Activities of enzymes of amino-acid metabolism in Pennisetum seedlings grown in the presence of 2-chloroethylphosphonic acid

The plant growth regulator 2-chloroethylphosphonic acid (CEPA) slightly inhibited the elongation of growth in Pennisetum typhoides seedlings, but greatly stimulated the activity of alanine aminotransferase (GPT), asparate aminotransferase (GOT), as well as glutamate dehydrogenase (GLDH).     

Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism

In this paper, we report the identification of genes from pine (PpAAT), Arabidopsis (AtAAT) and rice (OsAAT) encoding a novel class of aspartate aminotransferase (AAT, EC 2.6.1.1) in plants. The enzyme is unrelated to other eukaryotic AATs from plants and animals but similar to bacterial enzymes. Phylogenetic analysis indicates that this prokaryotic-type AAT is closely related to cyanobacterial enzymes, suggesting it might have an endosymbiotic origin. Interestingly, most of the essential residues involved in the interaction with the substrate and the attachment of pyridoxal phosphate cofactor in the active site of the enzyme were conserved in the deduced polypeptide. The polypeptide is processed in planta to a mature subunit of 45 kDa that is immunologically distinct from the cytosolic, mitochondrial and chloroplastic isoforms of AAT previously characterized in plants. Functional expression of PpAAT sequences in Escherichia coli showed that the processed precursor is assembled into a catalytically active homodimeric holoenzyme that is strictly specific for aspartate. These atypical genes are predominantly expressed in green tissues of pine, Arabidopsis and rice, suggesting a key role of this AAT in nitrogen metabolism associated with photosynthetic activity. Moreover, immunological analyses revealed that the plant prokaryotic-type AAT is a nuclear-encoded chloroplast protein. This implies that two plastidic AAT co-exist in plants: a eukaryotic type previously characterized and the prokaryotic type described here. The respective roles of these two enzymes in plant amino acid metabolism are discussed.     

Metabolic regulation of Drosophila apoptosis through inhibitory phosphorylation of Dronc

Apoptosis ensures tissue homeostasis in response to developmental cues or cellular damage. Recently reported genome‐wide RNAi screens have suggested that several metabolic regulators can modulate caspase activation in Drosophila. Here, we establish a previously unrecognized link between metabolism and Drosophila apoptosis by showing that cellular NADPH levels modulate the initiator caspase Dronc through its phosphorylation at S130. Depletion of NADPH removed this inhibitory phosphorylation, resulting in the activation of Dronc and subsequent cell death. Conversely, upregulation of NADPH prevented Dronc‐mediated apoptosis upon DIAP1 RNAi or cycloheximide treatment. Furthermore, this CaMKII‐mediated phosphorylation of Dronc hindered Dronc activation, but not its catalytic activity. Blockade of NADPH production aggravated the death‐inducing activity of Dronc in specific neurons, but not in the photoreceptor cells of the eyes of transgenic flies; similarly, non‐phosphorylatable Dronc was more potent than wild type in triggering specific neuronal apoptosis. Our observations reveal a novel regulatory circuitry in Drosophila apoptosis, and, as NADPH levels are elevated in cancer cells, also provide a genetic model to understand aberrations in cancer cell apoptosis resulting from metabolic alterations.     

Enzyme activities in Pennisetum seedlings germinated in the presence of abscisic and gibberellic acids

Effects of abscisic acid (ABA) and gibberellic acid (GA₃), alone and in combination, on growth and activity of alanine aminotransferase (GPT), aspartate aminotransferase (GOT), and glutamate dehydrogenase (GLDH) were studied in aerial parts of Pennisetum typhoides seedlings. ABA inhibited growth and activity of GLDH, but stimulated the activity of GPT and weakly that of GOT. GA₃, on the other hand, did not affect the activity of any of the enzymes tested, but in combination with ABA tended to antagonise the efrect of the latter.     

Glyoxylate metabolism and fatty acid oxidation in mango fruit during development and ripening

Throughout the development (maturation) of mango fruit the contents of citric and glyoxylic acids increased steadily. As the fruit matured the levels of isocitrate lyase, malate lyase and alanine: glyoxylate aminotransferase increased and reached maximum values prior to the time of harvesting. At and after harvest the levels of malate lyase and alanine : glyoxylate aminotransferase began to decrease but that of isocitrate lyase remained high until after the harvest when it decreased. The level of glyoxylate reductase was highest in the early developmental stage but declined as the fruit matured and ripened. As the fruit ripened, after harvest, the amounts of citric and glyoxylic acids decreased concomitant with a considerable increase in the levels of isocitrate dehydrogenase, malic dehydrogenase, malic enzyme and glyoxylate dehydrogenase.Fatty acid oxidizing capacity of mitochondria isolated from immature (developing) and postclimacteric fruit pulps was much less than that observed with mitochondria from preclimacteric and climacteric fruit. Glyoxylate stimulated the oxidation of caprylic, lauric, myristic and palmitic acids and inhibited the activity of isocitrate dehydrogenase in vitro.     

