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PubMed2006

Active ammonia absorption in the midgut of the Tobacco hornworm Manduca sexta L.: transport studies and mRNA expression analysis of a Rhesus-like ammonia transporter.

In this study the mid- and hindgut of Manduca sexta larvae were tested for their ammonia transport properties using a custom-made Ussing chamber. In the presence of 0.1 mmoll(-1) ammonia on both sides of the isolated epithelium, active transepithelial ammonia absorption (aTEPA) was observed in all midgut sections, with greatest transport rates (ca. 140 nmol cm(-2)h(-1)) detected in the median midgut. The hindgut showed no aTEPA. In the median midgut inhibition of energy metabolism by azide blocked aTEPA completely, whereas inhibition of vacuolar H(+)-ATPase by bafilomycin A(1) reduced the active transport by 50%. The imposition of a luminal-directed NH(3)-gradient (pH 6.5 apical, pH 8.5 basal) lowered the aTEPA by approximately 50% but did not reverse its direction. Apical addition of amiloride reduced aTEPA by 90%, suggesting a role of carrier-mediated ammonia transport across the apical membrane via a member of the NHE family. Inhibition of the microtubule network by colchicine reduced aTEPA by ca. 50%. In contrast, blocking basal K(+) channels by Ba(2+) had no effect on aTEPA. Using molecular methods, evidence for intestinal expression of a Rhesus-like ammonia transporter (RhMS) was found with low mRNA expression in midgut tissues, but high expression levels in the hindgut, Malpighian tublules and ganglia.

Amiloride↗
PubMed1998

Effect of long-term ammonia starvation on the oxidation of ammonia and hydroxylamine by Nitrosomonas europaea.

Axenic cultures of the ammonia-oxidizing bacterium Nitrosomonas europaea were starved of ammonia (energy source) for up to 342 d. During this time the bacteria retained the ability to respond instantly to ammonia (1 mM) or hydroxylamine (0.1 mM) amendment by oxidizing it to nitrite without initial protein synthesis. In vivo, the ability to oxidize amended ammonia stayed almost constant during the starvation period, but a drop in the hydroxylamine oxidation rate (to 33%) was observed after 4 wk of starvation when exogenous hydroxylamine was supplied as sole energy source. In contrast, it has been shown that the level and in vitro activity of hydroxylamine oxidoreductase were not significantly affected during the starvation period. Only minor changes were detected between the protein patterns on one-dimensional SDS-PAGE of growing and starved cells. Thus, it is concluded that the activities of the energy-generating enzymes in N. europaea were not affected during long-term ammonia starvation.

Ammonia↗
PubMed1983

Determination of blood ammonia using the Ammonia Checker.

A new diffusion method employing bromocresol green for determination of blood ammonia (Ammonia CheckerR) using disposable reagent test-plates and a pocket-size colorimeter with direct read-out of results was compared with an enzymatic method. The values obtained with the Ammonia Checker were slightly lower than those with the enzymatic method. Instead of arterial blood, capillary blood may be used for ammonia determination, but thorough cleansing of the fingertip used is necessary because of the high ammonia content of sweat.

Ammonia↗
PubMed2003

Structural studies of ammonia and metallic lithium-ammonia solutions.

The technique of hydrogen/deuterium isotopic substitution has been used to extract detailed information concerning the solvent structure in pure ammonia and metallic lithium-ammonia solutions. In pure ammonia we find evidence for approximately 2.0 hydrogen bonds around each central nitrogen atom, with an average N-H distance of 2.4 A. On addition of alkali metal, we observe directly significant disruption of this hydrogen bonding. At 8 mol % metal there remains only around 0.7 hydrogen bond per nitrogen atom. This value decreases to 0.0 for the saturated solution of 21 mol % metal, as all ammonia molecules have then become incorporated into the tetrahedral first solvation spheres of the lithium cations. In conjunction with a classical three-dimensional computer modeling technique, we are now able to identify a well-defined second cationic solvation shell. In this secondary shell the nitrogen atoms tend to reside above the faces and edges of the primary tetrahedral shell. Furthermore, the computer-generated models reveal that on addition of alkali metal the solvent molecules form voids of approximate radius 2.5-3.0 A. Our data therefore provide new insight into the structure of the polaronic cavities and tunnels, which have been theoretically predicted for lithium-ammonia solutions.

