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The mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Evidence for a carboxyl group at the active site.

Ethanolamine ammonia-lyase is an adenosylcobalamin-dependent enzyme that catalyzes the rearrangement of ethanolamine and other vicinal amino alcohols to oxo-compounds and ammonia. Treatment of this enzyme with the sulfhydryl group-blocking reagent methyl methanethiosulfonate produces a species with diminished catalytic activity. When methyl methanethiosulfonate -treated ethanolamine ammonia-lyase was incubated with a carboxyl-blocking reagent consisting of glycine ethyl ester plus a water-soluble carbodiimide, the enzyme lost more than 80% of its residual activity, while at the same time glycine ethyl ester was incorporated into it at a stoichiometry of 6 mol/mol of enzyme. Both the loss of activity and the incorporation of glycine ethyl ester were prevented if ethanolamine was included in the glycine ethyl ester-containing incubation mixture. These results suggest that an active site carboxyl group plays a role in the mechanism of catalysis by ethanolamine ammonia-lyase, and that this carboxyl group is amidated when the enzyme is incubated with glycine ethyl ester plus carbodiimide.

Ammonia-Lyases↗

Phenylalanine ammonia lyase in the management of phenylketonuria: the relationship between ingested cinnamate and urinary hippurate in humans.

The hypothesis that the action of an oral preparation of the enzyme phenylalanine ammonia lyase, used to lower blood phenylalanine levels in phenylketonuria, is by removal of phenylalanine from hydrolysed protein in the lumen of the gut has been examined. Phenylalanine ammonia lyase mediates the conversion of phenylalanine to cinnamate. If cinnamate is produced in the gut it will be absorbed and converted to hippurate in the liver. However, treatment with phenylalanine ammonia lyase does not result in raised urinary hippurate ammonia lyase does not result in raised urinary hippurate levels while ingestion of cinnamate does.

Adolescent↗

Detoxification of ammonia by immobilized urea cycle enzymes.

Detoxication of ammonia was performed by dialysis of ammonia through a hollow fiber unit and by conversion to urea in a column reactor packed with immobilized urea cycle enzymes. Enzymes in the urea cycle could be immobilized by covalently binding on CNBr-activated Sepharose 4B. Ammonia was sequentially converted to urea by immobilized urea cycle enzymes with an ATP-regenerating system. Optimum conditions for the cyclic reaction were obtained theoretically and experimentally for performing continuous urea synthesis in a packed column reactor. Detoxication of ammonia of similar concentration to that in the blood in hepatic insufficiency was analyzed and performed experimentally in an extracorporeal model.

Adenosine Triphosphate↗

Phenylalanine ammonia-lyase. Induction and purification from yeast and clearance in mammals.

Yeast phenylalanine ammonia-lyase (EC 4.3.1.5) catalyzes the deamination of L-phenylalanine to form trans-cinnamic acid and tyrosine to trans-coumaric acid. Maximal enzyme activity in Rhodotorula glutinis (2 units/g, wet weight, of yeast) was induced in late-log phase (12 to 14 hours) of growth in a culture medium containing 1.0% malt extract, 0.1% yeast extract, and 0.1% L-phenylalanine. A highly purified enzyme was obtained by fractionation with ammonium sulfate and sodium citrate followed by chromatography on DEAE-cellulose and Sephadex G-200. The active preparation yielded a major component on three different polyacrylamide gel electrophoretic systems. Antisera to phenylalanine ammonia-lyase was raised in rabbits and detected by double immunodiffusion. The antigen-antibody complex was enzymatically active in vitro. The biological half-life of the enzyme was approximately 21 hours in several mammalian species (mice without and with BW10232 adenocarcinoma and B16 melanoma, rats, and monkeys) after a single injection; however, upon repeated administration, phenylalanine ammonia-lyase had a much shorter biological half-life. The onset of rapid clearance occurred earlier in tumor-bearing than in nontumor-bearing mice indicating a direct or indirect influence by the tumor on the biological half-life of phenylalanine ammonia-lyase.

Ammonia-Lyases↗

Chronic lung disease secondary to ammonia inhalation injury: a report on three cases.

