Effect of trophic conditions and humic acid on alanine transamination in wheat plants

In dialyzed extracts from winter wheat plants an intensive enzymatical transamination reaction occurred between L-α-alanine and α-ketoglutaric acid (L-α-alanine + + 2-oxoglutarate = pyruvate + L-glutamate, EC 2.6.1.2) as well as a weak nonenzymatical transamination reaction, practically immeasurable. Pyridoxal-5’-phosphate strongly affected the reaction rate. Besides the transamination product-glutamate, γ-aminobutyric acid was formed in this reaction. This amino acid could have originated neither via proteolysis of the enzyme extract, nor via decarboxylation from glutamate formed, nor via transamination of succinic-γ-semialdehyde after α-ketoglutarate decarboxylation. This was the only case of its formation in the transamination reactions investigated in our laboratory — it originated from the alanine-glutamate reaction only, and the mechanism of its biosynthesis cannot be elucidated for the present. Dialysates from shoots exhibited a significantly higher enzymatic activity in comparison with those from roots. The effect of trophic conditions (Knop’s nutrient solution, a water solution of potassium humate, water) was not revealed when calculating per dry weight unit. However, when calculating per protein unit an increased activity was found in the dialysates from shoots of both nutrient — deficient variants. Roots of plants cultivated in potassium humate had the lowest activity. The discussion concerns the possibility of an adaptive use of this transamination for increasing the essential glutamate level in green parts of the plants cultivated under unfavourable nutritive conditions, and also deals with a further characteristic of the differing metabolism of plants cultivated in humate.     

Effect of trophic conditions on aspartate transamination in wheat plants

The enzymatic transamination reactions between aspartic and α-ketoglutaric acid and between aspartic and pyruvic acid were studied in fresh dialysed extracts of young wheat plants cultivated under various trophical conditions, in mineral solution (Knop), in the solution of an soil organic substance (potassium humate) and without nutrients (H₂O). Simultaneously, the level of endogenic aspartic acid, glutamic acid and the growth values were determined. The enzymatic reactions were characterized by determining the optimum pH, the time course, and the effect of coenzyme and of inhibitors. The activity of the aspartate-glutamate transaminase from the root system of plants was considerably higher than the activity of the overground organs. The enzymatic activity from both parts of the plant was inversely proportional to the growth rate: intensive growth of the plants from the Knop variant was connected with their low enzymatic activity; the level of endogenic glutamic acid was high. The slow growth of the plants without nutrients was connected with a higher enzymatic activity; the level of endogenic glutamic acid was low. The plants from the potassium humate variant had an intermediate position between these two variants from the point of view of growth as well as from that of enzymatic activity. The plants with insufficient nutrition (slow growth, low level of endogenic glutamic acid) apparently have a low capacity for supplementing the glutamic acid deficit, which is essential for the metabolic processes, by increasing the activity of the reactions leading to glutamic acid synthesis (Asp-Glu) and, on the other hand, by decreasing the reactions utilizing it (Glu-Ala). For wheat plants the active aspartate-glutamate reaction is obviously physiologically more important than the direct reaction glutamate-aspartate and the reaction aspartate-alanine which in all cases had a very low activity.     

The effect of humic acid on transamination in winter wheat plants

A very low, for the most part unmeasurable glutamic-aspartio transminase activity and a very high glutamic-alanine transaminase activity was found in the overground parts and roots of young wheat plants. The roots had a higher glutamic-alanine transaminase activity than the overground parts in the first and second leaf stage. Plants cultivated in Knop’s nutrient solution (variant with humate and without) showed a higher glutamic-alanine transaminase activity than poorly growing plants, cultivated in distilled water (with humate and without). In plants cultivated in nutrient solutions, transaminase activity increased with the age of the wheat plants. As in the previous experiments, the effect of humate was only significant, in the roots of plants cultivated in distilled water with humate, where transamination activity was greater than in the control without humate. The roots of this variant with a stimulatory growth effect showed a large accumulation of free sugars in the previous experiments. The connection between these effects of humate on the roots of young winter wheat plants is discussed.     

