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High frequency discharge experiment. I: Formation of organic compounds from methane and ammonia.

Investigations of the synthesis of organic compounds from methane and ammonia were carried out by using high frequency discharges. The radical of CN was observed in the early phase of the discharge. Therefore it is considered that the CN radical contributes to the successive reaction steps. An important intermediate, a tetramer of hydrogen cyanide was observed to be formed at room temperature. The amounts of this compound progressively increased with time. It was speculated that the purines which were identified in the refluxed sample were synthesized through the above tetramer. Compounds which are thought to be heterocyclic (max:250-265nm) were initially formed in the sample trapped at liquid nitrogen temperature. After hydrolysis with hydrochloric acid, Gly, Asp Ala, etc. were observed in the sample. Overall yield of organic compounds was estimates at 5-6% based on input carbon of methane.

Ammonia↗

The effect of nitrogen limitation on catabolite repression of amidase, histidase and urocanase in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, the synthesis of histidase, urocanase and amidase is severly repressed when succinate is added to a culture growing in pyruvate + ammonium salts medium. When growth is nitrogen-limited, catabolite repression by succinate of histidase and urocanase synthesis does not occur but succinate repression of amidase synthesis persists. Amidase synthesis is not regulated in the same way as histidase synthesis by the availability of other nitrogen compounds for growth. Growth of P. aeruginosa strain PACI in succinate + histidine media is nitrogen-limited since this strain is defective in a histidine transport system. When methyl-ammonium chloride is added to succinate + histidine media, growth inhibition occurs. Mutants isolated from succinate + histidine + methylammonium chloride plates were found to be resistant to catabolite repression by succinate even in ammonium salts media. It is suggested that the hut genes of P. aeruginosa may be regulated in the same way as in Klebsiella aerogenes, by induction by urocanate and activation by either the cyclic AMP-dependent activator protein or by glutamine synthetase.

Amidohydrolases↗

Three-dimensional molecular illustrations I: Isoelectron density contours and isoelectrostatic energy contours.

A method of depicting dimensional illustrations of molecules in vacuo that are sensitive to small electronic perturbations was attempted. This method would be useful in determining the effects of either perturbing groups from other molecules or changes produced by the addition or modification of an existing atom or chemical group on the same molecule. Isoelectron density contours for small molecules such as benzene, ammonia, and formaldehyde were first considered using the CNDO/2 molecular approximation method and then extended to the use of deorthogonalized CNDO/2 eigenvectors. These methods were similar in molecular projections but insensitive to electronic alterations. Therefore, the electrostatic potential energy was considered in developing contour surfaces of several of the molecules studied. In this case, acute and visually discernible changes were evidenced by electron exchange in the three-dimensional illustration of formaldehyde. The effect on the two-dimensional contour map of ammonia was strikingly altered by the addition of a proton, further substantiating the sensitivity of electrostatic contours to perturbing influences. These methods are considered and amplified in this report.

Ammonia↗

Rapid and selective determination of ammonium by fluorimetric flow injection analysis.

Selective and sensitive procedures for the determination of ammonium in river water and diluted urine were developed by using flow injection analysis equipment. The methods are based on the derivatization of ammonia with o-phthaldehyde (OPA) and thioglycolate under alkaline conditions. The formed isoindole derivative is detected fluorimetrically at an excitation wavelength of 415 nm and an emission wavelength of 485 nm. The derivatization only takes 15 to 20 s at room temperature to achieve the maximum sensitivity. The optimized OPA reagent shows a surprisingly high selectivity for ammonium in the presence of many primary amines. With respect to the analysis of turbid and fluorescent sample solutions the selectivity can be improved by separating the ammonia through a microporous membrane from the OPA reagent. Without this separation step ammonia can be detected in the range between 0.05 and 100 microM with excellent linearity. After the insertion of an optimized membrane separation cell ammonia can be determined in the linear range between 0.2 microM and 20 mM.

Amines↗

Carbamoyl phosphate synthetase I of human liver. Purification, some properties and immunological cross-reactivity with the rat liver enzyme.

The purification of mitochondrial carbamoyl phosphate synthetase I (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) from small samples of human liver is described. The enzyme is composed of a single polypeptide of Mr 160 000 +/- 500 as shown by SDS-polyacrylamide gel electrophoresis in the presence of reducing agents. The synthetase migrates in polyacrylamide gradient gels in the absence of detergents at a rate corresponding to a Mr of 165 000. Estimates of the molecular weight of the native enzyme by gel filtration and density gradient centrifugation yield a value of 178 000. The results indicate that the enzyme exists predominantly as monomeres. Amino acids composition, isoelectric point, stability, Km values and the ability to catalyze partial reactions have been measured and compared with known properties of carbamoyl phosphate synthetases from other sources. From the available data a high degree of evolutionary conservation of the ammonia-dependent synthetase is suggested. This is also supported by the demonstration of extensive immunological cross-reactivity between the human and rat enzymes.