Inhalation of highly hydrosoluble toxicants, like ammonia, can be associated with chronic lung diseases, which have been partially characterized. We present the case of three patients who were evaluated 2 years after massive exposure to ammonia in occupational settings. They presented with chronic dyspnea, and clinical pictures consistent with restrictive lung dysfunction, obstructive lung disease, and bronchial hyper-reactivity and small airways disease, respectively. The findings in 94 reported cases of inhalation injury due to massive exposure to ammonia are reviewed; in 35 cases follow-up for at least 1 year was available. The range of chronic pulmonary diseases associated with ammonia inhalation injury is reviewed, and suggestions for appropriate diagnostic evaluation are made.

Accidents, Occupational↗

Start-up and the effect of gaseous ammonia additions on a biofilter for the elimination of toluene vapors.

Biotechnological techniques, including biofilters and biotrickling filters are increasingly used to treat air polluted with VOCs (Volatile Organic Compounds). In this work, the start-up, the effect of the gaseous ammonia addition on the toluene removal rate, and the problems of the heat accumulation on the performance of a laboratory scale biofilter were studied. The packing material was sterilized peat enriched with a mineral medium and inoculated with an adapted consortium (two yeast and five bacteria). Start-up showed a short adaptation period and an increased toluene elimination capacity (EC) up to a maximum of 190 g/m3/h. This was related to increased CO2 outlet concentration and temperature gradients between the packed bed and the inlet (Tm-Tin). These events were associated with the growth of the microbial population. The biofilter EC decreased thereafter, to attain a steady state of 8 g/m3/h. At this point, gaseous ammonia was added. EC increased up to 80 g/m3/h, with simultaneous increases on the CO2 concentration and (Tm-Tin). Two weeks after the ammonia addition, the new steady state was 30 g/m3/h. In a second ammonia addition, the maximum EC attained was 40 g/m3/h, and the biofilter was in steady state at 25 g/m3/h. Carbon, heat, and water balances were made through 88 d of biofilter operation. Emitted CO2 was about 44.5% of the theoretical value relative to the total toluene oxidation, but accumulated carbon was found as biomass, easily biodegradable material, and carbonates. Heat and water balances showed strong variations depending on EC. For 88 d the total metabolic heat was -181.2 x 10(3) Kcal/m3, and water evaporation was found to be 56.5 kg/m3. Evidence of nitrogen limitation, drying, and heterogeneities were found in this study.

Ammonia↗

Modeling the partial nitrification in sequencing batch reactor for biomass adapted to high ammonia concentrations.

Partial nitrification has proven to be an economic way for treatment of industrial N-rich effluent, reducing oxygen and external COD requirements during nitrification/denitrification process. One of the key issues of this system is the intermediate nitrite accumulation stability. This work presents a control strategy and a modeling tool for maintaining nitrite build-up. Partial nitrification process has been carried out in a sequencing batch reactor at 30 degrees C, maintaining strong changing ammonia concentration in the reactor (sequencing feed). Stable nitrite accumulation has been obtained with the help of an on-line oxygen uptake rate (OUR)-based control system, with removal rate of 2 kg NH4 (+)-N x m(-3)/day and 90%-95% of conversion of ammonium into nitrite. A mathematical model, identified through the occurring biological reactions, is proposed to optimize the process (preventing nitrate production). Most of the kinetic parameters have been estimated from specific respirometric tests on biomass and validated on pilot-scale experiments of one-cycle duration. Comparison of dynamic data at different pH confirms that NH3 and NO2- should be considered as the true substrate of nitritation and nitratation, respectively. The proposed model represents major features: the inhibition of ammonia-oxidizing bacteria by its substrate (NH3) and product (HNO2), the inhibition of nitrite-oxidizing bacteria by free ammonia (NH3), the INFluence of pH. It appears that the model correctly describes the short-term dynamics of nitrogenous compounds in SBR, when both ammonia oxidizers and nitrite oxidizers are present and active in the reactor. The model proposed represents a useful tool for process design and optimization.

Adaptation, Physiological↗

Effect of free ammonia and free nitrous acid concentration on the anabolic and catabolic processes of an enriched Nitrosomonas culture.