Succinate metabolism related to lipid synthesis in the housefly Musca domestica L

The metabolism of succinate was examined in the housefly Musca domestica L. The labeled carbons from [2,3-¹⁴C]succinate were readily incorporated into cuticular hydrocarbon and internal lipid, whereas radioactivity from [1,4-¹⁴C]succinate was not incorporated into either fraction. Examination of the incorporation of [2,3-¹⁴C]succinate, [1-¹⁴C]acetate, and [U-¹⁴C]proline into hydrocarbon by radio-gas-liquid chromatography showed that each substrate gave a similar labeling pattern, which suggested that succinate and proline were converted to acetyl-CoA prior to incorporation into hydrocarbons. Carbon-13 nuclear magnetic resonance showed that the labeled carbons from [2,3-¹³C]succinate enriched carbons 1, 2, and 3 of hydrocarbons with carbon-carbon coupling showing that carbons 2 and 3 of succinate were incorporated as an intact unit. Radio-high-performance liquid chromatographic analysis of [2,3-¹⁴C]succinate metabolism by mitochondrial preparations showed that in addition to labeling fumarate, malate, and citrate, considerable radioactivity was also present in the acetate fraction. The data show that succinate was not converted to methylmalonate and did not label hydrocarbon via a methylmalonyl derivative. Malic enzyme was assayed in sonicated mitochondria prepared from the abdomens and thoraces of 1- and 4-day-old insects; higher activity was obtained with NAD⁺ in mitochondria prepared from thoraces, whereas NADP⁺ gave higher activity with abdomen preparations. These data document the metabolism of succinate to acetyl-CoA and not to a methylmalonyl unit prior to incorporation into lipid in the housefly and establish the role of the malic enzyme in this process.     

Aspartate metabolism in endothelial cells activates the mTORC1 pathway to initiate translation during angiogenesis

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Changes in the activity of enzymes involved with primary nitrogen metabolism due to ectomycorrhizal symbiosis on jack pine seedlings

Jack pine (Pinus banksiana Lamb.) seedlings were inoculated with either one of the ectomycorrhizal (ECM) fungi, Laccaria bicolor (Maire) Orton or Pisolithus tinctorius (Pers.) Coker and Couch, and grown for 16 weeks in a growth chamber along with non-ECM controls. Five enzymes involved with the assimilation of nitrogen or the synthesis of amino acids were measured in the 3 jack pine root systems as well as in the pure fungal cultures. Pisolithus tinctorius in pure culture had no detectable activity of nitrate reductase (NR. EC 1.6.6.1), glutamate dehydrogenase (GDH. EC 1.4.1.2), glutamate decarboxylase (GDCO. EC 4.1.1.15) or glutamate oxoglutarate aminotransferase (GOGAT, EC 1.4.1.13) but did have some glutamine synthetase (GS, EC 6.3.1.2) activity. Laccaria bicolor in pure culture had no NR activity, small levels of GDCO activity, and high GS, GDH and GOGAT activity. The high levels of enzymatic activity present in L. bicolor indicate that it may play a greater role in the nitrogen metabolism of its host plant than P. tinctorius. ECM infection clearly altered the enzymatic activity in jack pine roots but the nature of these changes depended on the fungal associate. Non-ECM root systems had higher specific activities than ECM root systems for NR, GS, GDH and GDCO but GOGAT activites were the same for both the ECM and non-ECM roots. Root systems infected with L. bicolor had significantly greater NR and GDCO activity than those infected with P. tinctorius. Differences in the GS activity of the two fungi in pure culture corresponded to the GS activity of jack pine roots in symbiotic association with these fungi. While the free amino acid profiles in roots were significantly affected by ECM infection, the profile of free amino acids exported to the stem was the same for all treatments. High asparagine and low glutamine in roots infected with P. tinctorius indicates that asparagine synthetase (EC x.x.x.x) activity should be higher within this symbiotic association than in the L. bicolor association or in the non-mycorrhizal roots.     

Glutamate metabolism directs energetic trade-offs to shape host-pathogen susceptibility in Drosophila

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Induction of TNF-like factor by murine macrophage-like cell line J774.1 on treatment with Sarcophaga lectin

On stimulation with Sarcophaga lectin, the mouse macrophage-like cell line J774.1 secreted a factor like the tumor necrosis factor (TNF) into the culture medium. This factor was a protein with a molecular weight of 40000-45000, and was cytotoxic to L-929 cells, but not to normal embryonic fibroblasts. This factor was effective on both the ascites form and solid form of sarcoma 180 transplanted into ICR mice.     

