Ammonium-grown Cells Research Articles

Urease of blue-green algae (Cyanobacteria)Anabaena doliolum andAnacystis nidulans

Urease fromAnabaena doliolum andAnacystic nidulans showed maximum activity at pH 7.0–7.4 at 40°C when measured in cell-free, phosphate-buffered extracts. It is a soluble enzyme located in cytoplasm. The apparent Km forA. doliolum urease was 120 μM. Anacystis nidulans urease exhibited biphasic kinetics (Km=250 μM and 1.66 mM). Enzyme, fully expressed in cells grown with urea, nitrate, or N2, was repressed in ammonia-grown cells, but ammonia did not inhibit the activity in vitro. Incubation of algal cells in N2 medium with chloramphenicol for 12 h caused degradation of urease. Cu2+ at 1 μM inhibited the enzyme activity by 50%, whereas Co2+ and Ni2+ up to 20 μM had no effect.p-Hydroxymercuribenzoate appeared to be a more powerful inhibitor of urease than acetohydroxamic acid.

Morphogenesis and free amino acid composition of the ascomyceteSphaerostilbe repensas influenced by nitrogen and calcium

Abstract Sphaerostilbe repens utilizes nitrate and ammonium as nitrogen sources. Differentiation of mycelium into rhizomorphs and coremia was reduced in the presence of nitrate and completely inhibited in the absence of calcium. The most abundant free amino acids were, in decreasing order: alanine, glutamine, glutatomic acid, serine, aspartic acid, γ-aminobutyric acid, arginine and threonine. These compounds represented 90% of the total amino acid pool. The free amino acid composition did not vary with cultural conditions although concentrations of individual amino acids differed. In ammonium-grown cells, γ-aminobutyric acid increased in concentration and glutamate, aspartate and alanine decreased. Calcium-deficient media reduced amino acid concentrations, especially of arginine and ornithine. Amino acid contents increased during the growth period and were higher in rhizomorphs than in vegetative mycelia.

Utilization of nitrate, nitrite and ammonium by Chlamydomonas reinhardii

In phototrophically grown Chlamydomonas cells, ammonium strongly inhibited the utilization of nitrate or nitrite. Under darkness, or in the presence of an uncoupler or inhibitor of the non-cyclic photosynthetic electron flow, the utilization of nitrate, nitrite or ammonium was suppressed. L-Methionine-D,L-sulfoximine (MSX) or azaserine, which blocks the assimilation of ammonium, inhibited the consumption of nitrate, but not nitrite, by the cells. Ammonium produced an immediate inhibition of the permease for nitrate in Chlamydomonas growing with nitrate, while ammonium-grown cells lacked this permease. The synthesis of nitrate-reductase activity was dependent on an active permease. In N-starved Chlamydomonas cells, previously treated with MSX, the permease for nitrate was insensitive to inhibition by ammonium, and a significant amount of nitrate reductase was synthetized. These cells photoproduce ammonium by reducing nitrate. Nitrogen-repleted cells, treated with MSX, actively photoproduced ammonium by reducing nitrite, but not nitrate.

Role of L-lysine-alpha-ketoglutarate aminotransferase in catabolism of lysine as a nitrogen source for Rhodotorula glutinis.

Wild-type and saccharopine dehydrogenaseless mutant strains of Rhodotorula glutinis grew in minimal medium containing lysine as the sole nitrogen source and simultaneously accumulated, in the culture supernatant, large amounts of a product identified as alpha-aminoadipic-delta-semialdehyde. The saccharopine dehydrogenase and pipecolic acid oxidase levels remained unchanged in wild-type cells grown in the presence of ammonium or lysine as the nitrogen source. Lysine-alpha-ketoglutarate aminotransferase activity was demonstrated in ammonium-grown cells. This activity was depressed in cells grown in the presence of lysine as the sole source of nitrogen.

The Uptake of Nitrite by the DiatomPhaeodactylum: Interactions between Nitrite and Nitrate

Freshly harvested ammonium-grown cells of the diatom Phaeodactylum tricornutum cannot take up nitrite but they acquire the ability to do so after a period (about 3 h) of nitrogen deprivation under conditions that allow photosynthesis. Addition of cycloheximide (1.0 μg ml−1) prevents development as does incubation in Na+ or K+-free medium. Nitrite uptake is dependent on the presence of Na+ in the medium and is inhibited by ammonium and nitrite uptake but it is concluded that the inhibition of nitrite uptake by nitrate is not of the competitive type.