Amino Acids↗

Ammonia and disinhibition in cat motor cortex by ammonium acetate, monofluoroacetate and insulin-induced hypoglycemia.

Ammonia intoxication abolished the suppression of action potential generation by cortical postsynaptic inhibition due to the inactivation of neuronal Cl- extrusion. The disinhibition by ammonia intoxication occurred when ammonia concentrations in the cerebral cortex were increased to 320% of normal. Fluoroacetate poisoning and insulin-induced hypoglycemia, which are known to increase ammonia concentrations in the CNS and previously have been shown to inactivate Cl- extrusion in spinal motoneurons, abolished the suppression of action potential generation by cortical postsynaptic inhibition like ammonia intoxication. This disinhibition occurred at unchanged cerebral ammonia concentrations. The effect of fluoroacetate and insulin induced hypoglycemia on cortical postsynaptic inhibition is either due to a direct, i.e. not ammonia mediated, inactivation of neuronal Cl- extrusion or due to a disturbance of the synaptic mechanisms mediated by the transmitter of cortical inhibition, GABA. Toxic-metabolic encephalopathies which increase cerebral ammonia concentrations beyond 320% of normal may produce a dysfunction of the CNS due to inactivation of neuronal Cl- extrusion leading to ineffective cortical inhibition. However, in fluoroacetate poisoning and insulin-induced hypoglycemia increased ammonia concentrations in the CNS have only a secondary role in initiating a dysfunction of the CNS since disinhibition occurs before ammonia concentrations increase.

Acetates↗

In vivo activity of the nitrogen control transcription factor NtcA is subjected to metabolic regulation in Synechococcus sp. strain PCC 7942.

The cyanobacterial protein NtcA is a global transcriptional regulator of genes involved in nitrogen assimilation that are subjected to ammonium-promoted repression and is itself controlled by positive autoregulation. Strain CSI70 derived from Synechococcus sp. strain PCC 7942 was constructed to overexpress an additional ntcA gene copy from a constitutive promoter. This strain exhibited high levels of the NtcA protein both in the presence and in the absence of ammonium. However, expression of the NtcA-dependent nir operon and glnA gene (tested by RNA/DNA hybridization and enzyme activity) was still subjected to nitrogen regulation. These results indicate in vivo regulation of the activity of NtcA at activation of transcription of nitrogen-regulated genes as a function of the nitrogen status of the cell.

Bacterial Proteins↗

Ammonia metabolism in Aedes aegypti.

We investigated the mechanisms by which Aedes aegypti mosquitoes are able to metabolize ammonia. When females were given access to solutions containing NH(4)Cl or to a blood meal, hemolymph glutamine and proline concentrations increased markedly, indicating that ammonium/ammonia can be removed from the body through the synthesis of these two amino acids. The importance of glutamine synthetase was shown when an inhibitor of the enzyme was added to the meal causing the glutamine concentration in hemolymph to decrease significantly, while the proline concentration increased dramatically. Unexpectedly, we found an important role for glutamate synthase. When mosquitoes were fed azaserine, an inhibitor of glutamate synthase, the glutamine concentration increased and the proline concentration decreased significantly. This confirms the presence of glutamate synthase in mosquitoes and suggests that this enzyme contributes to the production of glutamate for proline synthesis. Several key enzymes related to ammonium/ammonia metabolism showed activity in homogenates of mosquito fat body and midgut. The mosquito genes encoding glutamate dehydrogenase, glutamine synthetase, glutamate synthase, pyrroline-5-carboxylate synthase were cloned and sequenced. The mRNA expression patterns of these genes were examined by a real-time RT-PCR in fat body and midgut. The results show that female mosquitoes have evolved efficient mechanisms to detoxify large loads of ammonium/ammonia.

Aedes↗

Ammonium-nitrogen transformation and nitrogen retention in broiler manure supplemented with a soil amendment containing nitrifying bacteria.