Journal Article↗
PubMed2005

Liquid-vapor interfacial properties of water-ammonia mixtures: dependence on ammonia concentration.

The equilibrium and dynamical properties of the liquid-vapor interfaces of water-ammonia mixtures are investigated by means of molecular-dynamics simulations. Altogether, we have simulated seven different systems of different concentration of ammonia. The inhomogeneous density, anisotropic orientational profiles, surface tension, and the pattern of hydrogen bonding are calculated for both water and ammonia molecules in order to characterize the location, width, thermodynamic aspects, and microscopic structure of the liquid-vapor interfaces of each of the water-ammonia systems. The dynamical aspects of the interfaces are investigated in terms of the anisotropic diffusion and dipole orientational relaxation of water and ammonia molecules. The properties of the interfaces are compared with those of the corresponding bulk phases. The present theoretical results are also compared with experimental findings wherever available.

Journal Article↗
PubMed2000

Mathematical estimations of hyper-ammonia producing ruminal bacteria and evidence for bacterial antagonism that decreases ruminal ammonia production(1).

Mixed ruminal bacteria (MRB) from cattle fed hay produced ammonia from protein hydrolysate twice as fast as MRB from cattle fed mostly grain, and a mathematical model indicated that cattle fed hay had approximately four-fold more hyper ammonia-producing ruminal bacteria (HAB). HAB had a high maximum velocity of ammonia production (V(max)) and low substrate affinity (high K(m)), but simulations indicated that only large changes in V(max) or K(m) would cause a large deviation in HAB numbers. Some carbohydrate-fermenting ruminal bacteria produced ammonia at a slow rate (CB-LA), but many of the isolates had almost no activity (CB-NA). The model indicated that the ratio of CB-LA to CB-NA had little impact on HAB numbers. Validations based on predicted ratios of HAB, CB-LA and CB-NA over-predicted the specific activity of ammonia production by MRB, but co-culture incubations indicated that washed MRB from cattle fed grain could inhibit HAB. Because autoclaved MRB had virtually no effect on HAB and the incubations were always carried out at pH 7.0, the inhibition was not simply a chemical effect (e.g. low pH).

Journal Article↗
PubMed1991

Protein damage and lipid peroxidation: effects of diethyl maleate, bromotrichloromethane and vitamin E on ammonia, urea and enzymes involved in ammonia metabolism.

Changes in ammonia and urea were investigated as potential marker products of free radical damage to protein and subsequent metabolism of those damaged proteins in vivo. Both serum and liver lipid peroxidation products as measured by thiobarbituric-acid-reactive substances (TBARS) were increased by feeding rats a vitamin-E-deficient diet. The acute injection of diethyl maleate and bromotrichloromethane (DEM/BrCCl3) increased TBARS in liver of rats fed a vitamin-E-deficient diet. The concentrations of ammonia and urea in the serum and liver did not correlate with lipid peroxidation. The activities of liver glutaminase and arginase were decreased by DEM/BrCCl3 treatment in rats fed vitamin-E-deficient diet. Glutamate-ammonia ligase activity was decreased by vitamin-E-deficient diet but not by DEM/BrCCl3 treatment. Ornithine carbamoyltransferase, arginosuccinate synthase, argininosuccinate lyase and glutamate dehydrogenase (NAD(P)+) were not affected by dietary vitamin E or by DEM/BrCCl3. The data suggest that the concentrations of ammonia and urea, major by-products of nitrogen metabolism, are unchanged by the oxidant damage and lipid peroxidation, and that their control in vivo is a dynamic equilibrium of various metabolic pathways.

Ammonia↗
PubMed1989

Ammonia assimilation by Aspergillus nidulans: [15N]ammonia study.