Purification and characterization of demolybdo nitrate reductase (NADH-cytochrome c oxidoreductase) of Chlorella vulgaris.

Chlorella vulgaris was cultured on an ammonia-mineral salts medium until the nitrate reductase content reached a minimal level. These ammonia-grown cells were then induced by nitrate in the absence of molybdenum and of tungsten. A demolybdo nitrate reductase developed and reached high levels. This protein contained very little nitrate-reducing capacity, but had the full cytochrome c-reducing capacity of normal nitrate reductase. It was purified to homogeneity by the same procedures previously developed for the purification of nitrate reductase. The purified enzyme contained 1 molecule of heme and 1 molecule of FAD/subunit, but no detectable molybdenum or tungsten. This cytochrome c reductase was completely inhibited by antibodies raised against purified nitrate reductase of Chlorella. Mixtures prepared from normal nitrate reductase and the demolybdoenzyme could not be resolved by disc gel electrophoresis or by centrifugation in a density gradient. By a two-step enzyme induction (1, incubation with nitrate in absence of Mo; 2, incubation with Mo in absence of nitrate) the process of nitrate reductase synthesis could be cleanly separated from growth into two steps: Step 1, induction of cytochrome c reductase, was completely inhibited by cycloheximide. Step 2 was unaffected by cycloheximide, and most of the nitrate reductase synthesized accumulated in the form of the reversibly inactivated HCN complex of the enzyme.

Regulatory Properties of Glutamine Synthetase from the Nitrogen-Fixing Phototrophic Bacterium Rhodopseudomonas palustris

Abstract Rhodopseudomonas palustris, Glutamine Synthetase Regulation, Adenylylation Glutamine synthetase from Rhodopseudomonas palustris is regulated via an adenylylation/deadenylylation mechanism. The enzyme purified from ammonia-grown cells, released AMP upon treatment with phosphodiesterase, along with drastic changes in its pH and metal dependency. Kinetic parameters for enzyme-substrate interaction were also dependent on the adenylylation state of the enzyme, as was the influence of several nitrogenous feedback inhibitors on the catalytic activity. The adenylylation state of the enzyme was modified in vivo by the availability of ammonia.

Molecular characterization of glutamine synthetase from the nitrogen-fixing phototrophic bacterium Rhodopseudomonas palustris.

The phototrophic bacterium Rhodopseudomonas palustris assimilated ammonium via glutamine synthetase and glutamate synthase. Diazotrophic and ammonium-grown cells had high levels of both enzymes, whereas enzymes of alternative assimilatory pathways were absent or had only low activities. Glutamine synthetase was purified to electrophoretic homogeneity within three steps by dye-ligand and ion exchange chromatography. Electron microscopy revealed a dodecameric molecular entity which was in accordance with parameters derived from electrophoretic techniques. The molecular weight of the enzyme monomer was 558000; that of the dodecamer 670000. The amino acid composition of R. palustris glutamine synthetase was determined and compared by a statistical method with other known enzyme compositions from prokaryotic and eukaryotic origins.

Uptake of Nitrate by the DiatomPhaeodactylum tricornutum

Ammonium-grown cells of P. tricornutum lack ability to take up nitrate. This ability develops during 3 h of nitrogen-deprivation; development requires concurrent photosynthesis, and is inhibited by cycloheximide. Nitrate is accumulated by cells with a developed nitrate uptake mechanism. Nitrate uptake is inhibited by 10−5 M CCCP and, in darkness, by anaerobiosis; it therefore presumably requires ATP which can be supplied by either photophosphorylation or oxidative phosphorylation.

Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii.