The effects of free ammonia (FA; NH(3)) and free nitrous acid (FNA; HNO(2)) concentrations on the metabolisms of an enriched ammonia oxidizing bacteria (AOB) culture were investigated using a method allowing the decoupling of growth and energy generation processes. A lab-scale sequencing batch reactor (SBR) was operated for the enrichment of an AOB culture. Fluorescent in-situ hybridization (FISH) analysis showed that 82% of the bacterial population in the SBR bound to the NEU probe specifically designed for Nitrosomonas europaea. Batch tests were carried out to measure the oxygen and ammonium consumption rates by the culture at various FA and FNA levels, in the presence or absence of inorganic carbon (CO(2), HCO(3) (-), and CO(3) (2-)). It was revealed that FA of up to 16.0 mgNH(3)-N . L(-1), which was the highest concentration used in this study, did not have any inhibitory effect on either the catabolic or anabolic processes of the Nitrosomonas culture. In contrast, FNA inhibited both the growth and energy production capabilities of the Nitrosomonas culture. The inhibition on growth initiated at approximately 0.10 mgHNO(2)-N . L(-1), and the data suggested that the biosynthesis was completely stopped at an FNA concentration of 0.40 mgHNO(2)-N . L(-1). The inhibition on energy generation initiated at a slightly lower level but the Nitrosomonas culture was still oxidizing ammonia at half of the maximum rate at an FNA concentration of 0.50-0.63 mgHNO(2)-N . L(-1). The affinity constant of the Nitrosomonas culture with respect to ammonia was determined to be 0.36 mgNH(3)-N . L(-1), independent of the presence or absence of inorganic carbon.

Ammonia↗

Is increased ammonia liberation after bleeding in the digestive tract the consequence of complete absence of isoleucine in hemoglobin? A study in pigs.

A variable protein-induced toxicity has been reported in liver disease. The aim of this study was to establish the cause of increased ammonia liberation in the gut after intraluminal bleeding. Therefore, blood was sampled from catheterized piglets [20 +/- 0.8 kg (means +/- S.E.); n = 10] to determine ammonia, urea and amino acid levels before and 1, 2, 3 and 6 hr after a standard pig meal (750 gm, 12% protein). After 1 week, this procedure was repeated after ingestion of an isonitrogenous amount of bovine whole blood (400 ml). In a second series of experiments, the same procedures were performed after ingestion of plasma, whole blood, erythrocytes and feed. Electromagnetically measured total intestinal, small and large bowel flow was not significantly influenced by the type of meal ingested. Portal ammonia release was significantly increased 2-fold after a blood meal, whereas intestinal glutamine utilization did not increase. Plasma urea levels were increased 200 to 300% after whole blood and erythrocytes, whereas after ingestion of plasma, urea levels were similar to values in controls. Glutamine utilization was not different among the various groups and occurred predominantly in the small bowel. In the fasted state, small bowel glutamine utilization paralleled ammonia production. In the fed state, this equimolar relationship could not be assessed because luminal glutamine utilization could not be determined. Isoleucine levels decreased to 25% of fasting levels. Analysis of blood constituents revealed a complete lack of isoleucine in the hemoglobin molecule. Net total alpha-amino-nitrogen absorption was doubled after a blood meal.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗

Effect of ammonia on survival and adenylate energy charge in the shrimp Palaemonetes varians.

Shrimps Palaemonetes varians were submitted to lethal and sublethal concentrations of ammonia in order to measure lethality, ATP and adenylate nucleotide levels, and adenylate energy charge (AEC) index. LC50 was of about 3 mg/liter of ammonia. Adenylate measurements were performed over a period of 14 days and for two different concentrations. A population submitted to 0.5 mg/liter of ammonia exhibited high survival and a marked consumption of ATP, whereas high mortality and disordered ATP metabolism are the characteristics of the population submitted to 3 mg/liter of ammonia. An homeostatic model was applied in order to explain the significance of the AEC signal. Here, AEC is proposed as a measure of the limits of active response of an organism to environmental stress.

Adenosine Diphosphate↗

Acute toxicity of lead on tolerance, oxygen consumption, ammonia-N excretion, and metal accumulation in Penaeus indicus postlarvae.