15N kinetic labelling studies were done on liquid cultures of wild-type Aspergillus nidulans. The labelling pattern of major amino acids under 'steady state' conditions suggests that glutamate and glutamine-amide are the early products of ammonia assimilation in A. nidulans. In the presence of phosphinothricin, an inhibitor or glutamine synthetase, 15N labelling of glutamate, alanine and aspartate was maintained whereas the labelling of glutamine was low. This pattern of labelling is consistent with ammonia assimilation into glutamate via the glutamate dehydrogenase pathway. In the presence of azaserine, an inhibitor of glutamate synthase, glutamate was initially more highly labelled than any other amino acid, whereas its concentration declined. Isotope also accumulated in glutamine. Observations with these two inhibitors suggest that ammonia assimilation can occur concurrently via the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways in low-ammonia-grown A. nidulans. From a simple model it was estimated that about half of the glutamate was synthesized via the glutamate dehydrogenase pathway; the other half was formed from glutamine via the glutamate synthase pathway. The transfer coefficients of nine other amino acids were also determined.

Amino Acids↗
PubMed1992

Uptake of ammonia by Saccharomyces cerevisiae carrying the plasmid pCYG4 related with ammonia assimilation.

Batch culture experiments involving ammonia uptake in Saccharomyces cerevisiae BC55 pCYG4 have been carried out. This strain carries the plasmid pCYG4 that directs substantial overproduction of NADP-GDH, conferring an 11-fold increase in activity. The wild type cells had a specific growth rate greater than BC55 pCYG4. The ammonia uptake was practically the same until 15 h of growth. However, the amount of ammonia hydroxide added during growth (60 h) was two and half times greater in the BC55 pCYG4 than wild type cells. The results suggest that the presence of the plasmid pCYG4 can increase the amount of ammonia taken by the cells, but not the amount of biomass.

Ammonia↗
PubMed2006

Rapid exchange of fluoroethylamine via the Rhesus complex in human erythrocytes: 19F NMR magnetization transfer analysis showing competition by ammonia and ammonia analogues.

A remarkable recent discovery in red blood cell function is that the Rhesus antigen complex that for so long was considered to be simply a means of cell recognition is also the ammonia transporter. It catalyzes transmembrane exchange of ammonia on the subsecond time scale, and yet because of a lack of rapid-exchange methodology its kinetics had not been characterized. The flux of ammonia varies appreciably in diverse clinical states, and a convenient method for its characterization would be of basic and of clinical diagnostic value. Fluoroethylamine is water-soluble and when added to a suspension of human red blood cells (RBCs) displays the experimentally useful property of giving separate 19F NMR spectral peaks for the populations inside and outside the cells. By using two-site, one-dimensional magnetization exchange spectroscopy (1D-EXSY), the transmembrane exchange of fluoroethylamine was measured; it was found to occur on the subsecond time scale with an apparent first-order rate constant for efflux, under the equilibrium exchange conditions, of 3.4 s(-1). The method was used to characterize the concentration, temperature, and pH dependence of the exchange rate constant. We determined the extent of competitive inhibition exhibited by ammonia and two molecules that contain an amine group (ethylamine and methylamine). Inhibition of the exchange by incubating the suspension with anti-RhAG antibody, and no inhibition by anti-RhD antibody, suggested specificity of exchange via the RhAG protein of the Rh complex.

Ammonia↗
PubMed1978

The role of ammonia, L-glutamate, and cyclic adenosine 3',5'-monophosphate in the regulation of ammonia assimilation in Rhizobium japonicum.

The effects of three factors (ammonia, L-glutamate, and cyclic adenosine 3',5'-monophosphate) on the ammonia assimilatory processes in aerobically grown Rhizobium japonicum colony derivatives were examined. Ammonia repressed glutamine synthetase activity and increased the average state of adenylylation of this enzyme. The addition of L-glutamate drastically decreased growth and strongly repressed glutamate synthase levels. Glutamine synthetase repression and adenylylation state were also increased by L-glutamate. The presence of cyclic AMP led to the repression of all three NH+4 assimilatory enzymes.

Ammonia↗
PubMed1992

Mechanism of C-3 hydrogen exchange and the elimination of ammonia in the 3-methylaspartate ammonia-lyase reaction.