Two Azotobacter vinelandii strains capable of growing on N2(Nif+) were isolated from two different mutant strains that lacked dinitrogenase activity (Nif-). Extracts of N2-grown cells of the two Nif+ strains lacked significant amounts of the "conventional" dinitrogenase protein subunits, as determined by two-dimensional gel electrophoresis. Instead, the extracts contained at least four new proteins that appeared to be ammonia-repressible (i.e., they were not detected in extracts of ammonia-grown cells). Based on the results of genetic backcrosses, the two Nif+ strains were shown to be pseudorevertants. Both Nif+ pseudorevertant strains were able to grow in N-free media lacking molybdenum but containing tungsten (conditions that prevented growth of the wild-type strain). The four new proteins were observed in extracts of N2-fixing cells of the Nif+ pseudorevertants regardless of whether the cells were grown in the presence of molybdenum-starved wild-type A. vinelandii cells grown under N2-fixing conditions. Under conditions of molybdenum deprivation, Nif- mutant strains of several different phenotypic classes underwent phenotypic reversal to Nif+, as shown by their ability to incorporate 15N2 and to grow in N-free media. These results provide evidence that A. vinelandii possesses an alternative N2-fixation system that is expressed during conditions of molybdenum deficiency.

Synthesis of Nitrate Reductase in Chlorella

Antiserum was prepared against nitrate reductase (EC 1.6.6.1) purified to homogeneity from Chlorella vulgaris Beijerinck. Both crude antiserum and anti-nitrate reductase antibodies prepared from it were used as re-agents to study the synthesis of nitrate reductase. Cell extracts from cultures which were grown with ammonia salts as the sole source of nitrogen contained almost no active enzyme. These extracts did contain material which binds to antibody and is thus immunologically related to purified nitrate reductase. The presence of this cross reacting material in cell extracts was detected by the ability of these extracts to (a) lower the titer of antisera; (b) form a biphasic precipitin curve with purified antibody; and (c) increase the peak height of a standard amount of purified nitrate reductase in rocket immunoelectrophoresis assay. These results suggest that ammonia-grown cells contain nitrate reductase precursor protein.

Ammonium inhibition of nitrate uptake by the diatom, Phaeodactylum tricornutum

Summary Cells of Phaeodactylum tricornutum have a mechanism for uptake of nitrate which leads to its accumulation. The uptake system was absent from ammonium-grown cells but became active during incubation for 2 h with 1.0 μM nitrate. Nitrate uptake into the cells was stopped by addition of 0.3 mM ammonium chloride; it recommenced as soon as the added ammonium had disappeared. It is concluded that ammonium (or a product of its assimilation) can inhibit nitrate metabolism by inhibiting uptake of nitrate.

Post-transcriptional Control of Nitrate Reductase Formation in Green Algae

Cycloheximide (2·0 μg ml−1) inhibits the incorporation of [14C]phenylalanine and [14C]adenine into insoluble compounds in Ankistrodesmus braunii. 6-Methylpurine (1·0 mM) inhibits only the incorporation of [14C]adenine and it is concluded that it inhibits RNA synthesis. When ammonium-grown cells of Ankistrodesmus or Chlorella are nitrogen-starved or when ammonium-grown cells of Dunalitlla are resuspended in nitrate medium, the appearance of nitrate reductase in these organisms is not inhibited by 6-methylpurine. The appearance of nitrate reductase activity in Ankistrodesmus or Chlorella is inhibited by 6-methylpurine when ammonium-grown organisms are preincubated with this substance for 1-2 h before nitrogen starvation. It is concluded that cells growing with ammonium and lacking nitrate reductase activity nevertheless contain preformed mRNA for nitrate reductase synthesis.

Repression, Derepression and Activation of Nitrogenase in Azotobacter Vinelandii

Ammonium ion repressed nitrogenase in cells fixing N2 gas. Immunological tests and electrophoresis in various gels show that components I (Fe-Mo-S protein) was completely repressed by ammonium, whereas component II (Fe-S protein) apoprotein was not markedly affected. Component II from ammonium-grown cells, however, was inactive since it did not cross react with component I to reduce C2H2 to C2H4. The inactive component II apoprotein is immunologically identical to its active counterpart from cells fixing N2. Identical protein patterns were also observed in various gel-electrophoresis systems. Oxygen-inactivated component II may be reactivated with FeSo4. This salt is preferable to ferrous ammonium sulphate which inactivated component I. Immunodiffusion under aerobic conditions shows that purified component I is composed of aggregated and non-aggregated forms which are antigenically distinct. The aggregate was dissociated by treatment with sodium dodecyl sulphate (SDS) into a single antigenic species which was further resolved into two subunits on SDS disc polyacrylamide gel electrophoresis.