The effect of a soil amendment on ammonium nitrogen transformation and nitrogen retention in broiler manure was evaluated. Prior to incubation, broiler manure was mixed with autoclaved soil or non-autoclaved soil in different ratios to make 1 kg mixtures; broiler manure:non-autoclaved soil=9:1, 5:5, and 1:9 or broiler manure:autoclaved soil=9:1, 5:5, and 1:9. The non-autoclaved soil treatment reduced either numerically or significantly NH(4)(+)-N concentration compared to the autoclaved soil treatment during the 8-wk incubation. Total-N concentration of the non-autoclaved soil treatments was lower than the autoclaved soil treatments from 4 to 8 wk. The lowest manure to non-autoclaved soil treatment (M:S=1:9) had considerably more nitrite and nitrate; however, the higher ratio manure to non-autoclaved soil treatments (M:S=9:1 and 5:5) had slightly higher total nitrite and nitrate levels compared to the same ratio of autoclaved soil treatments. The moisture level of the 9:1, 5:5, and 1:9 M:S treatments were approximately 70, 45, and 30%, respectively. The results indicated that nitrifying bacteria in the non-autoclaved soil reduced the ammonium nitrogen concentrations of poultry manure by converting NH(3) or NH(4)(+) to NO(2)(-) or NO(3)(-). However, the higher moisture levels in treatments with greater manure to soil ratios (M:S=9:1 and 5:5) created anaerobic conditions that allowed for denitrification and greater N losses.

Agriculture↗

Biochemical background and compartmentalized functions of cytosolic glutamine synthetase for active ammonium assimilation in rice roots.

Rice plants in paddy fields prefer to utilize ammonium as a major nitrogen source. Glutamine synthetase (GS) serves for assimilation of ammonium in rice root, and ameliorates the toxic effect of ammonium excess. Among the three isoenzymes of the cytosolic GS1 gene family in rice, OsGLN1;1 and OsGLN1;2 were abundantly expressed in roots. Analysis of the purified enzymes showed that OsGLN1;1 and OsGLN1;2 can be classified into high-affinity subtypes with relatively high V(max) values, as compared with the major high-affinity isoenzyme, GLN1;1, in Arabidopsis. Low-affinity forms of GS1 comparable to those in Arabidopsis (GLN1;2 and GLN1;3) were absent in rice roots. The OsGLN1;1 and OsGLN1;2 transcripts showed reciprocal responses to ammonium supply in the surface cell layers of roots. OsGLN1;1 accumulated in dermatogen, epidermis and exodermis under nitrogen-limited condition. By contrast, OsGLN1;2 was abundantly expressed in the same cell layers under nitrogen-sufficient conditions, replenishing the loss of OsGLN1;1 following ammonium treatment. Within the central cylinder of elongating zone, OsGLN1;1 and OsGLN1;2 were both induced by ammonium, which was distinguishable from the response observed in the surface cell layers. The high-capacity Gln synthetic activities of OsGLN1;1 and OsGLN1;2 facilitate active ammonium assimilation in specific cell types in rice roots.

Cell Compartmentation↗

Gln49 and Ser174 residues play critical roles in determining the catalytic efficiencies of plant glutamine synthetase.

Two essential residues playing critical roles in determining the substrate specificities of cytosolic glutamine synthetase (GS1) have been identified from the alignment of high-affinity (GLN1;1 and GLN1;4) and low-affinity (GLN1;2 and GLN1;3) GS1 isoenzymes in Arabidopsis, and confirmed by site-directed mutagenesis. The results indicated that either K49Q or A174S mutation is sufficient to increase the catalytic efficiencies of GLN1;3 by decreasing its Km values for ammonium. In contrast, replacement of Gln49 and Ser174 by lysine and alanine, respectively, was detrimental to glutamine synthetic activities in GLN1;4. The results suggested that Gln49 and Ser174 in the high-affinity GS1 isoenzymes are interchangeable with Lys49 and Ala174 in the low-affinity variants at the corresponding positions.

Alanine↗

Nitrogen regulation of glutamine synthetase in Neurospora crassa.

A higher activity of glutamine synthetase (EC 6.3.1.2) was found in Neurospora crassa when NH4+ was limiting as nitrogen source than when glutamate was limiting. When glutamate, glutamine or NH4+ were in excess, a lower activity was found. Immunological titration and sucrose gradient sedimentation of the enzyme established that under all these conditions enzyme activity corresponded to enzyme concentration and that the octamer was the predominant oligomeric form. When N. crassa was shifted from nitrogen-limiting substrates to excess product as nitrogen source, the concentration of glutamine synthetase was adjusted with kinetics that closely followed dilution by growth. When grown on limiting amounts of glutamate, a lower oligomer was present in addition to the octameric form of the enzyme. When the culture was shifted to excess NH4+, glutamine accululated at a high rate; nevertheless, there was only a slow decrease in enzyme activity and no modification of the oligomeric pattern.

Culture Media↗

The reduction of nitrate to ammonium by a Clostridium sp. isolated from soil.