The enzyme 3-methylaspartate ammonia-lyase (EC 4.3.1.2) catalyzes the exchange of the C-3 hydrogen of the substrate, (2S,3S)-3-methylaspartic acid, with solvent hydrogen. The mechanism of the exchange reaction was probed using (2S,3S)-3-methylaspartic acid and its C-3-deuteriated isotopomer. Incubations conducted in tritiated water allowed the rate of protium or deuterium wash-out from the substrates to be measured as tritium wash-in. The primary deuterium isotope effects for the exchange under essentially Vmax conditions ( [S] much greater than Km) were 1.6, 1.5, and 1.5 at pH 9.0, 7.6, and 6.5. The deamination reaction, measured spectrophotometrically on the same incubations, showed isotope effects of 1.7, 1.6, and 1.4 at pH 9.0, 7.6, and 6.5, in agreement with the values of DV and D(V/K) reported previously [Botting, N.P., Akhtar, M., Cohen, M.A., & Gani, D. (1988) Biochemistry 27, 2956-2959]. The ratio of the rate of exchange to the rate of deamination, however, varied widely with pH. Together with the identical values of the primary isotope effects for the two reactions, this result indicates that the partition between reaction pathways occurs after the slowest steps in the common part of the reaction coordinate pathway, almost certainly after the cleavage of the C-N bond at the level of the enzyme-ammonia-mesaconic acid complex, and not at the putative carbanion level as was previously suggested. The enzyme requires both K+ and Mg2+ ions for activity, although ammonium ion is also able to bind in the K+ site and act as an activator. Variation of the metal ion concentration alters the magnitude of the primary deuterium isotope effects. The variation of potassium ion concentration causes the most marked changes: at 1.6 mM K+, DV and D(V/K) are 1.7, whereas at 50 mM K+, DV and D(V/K) are reduced to 1.0. The isotope effects are also reduced at low K+ concentration due to the emergence of a slow-acting high K+ affinity monopotassium form of the enzyme. The binding order and role of the metal ion cofactors and their influence in determining the formal mechanism of the reaction is discussed, and the failure of previous workers to observe primary deuterium isotope effects for the deamination process is explained. The product desorption order was tested by product inhibition, alternative product inhibition, and isotope exchange experiments. Ammonia and mesaconic acid debind in a random fashion.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammonia↗
PubMed1997

Maize phenylalanine ammonia-lyase has tyrosine ammonia-lyase activity.

A full-length cDNA encoding phenylalanine ammonia-lyase (PAL) from Zea mays L. was isolated and the coding region was expressed in Escherichia coli as a C-terminal fusion to glutathione S-transferase. After purification by glutathione-Sepharose chromatography, the glutathione S-transferase moiety was cleaved off and the resulting PAL enzyme analyzed. In contrast to PAL from dicots, this maize PAL isozyme catalyzed the deamination of both L-phenylalanine (PAL activity) and L-tyrosine (tyrosine ammonia-lyase activity). These results provide unequivocal proof that PAL and tyrosine ammonia-lyase activities reside in the same polypeptide. In spite of large differences in the Michaelis constant and turnover number of the two activities, their catalytic efficiencies are very similar. Also, both activities have the same pH and temperature optima. These results imply that maize can produce p-coumaric acid from both phenylalanine and tyrosine.

Amino Acid Sequence↗
PubMed1985

Reduced phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities and lignin synthesis in wheat grown under low pressure sodium lamps.

Wheat (Triticum aestivum L. cv Fremont) grown in hydroponic culture under 24-hour continuous irradiation at 560 to 580 micromoles per square meter per second from either metalhalide (MH), high pressure sodium (HPS), or low pressure sodium (LPS) lamps reached maturity in 70 days. Grain yields were similar under all three lamps, although LPS-grown plants lodged at maturity. Phenylalanine ammonia-lyase (PAL) and a tyrosine ammonia lyase (TAL) with lesser activity were detected in all extracts of leaf, inflorescence, and stem. Ammonia-lyase activities increased with age of the plant, and plants grown under the LPS lamp displayed PAL and TAL activities lower than wheat cultured under MH and HPS radiation. Greenhouse solar-grown wheat had the highest PAL and TAL activities. Lignin content of LPS-grown wheat was also significantly reduced from that of plants grown under MH or HPS lamps or in the greenhouse, showing a correlation with the reduced PAL and TAL activities. Ratios of far red-absorbing phytochrome to total phytochrome were similar for all three lamps, but the data do not yet warrant a conclusion about specific wavelengths missing from the LPS lamps that might have induced PAL and TAL activities in plants under the other lamps.