The estuaries and backwaters that are the potential breeding grounds of penaeid shrimps are subject to heavy metal pollution through industrial effluents and domestic sewage. In the present investigation, laboratory experiments were conducted to study the acute toxicity of lead on tolerance, oxygen consumption, ammonia-N excretion, and metal accumulation in Penaeus indicus postlarvae. Static bioassay tests were employed to determine tolerance limits. Oxygen consumption, ammonia-N excretion, and metal accumulation were determined in postlarvae by exposing them to different concentrations of lead for a period of 48 h. Oxygen consumption measurements were made by using a respiratory chamber equipped with an oxygen electrode and ammonia-N was determined with trione (dichloro-S-triamine 2,4,6(1H,3H,5H-trione)). Accumulation of metal was estimated by wet-ash method. The LC50 value for 96 h was 7.223 ppm and the regression equation Y=4.1638+0.9738X with correlation coefficient of 0.9613 was obtained by probit method. A decrease in oxygen consumption and ammonia-N excretion was observed in postlarvae with increasing concentration of lead. A concentration-dependent accumulation of metal was noticed in these postlarvae. Modifications in O:N ratios of postlarvae suggest that lead accumulation might have altered utilization patterns.

Ammonia↗

Effect of ammonia on taurine transport in C6 glioma cells.

Both short-term and prolonged treatment with ammonia stimulate Tau efflux from C6 glioma cells, which confirms earlier observations with different CNS preparations. In addition, prolonged, but not short-term treatment reversibly decreases Tau uptake. The results are consistent with in vivo observations showing robust extracellular accumulation of Tau in the CNS under hyperammonemic conditions. Enhancement of Tau efflux by short term ammonia treatment is associated with its passage via a channel (or channels) which in contrast to the release from control cells is (are) not inhibited by NA. However, the increased Tau efflux does not appear to involve active Tau transport. Insensitivity to NA distinguishes ammonia-dependent Tau efflux from C6 cells from that reported in other CNS-derived preparations. Tau release evoked by prolonged ammonia treatment likewise shows a NA-insensitive component, but also a component associated with activation of Tau transport in a reverse mode. Increased outward transport of Tau may be associated with transiently increased expression of TauT mRNA. Mutual relation of the two components, but also the identity of the NA-insensitive efflux route with the anion channels so far described remains to be analyzed.

Ammonia↗

Biotreatment of ammonia- and butanal-containing waste gases.

The biological removal of ammonia and butanal in contaminated air was investigated by using, respectively, a laboratory-scale filter and a scrubber-filter combination. It was shown that ammonia can be removed with an elimination efficiency of 83% at a volumetric load of 100 m3.m-2.h-1 with 4-16 ppm of ammonia. During the experiment percolates were analysed for nitrate, nitrite, ammonium and pH. It was found that the nitrification in the biofilter could deteriorate due to an inhibition of Nitrobacter species, when the free ammonia concentration was rising in the percolate. It should be easy to control such inhibition through periodic analysis of the liquid phase by using a filter-scrubber combination. Such a combination was studied for butanal removal. Butanal was removed with an elimination efficiency of 80% by a scrubber-filter combination at a volumetric load of 100 m3.m-2.h-1 and a high butanal input concentration. Mixing the filter material with CaCO3 and pH control of the liquid in the scrubber resulted in an increase of the elimination efficiency. These results, combined with previous results on the biofiltration of butanal and butyric acid, allow us to discuss the influence of odour compounds on the removal efficiency of such systems and methods for control. The results were used to construct a full-size system, which is described.

Air Pollutants↗

Substance P antagonist does not block the stimulation of rapidly adapting pulmonary stretch receptors by ammonia.

We investigated the effects of the substance P (SP) blocker [D-Pro2,D-Trp7,9]-SP on the response of rapidly adapting pulmonary stretch receptors (RARs) to SP administered into the right atrium, or ammonia vapor inhaled into the lungs in anesthetized, spontaneously breathing rabbits. Right atrial administration of SP (0.3, 1.0, and 3.0 micrograms/kg) caused an increase in the RAR activity, and this increase became more prominent as the dose of SP was increased. The RARs increased their activity following inhalation of vapor from 5 and 10% ammonia solutions, and the increase was concentration dependent. The excitatory responses of RAR activity to SP at different doses were greatly diminished or completely blocked by administration of the selective SP antagonist (300 and 500 micrograms/kg). However, the ammonia-induced RAR stimulation was not significantly altered by prior treatment with the SP blocker (300 and 500 micrograms/kg). These results suggest that the stimulation of RARs by ammonia does not occur as a result of the release of SP from sensory nerves in the airways and lungs.