Isolation and enzymological characterization of infected and uninfected cell protoplasts from root nodules of Glycine max

Infected and uninfected cell protoplasts were isolated from soybean ( Glycine max L. Merr. cv. Akisengoku) root nodules and purified by the use of nylon mesh filters and discontinuous Percoll gradients. Activities of the enzymes involved in carbon and nitrogen metabolism were measured in cytoplasmic fractions of purified protoplasts, as well as in the bacteroids isolated from infected cell protoplasts and in the cortical tissues after enzymatic digestion of the central zone of the nodules.
A high degree of purity of both infected and uninfected cells was demonstrated by microscopic observations and assays of β-hydroxybutyrate dehydrogenase (EC 1.1.1.30) and uricase (EC 1.7.3.3) activities and leghemoglobin contents.
As a whole, higher specific activities of enzymes of glycolysis were found in the cortical and uninfected cells than in the infected cells. The activities of glycolytic enzymes were extremely low in the bacteroids. Invertase (EC 3.2.1.26) was highly localized in the cortex. However, activity of sucrose synthase (EC 2.4.1.13) was highest in the cytosol of infected cells. Alcohol dehydrogenase (EC 1.1.1.1) and lactate dehydrogenase (EC 1.1.1.27) activities were much higher in uninfected than in infected cells. Specific activities of enzymes for nitrogen assimilation, that is, glutamine synthetase (EC 6.3.1.2), glutamate synthase (EC 1.4.1.14), aspartate (EC 2.6.1.1) and alanine (EC 2.6.1.2) aminotransferase were several-fold higher in uninfected cells than in the infected cells.
The results are discussed in relation to the possible cellular organization of carbon and nitrogen metabolism in soybean root nodules.     

Developmental changes of enzymes of malate metabolism in relation to respiration, photosynthesis and nitrate assimilation in peach leaves

The developmental profile of the activities of some enzymes involved in malate metabolism, namely phosphoenolpyruvate carboxylase (PEPC; EC 4. 1. 1. 31), NAD⁺-linked (EC 1. 1. 1. 37) and NADP⁺-linked (EC 1. 1. 1. 82) malate dehydrosenase (MDH), NAD⁺linked (EC 1. 1. 1. 39) and NADP⁺-linked (EC 1. 1. 1. 40) malic enzyme (ME), has been determined in leaves of peach [ Prunus persica (L.) Batsch cv. Maycrest], a woody C₃ species. In order to study the role of these enzymes, their activities were related to developmental changes of photosynthesis, respiration, and capacity for N assimilation. Activities of PEPC, NAD(P)⁺-MDH and NADP⁺-ME were high in young expanding leaves and decreased 2- to 3-fold in mature ones, suggesting that such enzymes play some role during the early stages of leaf expansion. In leaves of peach, such a role did not seem to be linked to C₃ photosynthesis or nitrate assimilation, in that photosynthetic O₂ evolution and activities of nitrate reductase (EC 1. 6. 6. 1) and glutamine synthetase (EC 6. 3. 1. 2) increased during leaf development. In contrast, leaf respiration strongly decreased with increasing leaf age. We suggest that in expanding leaves of this woody species the enzymes associated with malate metabolism have anaplerotic functions, and that PEPC may also contribute to the recapture of respiratory CO₂.     

Understanding the regulation of aspartate metabolism using a model based on measured kinetic parameters

The aspartate‐derived amino‐acid pathway from plants is well suited for analysing the function of the allosteric network of interactions in branched pathways. For this purpose, a detailed kinetic model of the system in the plant model Arabidopsis was constructed on the basis of in vitro kinetic measurements. The data, assembled into a mathematical model, reproduce in vivo measurements and also provide non‐intuitive predictions. A crucial result is the identification of allosteric interactions whose function is not to couple demand and supply but to maintain a high independence between fluxes in competing pathways. In addition, the model shows that enzyme isoforms are not functionally redundant, because they contribute unequally to the flux and its regulation. Another result is the identification of the threonine concentration as the most sensitive variable in the system, suggesting a regulatory role for threonine at a higher level of integration.     

Gabaculine as a tool to investigate the polyamine biosynthesis pathway in habituated callus of Beta vulgaris (L.)

The polyamine content of a habituated callus of Beta vulgaris (L.) is strongly diminished after treatment with gabaculine, a potent inhibitor of ornithine aminotransferase. The inhibitory effect of gabaculine is reversed if ornithine is supplied. This result may indicate that proline catabolism provides ornithine for polyamine synthesis.     

Macropinocytosis Renders a Subset of Pancreatic Tumor Cells Resistant to mTOR Inhibition

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