Ammonia-Lyases↗
PubMed1975

Mechanism of action of ethanolamine ammonia-lyase, an adenosylcobalamin-dependent enzyme. Proton nuclear magnetic resonance studies of the binding of adenine nucleosides and substrate to ethanolamine ammonia-lyase.

Proton NMR spectroscopy was used to study the binding of adenosine, 5'-deoxyadenosine, adenine, and ethanolamine to the adenosylcobalamin-dependent enzyme ethanolamine ammonia-lyase. Broadening of ligand resonances in the presence of ethanolamine ammonia-lyase indicated that adenosine, 5'-deoxyadenosine, and ethanolamine all formed complexes with the enzyme (KD(mM) = 3.5, 3.0, and 2.5 respectively). The methyl group of enzyme-bound 5'-deoxyadenosine rotated at a rate exceeding 10(7) revolutions/s. Adenine did not appear to bind to the enzyme. Rates of dissociation of nucleosides from the enzyme were fast on the NMR time scale, precluding measurements of rate constants for the binding reaction. The departure of ethanolamine was slow, however, permitting their determination. The values for these rate constants were: k1 = 4.4 times 10(5) M-1 S-1; k-1 = 1.1 times 10(3) S-1. Addition of 1 mol of cyanocobalamin/mol of active sites led to narrowing of the enzyme-broadened ligand resonances. With 5'-deoxyadenosine, linewidths still exceeded those of the free ligand, indicating that binding to enzyme was weakened but not abolished. The KD for this nucleoside in the presence of CNCbl was 8.0 mM. With ethanolamine and adenosine, however, linewidths reverted to values characteristic of the unbound ligand, indicating either that CNCbl greatly lowered the rate of dissociation of the ligand or displaced the ligand from the enzyme. A decision between these two possibilities could not be made from the data at hand, although analogy with the situation obtaining with 5'-deoxyadenosine suggests that adenosine is displaced from the enzyme by CNCbl. 5'-Deoxyadenosine inhibited catalytic activity of the enzyme, competing with adenosylcobalamin (Ki = 2.7 mM). Adenosine had no effect, despite NMR evidence indicating that it formed a complex with free enzyme. These experiments showed that ethanolamine ammonia-lyase possesses binding sites for adenine nucleosides, a class of compounds chemically related to the Cobeta-ligand of the cofactor, as well as for ethanolamine. Binding to the enzyme has now been demonstrated for all three categories of low molecular weight compounds thought to be involved in the reaction; namely, substrate (ethanolamine), corrin, and adenine nucleoside.

Adenine↗
PubMed1992

Reversible stepwise mechanism involving a carbanion intermediate in the elimination of ammonia from L-histidine catalyzed by histidine ammonia-lyase.

L-Histidine labeled with deuterium at the C-5' position of the imidazole ring, L-[5'-2H]histidine (His-5'-D), was used as a probe for investigating a stepwise reversible mechanism via a carbanion intermediate in the elimination of ammonia catalyzed by histidine ammonia-lyase (EC 4.3.1.3). The labeled L-histidine (His-5'-D) (2.45 mM) was incubated with histidine ammonia-lyase (200 units) from Pseudomonas fluorescens at pH 7.0 or 9.0 at 25.0 degrees C for 24 h. The time course of the reaction was examined to determine the rates of enzyme-catalyzed hydrogen exchange at C-5' of L-histidine and urocanic acid. The finding of the enzyme-catalyzed hydrogen exchange at C-5' of both L-histidine and urocanic acid in the presence of L-histidine provided a rational explanation for a stepwise reversible mechanism via a carbanion intermediate in the elimination reaction. The rate of increase in the concentration of urocanic acid exchanged with hydrogen (UA-5'-H) did not depend on the formation rate of urocanic acid and UA-5'-H was continuously formed at a constant rate (25.6 microM/h) even after the completion of urocanic acid formation. These observations suggested the presence of the reversible reaction of urocanic acid and a carbanion intermediate. Since there was only a minor contribution for the formation of UA-5'-H from L-histidine exchanged with solvent hydrogen (His-5'-H), the main pathway in the enzymatic reaction of His-5'-D must be the formation of UA-5'-D via a carbanion intermediate (carbanion-D). Regeneration of the carbanion-D from UA-5'-D by its reverse reaction and subsequent hydrogen incorporation at C-5' would contribute to a large extent for the formation of UA-5'-H. The stability of carbanion was also demonstrated to be approximately three times higher at pH 7.0 than at pH 9.0.