The effect of humus substances on the level of free amino acids in wheat plants

The presence of potassium humate (commercial sample of Humussäure Riedel-de Haën A. G., Seelze-Hannover) in distilled water led to an increase in the content of alanine, aspartic and glutamic acid in the overground parts of wheat plants as compared with the content of these substances in control plants grown in distilled water. In plants cultivated on nutrient solutions a higher level of alanine and glutamic acid and a considerably lower level of amides was found than in plants grown in distilled water and in water with humate. Generally the content of amino acids was higher in the overground parts than in the roots after a cultivation period of one week as well as of 14 day. In the overground parts of 1 days old plants the level of aspartic acid, asparagine and alanine was found to be higher and that of glutamine lower than in seven days old plants. In the roots of the examined plants a decrease of the amino acid content accurred almost in all cases after a cultivation period of 14 days as compared to one of 7 days.     

Targeted metabolomic analysis of amino acid response to L-asparaginase in adherent cells

L-asparaginase (L-ASP) is a therapeutic enzyme used clinically for the treatment of childhood acute lymphoblastic leukemia. L-ASP’s anticancer activity is believed to be associated primarily with depletion of asparagine, but secondary glutaminase activity has also been implicated in its anticancer mechanism of action. To investigate the effects of L-ASP on amino acid metabolism, we have developed an LC–MS/MS metabolomics platform for high-throughput quantitation of 29 metabolites, including all 20 proteinogenic amino acids, 6 metabolically related amino acid derivatives (ornithine, citrulline, sarcosine, taurine, hypotaurine, and cystine), and 3 polyamines (putrescince, spermidine, and spermine) in adherent cultured cells. When we examined the response of OVCAR-8 ovarian cancer cells in culture to L-ASP, asparagine was depleted from the medium within seconds. Interestingly, intracellular asparagine was also depleted rapidly, and the mechanism was suggested to involve rapid export of intracellular asparagine followed by rapid conversion to aspartic acid by L-ASP. We also found that L-ASP-induced cell death was more closely associated with glutamine concentration than with asparagine concentration. Time-course analysis revealed the dynamics of amino acid metabolism after feeding cells with fresh medium. Overall, this study provides new insight into L-ASP’s mechanism of action, and the optimized analytical method can be extended, with only slight modification, to other metabolically active amino acids, related compounds, and a range of cultured cell types.     

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids

Summary The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5–3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), a-ketoglutaric acid (a-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with -KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with -KGA than through reductive amination of -KGA.The experimental data proved that intact bacteroids posess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon.The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N₂-fixation, and sucrose available from leaves.     

Non-enzymic β-decarboxylation of aspartic acid

Summary Non-enzymic-decarboxylation of aspartic acid at 85° is catalyzed by Al³⁺ and pyridoxal. The reaction is optimum at pH 4.0. Both Al³⁺ and pyridoxal are specifically required because replacing these by other cations or by other vitamin B₆ derivatives greatly lowers the formation of alanine. Conversion of 8 µmoles of aspartic acid to alanine is optimum in presence of 1µmole of Al³⁺ and 5 µmoles of pyridoxal. Increasing the concentration of pyridoxal to more than 5 µmoles lowers the alanine formation by the latter being converted to pyruvate by transamination with the excess pyridoxal.Studies on the mechanism of decarboxylation suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The results are interpreted to suggest that the non-enzymic aspartate-decarboxylation process is closely related to and inseparable from the non-enzymic transamination process in a manner analogous to that reported for the highly purified asparate-decarboxylase. The possible significance of these results to prebiotic molecular evolution is briefly discussed.     

The amylolytic activity of Ca-deficient pumpkin plants (Cucurbita pepo L.)