Methylamine and ammonia transport in Saccharomyces cerevisiae

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the vmax for methylamine transport was similar 17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 muM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70 percent when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (Mea-R) simultaneously exhibited a decreased ability to transport ammonia.

Regulation of the nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenases of Saccharomyces cerevisiae.

Saccharomyces cerevisiae contains two distinct l-glutamate dehydrogenases. These enzymes are affected in a reciprocal fashion by growth on ammonia or dicarboxylic amino acids as the nitrogen source. The specific activity of the nicotinamide adenine dinucleotide phosphate (NADP) (anabolic) enzyme is highest in ammonia-grown cells and is reduced in cells grown on glutamate or aspartate. Conversely, the specific activity of the nicotinamide adenine dinucleotide (NAD) (catabolic) glutamate dehydrogenase is highest in cells grown on glutamate or aspartate and is much lower in cells grown on ammonia. The specific activity of both enzymes is very low in nitrogen-starved yeast. Addition of the ammonia analogue methylamine to the growth medium reduces the specific activity of the NAD-dependent enzyme and increases the specific activity of the NADP-dependent enzyme.

The appearance of nitrate reductase activity in nitrogen-starved cells of Ankistrodesmus braunii

Nitrate reductase activity was detectable in ammonium-grown cells of Ankistrodesmus braunii after 50 minutes of nitrogen starvation. The rate of formation of nitrate reductase was stimulated by addition of nitrate and inhibited completely by cycloheximide (20 μg/ml). Nitrogen-starved cells assimilated added nitrate or nitrite rapidly and no nitrite or nitrate was detectable in either cells or culture medium from cultures subjected to nitrogen starvation. It is concluded that nitrate is not obligatory for the formation of nitrate reductase.

Control of pyruvate phosphoroclastic activity in extracts of Clostridium pasteurianum by ADP and acetyl phosphate

1. 1. The ATP requirement for the pyruvate phosphoroclastic reaction has been investigates in extracts of Clostridium pasteurianum, particularly with regard to control of the system. 2. 2. S-Adenosylmethione is not the active component in the ATP requirement in pyruvate metabolism in C. pasteurianum although it is known to be in Escherichia coli and Streptococcus faecalis. 3. 3. AMP and ADP can replace ATP, since enzymes in the extract establish an equilibrium mixture of the three. The active species has been shown to b ADP. 4. 4. Acetyl phosphate is a product inhibitor of the reaction. ADP, through its involvement in the acetate kinase (ATP: acetate phosphotransferase, EC 2.7.4.3) reaction, appears to exert its effect by influencing acetyl phosphate concentration. 5. 5. Arsenate removes the requirement for adenosine phosphate by preventing the accumulation of acetyl phosphate. 6. 6. Pyruvate consumption by extracts of ammonia-grown cells is not stimulated by the addition of ATP. Addition of arsenate, or ATP + ATPase (ATP phosphohydrolase, EC 3.6.1.4), stimulates pyruvate breakdown by preventing the accumulation of acetyl phosphate which otherwise occurs. 7. 7. Increasing ADP concentration counters some of the inhibitory effect of acetyl phosphate, indicating a direct control by ADP in addition to its involvement in acetyl phosphate breakdown. 8. 8. A scheme for the control of pyruvate phosphoroclastic activity in C. pasteurianum is proposed: acetyl phosphate acts as a coarse control and ADP as a fine regulator.

The development of nitrate reductase in Chlorella and its repression by ammonium

Chlorella vulgaris, grown with ammonium sulphate as nitrogen source, contains very little nitrate reductase activity in contrast to cells grown with potassium nitrate. When ammonium-grown cells are transferred to a nitrate medium, nitrate reductase activity increases rapidly and the increase is partially prevented by chloramphenicol and by p-fluorophenylalanine, suggesting that protein synthesis is involved. The increase in nitrate reductase activity is prevented by small quantities of ammonium; this inhibition is overcome, in part, by raising the concentration of nitrate. Although nitrate stimulates the development of nitrate reductase activity, its presence is not essential for the formation of the enzyme since this is formed when ammonium-grown cells are starved of nitrogen and when cells are grown with urea or glycine as nitrogen source. It is concluded that the formation of the enzyme is stimulated (induced) by nitrate and inhibited (repressed) by ammonium.