Ammonia↗
PubMed2005

Ammonia formation by the reduction of nitrite/nitrate by FeS: ammonia formation under acidic conditions.

One issue for the origin of life under a non-reducing atmosphere is the availability of the reduced nitrogen necessary for amino acids, nucleic acids, etc. One possible source of this nitrogen is the formation of ammonia from the reduction of nitrates and nitrites produced by the shock heating of the atmosphere and subsequent chemistry. Ferrous ions will reduce these species to ammonium, but not under acidic conditions. We wish to report results on the reduction of nitrite and nitrate by another source of iron (II), ferrous sulfide, FeS. FeS reduces nitrite to ammonia at lower pHs than the corresponding reduction by aqueous Fe+ 2. The reduction follows a first order decay, in nitrite concentration, with a half-life of about 150 min (room temperature, CO2, pH 6.25). The highest product yield of ammonia measured was 53%. Under CO2, the product yield decreases from pH 5.0 to pH 6.9. The increasing concentration of bicarbonate, at higher pH, interferes with the reaction. Comparing experiments under N2 CO2 shows the interference of bicarbonate. The reaction proceeds well in the presence of such species as chloride, sulfate, and phosphate, though the yield drops significantly with phosphate. FeS also reduces nitrate and, unlike with Fe+ 2, the reduction shows more reproducibility. Again, the product yield decreases with increasing pH, from 7% at pH 4.7 to 0% at pH 6.9. It appears that nitrate is much more sensitive to the presence of added species, perhaps not competing as well for binding sites on the FeS surface. This may be the cause of the lack of reproducibility of nitrate reduction by Fe+ 2 (which also can be sensitive to binding by certain species).

Ammonia↗
PubMed2001

Ammonia regulates VID30 expression and Vid30p function shifts nitrogen metabolism toward glutamate formation especially when Saccharomyces cerevisiae is grown in low concentrations of ammonia.

The GATA family proteins Gln3p and Gat1p mediate nitrogen catabolite repression (NCR)-sensitive transcription in Saccharomyces cerevisiae. When cells are cultured with a good nitrogen source (glutamine, ammonia), Gln3p and Gat1p are restricted to the cytoplasm, whereas with a poor nitrogen source (proline), they localize to the nucleus, bind to the GATA sequences of NCR-sensitive gene promoters, and activate transcription. The target of rapamycin-signaling cascade and Ure2p participate in regulating the cellular localization of Gln3p and Gat1p. Rapamycin, a Tor protein inhibitor, like growth with a poor nitrogen source, promotes nuclear localization of Gln3p and Gat1p. gln3 Delta and ure2 Delta mutants are partially resistant and hypersensitive to growth inhibition by rapamycin, respectively. We show that a vid30 Delta is more rapamycin-sensitive than wild type but less so than a ure2 Delta. VID30 expression is modestly NCR-sensitive, responsive to deletion of URE2, and greatly increases in low ammonia medium. Patterns of gene expression in a vid30 Delta suggest that the Vid30p function shifts the balance of nitrogen metabolism toward the production of glutamate, especially when cells are grown in low ammonia. CAN1, DAL4, DAL5, MEP2, DAL1, DAL80, and GDH3 transcription is down-regulated by Vid30p function with proline as the nitrogen source. An effect, however, that could easily be indirect.

Ammonia↗