We studied the significance of actual accessibility of ions for presence of α-amylase in roots and cotyledons of young pumpkin plants. We considered α-amylases to be—in agreement with published data—amylolytic enzymes which lose their activity after being dialysed against EDTA or a dilute solution of hydrochloric acid and which can be reactivated by addition of Ca²⁺. N connection with activity and Ca²⁺ nutrition appeared in cotyledons; it did, however, in roots: Ca-deficient plants revealed after preparation either completely inactive enzymes or enzymes with slight activity. It was, however, possible to reactivate them by addition of Ca²⁺ during preparation. So, using polyacrylamide disc electrophoresis we present evidence for the appearence of amylase with typical characteristics for α-amylases of other sources. Its relative mobility was the same for isolates from both roots and cotyledons. Its biosynthesis was not dependent upon metal which is associated with enzyme. No conclusion can be drawn as to whether Ca-deficient α-amylases are activein vivo; therefore it was impossible to judge whether α-amylase activity is subject to feed-back regulation.     

Chromatography as a means of selecting effective strains ofRhizobia

Summary 1. In 0.1 ml of bleeding sap of uninoculated plants aspartic acid was found to occur as the sole amino-acid usually in a small concentration. With larger quantities of sap we also found a spot in the chromatograms and in the electropherogram, that most probably is identical with threonine.2. Under the same conditions with a bleeding period of 4–7 hrs. aspartic acid was the only amino-acid detectable in the chromatogram of the bleeding sap of plants inoculated with ineffective strains.3. Sap of plants inoculated with effective strains contains aspartic acid asparagine, glutamine, hydroxyproline and threonine. Sometimes in addition to these substances 1 or 2 unknown substances were found, havingR _f-phenol values about identical with those of valine and leucine.4. Glutamic acid never was detected in the bleeding sap. We suggest, that glutamic acid formed in the nodules might be rapidly transformed into glutamine and transported in this form.5. The uptake of water is significantly higher with effective strains than with ineffective ones or with plants that have not been inoculated.6. With this technique it is possible to select effective strains within 3 weeks after planting.     

Growth and essential oil responses of <Emphasis Type="Italic">Nepeta</Emphasis> species to potassium humate and harvest time

Nepeta is a perennial herbal plant that belongs to the Lamiaceae mint family. Nepta has different species that are widely used in the agriculture, medicine, and pharmaceutical industries. A field experiment was conducted in 2015 and 2016 to determine the effects of potassium humate foliar application [0 (control) and 400 ppm] on growth, essential oil and constituents of three Nepta species [Nepeta cataria (catnip), Nepeta cataria var. citriodora (lemon catnip), and Nepeta grandiflora (giant catmint)] under Egyptian conditions. In all three species, the application of potassium humate increased growth indicators, essential oil content (%) and yield (mL/plant), and flavonoids in each of the two harvests. However, the height of giant catmint was significantly higher than that of lemon catnip, which was higher than that of catnip. Lemon catnip that received potassium humate and harvested second gave the highest essential oil content and yield. The major constituents were geraniol and nepetalactone in catnip; citronellol and geraniol in lemon catnip; and o-cymene, c-terpinene, p-cymene and carvacrol in giant catmint. The highest p-cymene in giant catmint, citronellol in lemon catnip, and geraniol in catnip were obtained from the application of potassium humate, while the highest o-cymene, c-terpinene and carvacrol in giant catmint, geraniol in lemon catnip, and nepetalactone in catnip were obtained from control plants. This study demonstrated the variations among species of nepeta, and how they respond to the application of potassium humate. The findings of this study can guide the customization of potassium humate applications to the three species for achieving desired growth and essential oil production outcomes.     

Functional analysis of novel alpha-1 antitrypsin variants G320R and V321F

Alpha-1 antitrypsin (AAT) gene is highly polymorphic, with a large number of rare variants whose phenotypic consequences often remain inconclusive. Studies addressing functional characteristics of AAT variants are of significant biomedical importance since deficiency and dysfunctionality of AAT are associated with liver and lung diseases. We report the results of the functional analysis of two naturally occurring AAT variants, G320R and V321F, previously identified in patients with lung disease. Neither of variants has been fully functionally characterized. In order to perform their functional analysis both variants were expressed in prokaryotic and eukaryotic systems and their intracellular localization, activity, stability, and polymerization were determined. The results of this study demonstrated that variants G320R and V321F have neither impaired activity against porcine pancreatic elastase nor propensity to form polymers. However, both variants had altered electrophoretic mobility and reduced thermostability when compared to M variant of the protein, indicating a slightly impaired secondary or tertiary structure.     

