophic, aerobic hydrogen-oxidizing bacterium. Activities of phosphofructokinase, aldolase, pyruvate kinase, 6-phosphogluconate dehydrase and 2-keto-3-deoxy-6-phosphogluconate aldolase, key enzymes of the Embden-Meyerhof and the Entner-Doudoroff pathways were not found in the extracts. All of the tricarboxylic acid cycle enzymes except α-ketoglutarate dehydrogenase, and reduced nicotinamide adenine dinucleotide oxidase were present. These metabolic defects are considered to be one of the reasons for the obligate autotrophy of strain TK-6.     

Biosynthesis of Glutamic Acid in Saccharomyces: Accumulation of Tricarboxylic Acid Cycle Intermediates in a Glutamate Auxotroph

Aconitaseless glutamic acid auxotroph MO-1-9B of Saccharomyces grew in glutamic acid-supplemented minimal medium, but failed to grow when glutamic acid was substituted by proline, arginine, ornithine, or glutamine. This mutant was also unable to utilize lactate or glycerol as a carbon source. Under a glutamic acid-limiting condition, by using acetate-1-(14)C as tracer, the mutant accumulated rather large amounts of (14)C-citric acid and (14)C-succinic acid when compared with the wild-type strain. Under excess glutamic acid supplementation, accumulation of citric acid and succinic acid was considerably reduced. When (14)C-glutamic acid-(U) was used as tracer, (14)C-alpha-ketoglutaric acid, (14)C-citric acid, and (14)C-succinic acid were accumulated in the mutant. The citric acid peak was the largest, followed by alpha-ketoglutaric acid and succinic acid. In the wild-type strain under similar conditions, only small amounts of (14)C-citric acid and (14)C-succinic acid and no (14)C-alpha-ketoglutaric acid were accumulated.     

Photosynthesis in the Higher Plant, Vicia faba: III. Serine, a Precursor of the Tricarboxylic Acid Cycle

Evidence is presented to support the hypothesis that serine, rather than 3-phosphoglycerate of the Calvin cycle, is a precursor of the tricarboxylic acid cycle during photosynthesis by the higher plant, Vicia faba. Identification of the serine intermediate is based upon a unique C₁ > C₂ > C₃ isotope distribution for that metabolite following the fixation of ¹⁴CO₂. This labeling pattern, while incompatible with an origin either in the Calvin cycle or the glycolate pathway, satisfies a critical criterion for the 3-carbon precursor of the anomalously labeled organic acids. The predominant carboxyl carbon atom labeling of serine reflects either a mixing of two pools of that metabolite, ie., C₁ = C₂ > C₃ and C₁ > C₂ = C₃, or a higher order of complexity in its synthesis. An anomalous C₁ = C₂ > C₃ < C₄ distribution for aspartate, however, suggests an origin by the carboxylation of a 3-carbon intermediate related to serine which has a C₁ = C₂ > C₃ distribution. The latter distribution has been proposed for the serine intermediate of the postulated formate pathway. This pathway is described by the generalized metabolic sequence: CO₂ → formate → serine → organic acids. Corresponding carbon atom distributions for citrate (C₁ > C₂), aspartate (C₂ > C₃), and serine (C₂ > C₃) belie a precursor-product relationship with alanine (C₂ = C₃), which is a molecular parameter of the Calvin cycle product, 3-phosphoglycerate.     

Photosynthesis in the Higher Plant Vicia faba: V. Role of Malate as a Precursor of the Tricarboxylic Acid Cycle

In the higher plant Vicia faba, anomalous labeling patterns in the organic acids and related amino acids of the tricarboxylic acid cycle which result from photosynthetic ¹⁴CO₂ fixation (in conjunction with an enzyme localization pattern unique to plant mitochondria) suggest that the tricarboxylic acid cycle functions primarily as a pathway leading to glutamic acid biosynthesis during autotrophic growth. The distribution of isotope in citrate indicates little recycling of oxaloacetate for the resynthesis of citrate. Rather, malate appears to provide both the C₂ and C₄ fragments for the synthesis of citrate, and [³H]formate and ¹⁴CO₂-labeling patterns implicate serine as the ultimate C₃ precursor of malate.     

Chloroplast and Cytoplasmic Enzymes: VIII. Amino Acid Composition of the Pea Leaf Aldolases

Pea leaf chloroplast aldolase contains seven more aspartyl (asx) residues and four fewer leucine and isoleucine residues than the cytoplasmic enzyme. The two forms are therefore primary isoenzymes, differing in amino acid sequence.     