Cultures of Clostridium KDHS2 reduced 15NO3- to 15NH4+ with a concurrent increase in molar growth yield of 15.7% compared with fermentatively grown bacteria. The bacteria exhibited a Ks (NO3-) of 0.5 mM and reduced NO3- maximally at a rate of 0.1 mumol h(-1) mg dry wt)-1. A partially purified nitrate reductase was obtained which had a Km (NO3-) of 0.15 mM. The reduction of 13NO3- to 13NH4+ by resting bacteria was not inhibited by NH4+, glutamate, glutamine, methionine sulphoximine or azaserine. Glutamine synthetase affected neither the synthesis nor the activity of the NO3(-)-reducing enzymes. The results are consistent with the hypothesis that NO3- reduction to NH4+ in this Clostridium sp. is dissimilative. SO32-, but not SO42-, inhibited the reaction, apparently at the level of NO2- reduction.

Clostridium↗

Glutamine biosynthesis and the utilization of succinate and glutamine by Rhizobium etli and Sinorhizobium meliloti.

Sinorhizobium meliloti 1021 and Rhizobium etli CE3 turn over nitrogen and carbon from glutamine to ammonium and CO2, respectively. Some of the ammonium released is assimilated back into glutamine, indicating that a glutamine cycle similar to that in Neurospora operates in Rhizobium. In addition, a previously unrecognized metabolic pathway in Rhizobium was discovered--namely, conversion of glutamine-carbon to gamma-hydroxybutyric acid and beta-hydroxybutyric acid. Additionally, some of the 2-oxoglutarate derived from glutamine catabolism in Rhizobium is converted to succinate in glutamine-containing medium. Both S. meliloti 1021 and R. etli CE3 oxidize succinate preferentially over glutamine when provided with both carbon sources. In contrast to Sinorhizobium meliloti 1021 and Rhizobium etli CE3, an S. meliloti double mutant that lacks both glutamine synthetase (GS) I and II preferentially oxidizes glutamine over succinate when supplied with both substrates. GSII activity is induced in wild-type S. meliloti 1021 and R. etli CE3 grown in succinate-glutamine medium, and this enzyme participates in the cycling of glutamine-carbon and -nitrogen. On the other hand, GSII activity is repressed in both micro-organisms when glutamine is the only carbon source. These findings show that, in medium containing both glutamine and succinate, glutamine synthesis helps drive the utilization of succinate. When glutamine is in excess as an energy-providing substrate its synthesis is restricted, allowing for more effective utilization of glutamine as an energy source.

Amino Acids↗

Lack of a regulatory function for glutamine synthetase protein in the synthesis of glutamate dehydrogenase and nitrite reductase in Escherichia coli K12.

Synthesis of glutamine synthetase (GS) in anaerobic batch cultures of Escherichia coli was repressed when excess NH4+ was available, but derepressed during growth with a poor nitrogen source. In wild-type bacteria there was only a weak inverse correlation between the activities of GS and glutamate dehydrogenase (GDH) during growth in various media. No positive correlations were found between the activities of GS and nitrite reductase, or between GS and cytochrome c552: both of these proteins were synthesized normally by mutants that contained no active GS. Although activities of GS and GDH were low in two mutants that are unable to synthesize cytochrome c552 or reduce nitrite because of defects in the nirA gene, the nirA defect was separated from the GS and GDH defects by transduction with bacteriophage P1. Attempts to show that catabolite repression of proline oxidase synthesis could be relieved during NH4+ starvation also failed. It is, therefore, unlikely that nitrite reduction or proline oxidation by E. coli are under positive control by GS protein. The regulation of the synthesis of enzymes for the utilization of secondary nitrogen sources in E. coli, therefore, different from that in Klebsiella aerogenes, but is similar to that in Salmonella typhimurium.

Culture Media↗

Modeling of nitrogen in river water using a detailed and a simplified model.

To model catchment surface water quantity and quality, different model types are available. They vary from detailed physically based models to simplified conceptual and empirical models. The most appropriate model type for a certain application depends on the project objectives and the data availability. The detailed models are very useful for short-term simulations of representative events. They cannot be used for long-term statistical information or as a management tool. For those purposes, more simplified (conceptual or meta-) models must be used. In this study, nitrogen dynamics are modeled in a river in Flanders. Nitrogen sources from agricultural leaching and domestic point sources are considered. Based on this input, concentrations of ammonium (NH4-N) and nitrate (NO3-N) in the river water are modeled in MIKE 11 by taking into consideration advection and dispersion and the most important biological and chemical processes. Model calibration was done on the basis of available measured water quality data. To this detailed model, a more simplified model was calibrated with the objective to more easily yield long-term simulation results that can be used in a statistical analysis. The results show that the conceptual simplified model is 1800 times faster than the MIKE 11 model. Moreover the two models have almost the same accuracy. The detailed models are recommended for short-term simulations unless there are enough data for model input and model parameters. The conceptual simplified model is recommended for long-term simulations.

Agriculture↗