The effect of simultaneous short term action of streptomycin and humate on plants

Humate (10 mg l^?1) supplemented to streptomycin solutions (0.1 and 1 mM) stimulates growth of germinating wheat and barley grains and of apical cuttings ofCrassula portulacea after 24 h treatment. It does not, however, prevent formation of albinic leaves. Albinism induced by the streptomycin alone and by streptomycin in presence of humate is irreversible and can be removed neither by an iron salt nor by a chelate added to the nutrition solution or applied on the leaves. Cells of plants treated with streptomycin and humate are larger than those of plants treated with the streptomycin alone. The same is true for plastids which in both cases are colourless and much smaller than chloroplasts of control plants. These plastids in a living or a fixed state have reduced ability to uptake stains. The albinic leaves are anatomically similar to chlorotic leaves of virus infected plants.     

The effect of 9-(2,3-dihydroxypropyl) adenine (DHPA) on seedling roots ofVicia faba L. in comparison with adenine,adenosine and some cytokinins

3-day-old seedlings ofVicia faba L. were put onto a diluted Knop’s solution containing the tested substances within the intentional concentration range and left there for 7 days. In comparison with controls in plain nutrient solution, the DHPA treated plants revealed a suppression of shoot and root development. The main root growth was less sensitive than root branching. 8 h exposure was sufficient to gain a distinct effect. Microscopically the block of mitosis and/or cytokinesis, the formation of binucleate cells and local tissue damage were revealed. Under the given conditions, the cytokinins (BAP, (9R)BAP, iP, (9R)iP) proved to be much more powerful inhibitors than DHPA, whereas adenine and adenosine were less impairing.     

An evolutionarily conserved alternate metal ligand is important for activity in α-isopropylmalate synthase from Mycobacterium tuberculosis

Members of the DRE-TIM metallolyase superfamily rely on an active-site divalent cation to catalyze various reactions involving the making and breaking of carbon–carbon bonds. While the identity of the metal varies, the binding site is well-conserved at the superfamily level with an aspartic acid and two histidine residues acting as ligands to the metal. Previous structural and bioinformatics results indicate that the metal can adopt an alternate architecture through the addition of an asparagine residue as a fourth ligand. This asparagine residue is strictly conserved in all members of the DRE-TIM metallolyase superfamily except fungal homocitrate synthase (HCS-lys) where it is replaced with isoleucine. The role of this additional metal ligand in α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) has been investigated using site-directed mutagenesis. Substitution of the asparagine ligand with alanine or isoleucine results in inactive enzymes with respect to α-isopropylmalate formation. Control experiments suggest that the substitutions have not drastically affected the enzyme's structure indicating that the asparagine residue is essential for catalysis. Interestingly, all enzyme variants retained acetyl CoA hydrolysis activity in the absence of α-ketoisovalerate, similar to the wild-type enzyme. In contrast to the requirement of magnesium for α-isopropylmalate formation, hydrolytic activity could be inhibited by the addition of magnesium chloride in wild-type, D81E, and N321A MtIPMS, but not in the other variants studied. Attempts to rescue loss of activity in N321I MtIPMS by mimicking the fungal HCS active site through the D81E/N321I double variant were unsuccessful. This suggests epistatic constraints in evolution of function in IPMS and HCS-lys enzymes.     

The influence of potassium humate on 137Cs accumulation by barley growing on soil with different fertility

The changes studied in 137Cs uptake by plants and its distribution between vegetative and generative organs of barley cultivated with the application of potassium humate. A relationship has been found between 137Cs accumulation size in barley at various ontogenesis stages and way of potassium humate application (treatment of seeds or plants), as well as availability of mineral nutrients in the soil. Changes in K+ and NH4+ concentrations in soil solution are shown to be of prevailing importance in regulating 137Cs uptake by plants compared with potassium humate effects.     