Administration, Inhalation↗

Effect of acute ammonia intoxication on energy stores in the cerebral reticular activating system.

Ammonia intoxication causes loss of consciousness. One postulated mechanism for this stipulates impaired energy metabolism in critical brain sites. The ascending reticular activating system in the brainstem may modulate consciousness. Accordingly, the present study, using micromethods, assessed energy stores in cells from the reticular activating system of mice acutely intoxicated with ammonia. In the early coma period (3.5 min after ammonia) phosphocreatine, adenosine triphosphate and glucose fell significantly while glycogen decreased later. Subsequently during coma, the high energy phosphates returned to normal and supranormal. The maximal fall in these metabolites was not accompanied by a rise in lactate, implying lack of local hypoxia or acidosis. The cells of the posterior coliculus in the same animals failed to show a significant fall in energy stores. These data suggest a selective effect of ammonia on energy metabolism in the cells of the reticular activating system of the brainstem.

Adenosine Triphosphate↗

Genes at different regulatory levels are required for the ammonia control of nodulation in Rhizobium meliloti.

The expression of the nodulation genes nodABC of Rhizobium meliloti, which determine early response functions to plant host signals, is regulated by the level of ammonia, the primary product of symbiotic nitrogen fixation. We show that the pathway that links the ammonia-induced signal to the transcriptional control of the nodABC genes involves at least two regulatory levels. The fluctuating nitrogen level is sensed and the signal is mediated by the members of the general nitrogen regulatory (ntr) system, then transmitted to the syrM-nodD3 genes representing the nod-specific level of ammonia regulation. At low ammonia concentration, the activator protein NtrC exerts its effect via nodD3. In conditions of nitrogen excess ntrR, involved in the repression of nod genes, may function in coordination with the syrM gene. Finally, the NodD3 protein may relay the nitrogen status signal to the transcriptional control of the nodABC genes.

Ammonia↗

Exercise-induced changes in blood ammonia levels in humans.

Five male and two female subjects each performed a maximal aerobic capacity (VO2 max) test, and two to four submaximal aerobic exercise bouts (requiring approximately 50 and 80% of the individual's measured VO2 max) on a motor-driven treadmill. Pre-exercise resting oxygen uptakes (VO2) and heart rates were determined and a venous blood sample drawn prior to each work session. These same measurements were repeated at 4, 15, 30, and 45 min of the resting recovery period that followed each exercise experiment. Additionally, at the 30th min of each 45-min submaximal exercise, another peripheral venous blood sample was drawn following determination of VO2 and heart rate. In all blood samples, the hematocrit and concentrations of ammonia, lactate, pyruvate, glucose, hemoglobin, and total plasma proteins were measured. A significant exponential relationship was observed betwen blood ammonia levels and VO2 for all sample periods (pre-exercise rest, exercise, and post-exercise recovery). Peripheral venous blood ammonia levels were significantly correlated with levels of pyruvate and lactate, as these latter substrates exhibited a similar exponential relationship with VO2 as was observed with ammonia.

Adult↗

Effects of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma cells. A flow-cytometry and cytochemical study.

The effect of prolonged exposure to ammonia on fluid-phase, receptor-mediated, and adsorptive (non specific) endocytosis in cultured neuroblastoma (Neuro-2a) cells were studied using fluorescein-labeled dextran, concanavalin A conjugated with fluorescein isothiocyanate, and cationized ferritin as tracers. Ammonia treatment increased the rate of endocytosis of cationized ferritin as well as the number of cell elements involved in the process. Moreover, the number of cytoplasmic components containing acid phosphatase activity was also found to increase following ammonia treatment. In contrast, flow-cytometric analyses showed that, under experimental conditions, exposure to ammonia did not alter the intralysosomal pH and had little effect on the fluid-phase and receptor-mediated endocytosis of fluorescein-labeled dextran and concanavalin-A fluorocrome, respectively.

Acetylcholinesterase↗