On the Metabolic Relationship between the Calvin Cycle and the Tricarboxylic Acid Cycle: IV. A Plant Survey for Anomalous Acetyl Coenzyme A

Leaves of 10 randomly selected plants representing eight dicotyledonous families were exposed to ¹⁴CO₂ for a 10-minute period in the light. Citrate and alanine were isolated, purified isotopically, and degraded to obtain the ¹⁴C-isotope distribution of corresponding carbon atoms, i.e. citrate (C-1,2) and alanine (C-2,3). The cited carbon atoms of alanine were equally labeled as is typical of a 3-carbon intermediate derived from photosynthetic 3-phosphoglycerate. The carbon atoms of citrate, equivalent to acetyl-CoA, were unequally labeled. The citrate (C-1,2) isotope ratio ranged from 1.20 to 1.78 for the various plants compared to a ratio of unity in the uniformly labeled control. The results infer that 3-phosphoglycerate produced in the chloroplast is not the singular precursor of mitochondrial citrate.     

大肠杆菌糖酵解途径和三羧酸循环启动子的表征及其应用

启动子是控制基因转录的重要元件,也是合成生物学研究和细胞工厂设计的关键环节。糖酵解途径和三羧酸循环是糖类分解代谢的中心代谢,受到包括启动子强度在内的严格调控。为了筛选一系列能满足合成生物学研究和细胞工厂设计需要的不同强度的内源性组成型启动子,利用报告基因——红色荧光蛋白m Cherry和在线分析软件,系统研究了大肠杆菌糖酵解和三羧酸循环中27个启动子的强度和核心结构元件。结果表明:这些启动子的强度范围变化很大,最强启动子Pgap A的强度是最弱启动子Pacn A强度的43. 6倍;启动子的-10序列和-35序列与它们的一致序列也不完全相同,两者之间的距离为17±3bp;但是,启动子的强度和启动子的结构特征基本一致。应用最强启动子Pgap A在重组大肠杆菌DH5αΔpck中分别表达磷酸烯醇式丙酮酸羧化酶基因和丙酮酸激酶基因,它们的酶活性分别提高了0. 32和1. 57倍,柠檬酸产量也提高了124. 7%和75. 5%。这些不同强度的启动子为大肠杆菌的合成生物学研究和细胞工厂设计奠定了一定的基础。     

Adaptation of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> to Al-Citrate: Involvement of Tricarboxylic Acid and Glyoxylate Cycle Enzymes and the Influence of Phosphate

The degradation of Aluminum-citrate by Pseudomonas fluorescens necessitated a major restructuring of the various enzymatic activities involved in the TCA and glyoxylate cycles. While a six-fold increase in fumarase (FUM EC 4.2.1.2) activity was observed in cells subjected to Al-citrate compared to control cells, citrate synthase (CS EC 4.1.3.7) activity experienced a two-fold increase. On the other hand, in the Al-stressed cells malate synthase (MS EC 4.1.3.2) activity underwent a five-fold decrease in activity. This modulation of enzymatic activities appeared to be evoked by Al stress, as the incubation of Al-stressed cells in control media led to the complete reversal of these enzymatic profiles. These observations were further confirmed by ¹H NMR and ¹³C NMR spectroscopy. No significant variations were observed in the activities of other glyoxylate and TCA cycle enzymes, like isocitrate lyase (ICL EC 4.1.3.1), malate dehydrogenase (MDH EC 1.1.1.37), and succinate dehydrogenase (SDH EC 1.3.99.1). This reconfiguration of the metabolic pathway appears to favour the production of a citrate-rich aluminophore that is involved in the sequestration of Al.     

STUDIES ON CELL METABOLISM AND CELL DIVISION : VIII. THE DIPHOSPHOPYRIDINE NUCLEOTIDE (COZYMASE) CONTENT OF EGGS OF ARBACIA PUNCTULATA

1. The diphosphopyridine nucleotide content of Arbacia eggs has been measured manometrically and found to be approximately 250–500 micrograms per gm. wet weight of eggs, the value varying with individual egg samples and with the state of development of the eggs. Of the total diphosphopyridine nucleotide present, approximately 25–40 per cent is in an alkali-stable, presumably the dihydro, form. 2. Tests for triosephosphate and glutamic acid dehydrogenases carried out on Arbacia egg cytolysates were negative.     

The Effect of Light on the Tricarboxylic Acid Cycle in Green Leaves: II. Intermediary Metabolism and the Location of Control Points

Long term feeding of acetate-2-¹⁴C, ¹⁴CO₂, citrate-1,5-¹⁴C, fumarate-2,3-¹⁴C, and succinate-2,3-¹⁴C to mung bean (Phaseolus aureus L. var. Mungo) leaves in the dark gave labeling predominantly in tricarboxylic acid cycle intermediates. Kinetics of the intermediates during dark/light/dark transitions showed a light-induced interchange of ¹⁴C between malate and aspartate, usually resulting in an accumulation of ¹⁴C in malate and a decrease of it in aspartate. ¹⁴C-Phosphoenolpyruvate also showed a marked decrease during illumination. Changes in other intermediates of the tricarboxylic acid cycle were relatively minor. The kinetic data have been analyzed using the Chance crossover theorem to locate control points during the dark/light/dark transitions. The major apparent control points are located at malate and isocitrate dehydrogenases, and less frequently at citrate synthase and fumarase. These findings are explained in terms of the light-induced changes in adenine nucleotides and nicotinamide adenine dinucleotides.     