Transaminierung von Phenylalanin,Asparaginsäure und Ornithin in Keimlingen des Mohnes,Papaver somniferum L.

The transamination activity in five or seven days-old poppy seedlings (Papaver somniferum L. cv “Váhovecký”) germinated in darkness or light was studied. The changes in L-phenylalanine:—(E.C.2.6.1.5), aspartic acid:—(E.C.2.6.1.1), and L-ornithine: α-oxoglutarate transaminase (E.C. 2.6.1.13) were followed and correlated with duration and conditions of cultivation.     

Acetylcholine in plants: Presence,metabolism and mechanism of action

Acetylcholine (ACh) has been detected in representatives of many taxonomic groups throughout the plant kingdom. The site of its synthesis in plants is probably young leaves. In some plant species choline acetyltransferase (ChAT) activity has been found. This enzyme showing properties similar to animal ChAT, probably participates in ACh synthesis from its precursors, choline and acetyl-Coenzyme A. Acetylcholinesterase (AChE) activity has also been found in many plant tissues. This enzyme decomposes ACh and exhibits properties similar to animal AChE. The presence of both ChAT and AChE in plant tissues suggests that ACh undergoes similar metabolism in plants as it does in animals. Exogenous ACh affects phytochrome-controlled plant growth and development. Mimicking red light (R), ACh stimulates adhesion of root tips to a glass surface and influences leaf movement and membrane permeability to ions. It also affects seed germination and plant growth. Moreover, ACh can modify some enzyme activity and the course of some metabolic processes in plants. Acetylcholine in the presence of calcium ions (Ca²⁺), like R stimulates swelling of protoplast isolated from etiolated wheat leaves. It is proposed that the primary mechanism of action of ACh in plant cells is via the regulation of membrane permeability to protons (H⁺), potassium ions (K⁺), sodium ions (Na⁺) and Ca²⁺.     

Hemolytic action of staphylococcal α-hemolysin on human erythrocytes in a Na+- and K+-containing suspending fluid

The hemolytic action of staphylococcal α-hemolysin on human erythrocytes was studied. In a Na⁺- and K⁺-containing suspending fluid α-hemolysin caused a rapid potassium release from the cells, which probably preceded hemoglobin release, and an influx of sodium. After storage of the cells for 4 days the same dose of α-hemolysin lyzed more cells. Lysis increased with the potassium concentration in the suspending fluid, while no correlation could be demonstrated between lysis and intracellular potassium concentration nor between lysis and potassium leakage from the cells. α-Hemolysin stimulated Mg²⁺-activated ATPase activity but did not change Na⁺-K⁺-Mg²⁺-ATPase activity. α-Hemolysin may cause increased membrane permeability for sodium ions and still more so for potassium ions, which may lead to hemolysis through swelling of the cells.     

G6PD Punjab,a dialysis sensitive variant of human glucose-6-phosphate dehydrogenase

Erythrocyte samples from 101 individuals, originally from Punjab and living at the time of investigation in England, were screened for glucose-6-phosphate dehydrogenase (G6PD) variants by Beutler’s fluorescent spot test and standard cellulose acetate gel (Cellogel) electrophoresis. All but 2 of the 40 males in the study were found to be indistinguishable from normal G6PD B. One of the variants had 2% of the normal activity and resembled G6PD Mediterranean in electrophoretic behaviour. The other variant showed 52% of the normal activity and migrated slower than G6PD B in Cellogel with about half of the normal band intensity. A set of physicochemical characteristics of the variant determined by conventional methods distinguished it from the variants reported so far. It was designated as G6PD Punjab, and the corresponding allele asG6PD PUN. The most striking feature of G6PD Punjab is a remarkable alteration in its electrophoretic behaviour after dialysis.     

Regulation of citrate synthase activity ofSaccharomyces cerevisiae

Citrate synthase activity ofSaccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product,¹⁴C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADPH were most inhibitory to the citrate synthasein vitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and α-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.