Differential Lowering by Manganese Treatment of Activities of Glycolytic and Tricarboxylic Acid (TCA) Cycle Enzymes Investigated in Neuroblastoma and Astrocytoma Cells Is Associated with Manganese-Induced Cell Death

Manganese (Mn) is a trace metal required for normal growth and development. Manganese neurotoxicity is rare and usually associated with occupational exposures. However, the cellular and molecular mechanisms underlying Mn toxicity are still elusive. In rats chronically exposed to Mn, their brain regional Mn levels increase in a dose-related manner. Brain Mn preferentially accumulates in mitochondria; this accumulation is further enhanced with Mn treatment in vivo. Exposure of mitochondria to Mn in vitro leads to uncoupling of oxidative phosphorylation. These observations prompted us to investigate the hypothesis that Mn induces alterations in energy metabolism in neural cells by interfering with the activities of various glycolytic and TCA cycle enzymes using human neuroblastoma (SK-N-SH) and astrocytoma (U87) cells. Treatments of SK-N-SH and U87 cells with MnCl2 induced cell death in these cells, in a concentration- and time-dependent manner, as determined by MTT assays. In parallel with the Mn-induced, dose-dependent decrease in cell survival, treatment of these cells with 0.01 to 4.0 mM MnCl2 for 48 h also induced dose-related decreases in their activities of hexokinase, pyruvate kinase, lactate dehydrogenase, citrate synthase, and malate dehydrogenase. Hexokinase in SK-N-SH cells was the most affected by Mn treatments, even at the lower range of concentrations. Mn treatment of SK-N-SH cells affected pyruvate kinase and citrate synthase to a lesser extent as compared to its effect on other enzymes investigated. However, citrate synthase and pyruvate kinase in U87 cells were more vulnerable than other enzymes investigated to the effects of Mn. The results suggest the two cell types exhibited differential susceptibility toward the Mn-induced effects. Additionally, the results may have significant implications in flux control because HK is the first and highly regulated enzyme in brain glycolysis. Thus these results are consistent with our hypothesis and may have pathophysiological implications in the mechanisms underlying Mn neurotoxicity.     

Dynamic Changes of Intracellular pH in Individual Lactic Acid Bacterium Cells in Response to a Rapid Drop in Extracellular pH

We describe the dynamics of changes in the intracellular pH (pH_i) values of a number of lactic acid bacteria in response to a rapid drop in the extracellular pH (pH_ex). Strains of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus, and Lactococcus lactis were investigated. Listeria innocua, a gram-positive, non-lactic acid bacterium, was included for comparison. The method which we used was based on fluorescence ratio imaging of single cells, and it was therefore possible to describe variations in pH_i within a population. The bacteria were immobilized on a membrane filter, placed in a closed perfusion chamber, and analyzed during a rapid decrease in the pH_ex from 7.0 to 5.0. Under these conditions, the pH_i of L. innocua remained neutral (between 7 and 8). In contrast, the pH_i values of all of the strains of lactic acid bacteria investigated decreased to approximately 5.5 as the pH_ex was decreased. No pronounced differences were observed between cells of the same strain harvested from the exponential and stationary phases. Small differences between species were observed with regard to the initial pH_i at pH_ex 7.0, while different kinetics of pH_i regulation were observed in different species and also in different strains of S. thermophilus.     

Fermentation Products of Solvent Tolerant Marine Bacterium Moraxella spp. MB1 and Its Biotechnological Applications in Salicylic Acid Bioconversion

As part of a proactive approach to environmental protection, emerging issues with potential impact on the environment is the subject of ongoing investigation. One emerging area of environmental research concerns pharmaceuticals like salicylic acid, which is the main metabolite of various analgesics including aspirin. It is a common component of sewage effluent and also an intermediate in the degradation pathway of various aromatic compounds which are introduced in the marine environment as pollutants. In this study, biotransformation products of salicylic acid by seaweed, Bryopsis plumosa, associated marine bacterium, Moraxella spp. MB1, have been investigated. Phenol, conjugates of phenol and hydroxy cinnamic acid derivatives (coumaroyl, caffeoyl, feruloyl and trihydroxy cinnamyl) with salicylic acid (3–8) were identified as the bioconversion products by electrospray ionization mass spectrometry. These results show that the microorganism do not degrade phenolic acid but catalyses oxygen dependent transformations without ring cleavage. The degradation of salicylic acid is known to proceed either via gentisic acid pathway or catechol pathway but this is the first report of biotransformation of salicylic acid into cinnamates, without ring cleavage. Besides cinnamic acid derivatives (9–12), metabolites produced by the bacterium include antimicrobial indole (13) and β-carbolines, norharman (14), harman (15) and methyl derivative (16), which are beneficial to the host and the environment.