Ammonia In Medium Research Articles

Different Rates of Synthesis and Degradation of Two Chloroplastic Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase Isoenzymes during Induction and Deinduction in Chlorella sorokiniana Cells

The kinetics of accumulation (per milliliter of culture) of the alpha- and beta- subunits, associated with chloroplast-localized ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzymes, were measured during a 3 hour induction of synchronized daughter cells of Chlorella sorokiniana in 29 millimolar ammonium medium under photoautotrophic conditions. The beta-subunit holoenzyme(s) accumulated in a linear manner for 3 hours without an apparent induction lag. A 40 minute induction lag preceded the accumulation of the alpha-subunit holoenzyme(s). After 120 minutes, the alpha-subunit ceased accumulating and thereafter remained at a constant level (i.e. steady state between synthesis and degradation). From pulsechase experiments, using (35)SO(4) and immunochemical procedures, the rate of synthesis of the alpha-subunit was shown to be greater than the beta-subunit during the first 80 minutes of induction. The alpha- and beta-subunits had different rates of degradation during the induction period (t((1/2)) = 50 versus 150 minutes, respectively) and during the deinduction period (t((1/2)) = 5 versus 13.5 minutes) after removal of ammonium from the culture. During deinduction, total NADP-GDH activity decreased with a half-time of 9 minutes. Cycloheximide completely inhibited the synthesis and degradation of both subunits. A model for regulation of expression of the NADP-GDH gene was proposed.

Electrochemical reactions on a titanium alloy by the rotating disk electrode method in aqueous ammonia media

Electrochemical reactions on a titanium alloy by the rotating disk electrode method in aqueous ammonia media

Preparation of ThO2-UO3Sols with Uranium Contents of 0–35% for Gelation into Microspheres in CC14-Ammonia Media

Preparation of ThO₂-UO₃Sols with Uranium Contents of 0–35% for Gelation into Microspheres in CC1₄-Ammonia Media

Regulation of Nitrate Reductase Synthesis in the Yeast Candida nitratophila

Summary: Immunoelectrophoresis, using antibodies raised against homogeneously pure nitrate reductase (NR; EC 1.6.6.2), was used to study the regulation of NR synthesis in Candida nitratophila. Extracts from ammonium-grown cultures did not contain cross-reacting material, but cross-reacting material was detected in cultures transferred to nitrogen-free and nitrate medium. The appearance of NR activity in these extracts appeared to be the result of de novo protein synthesis. The possible role of nitrate in the stimulation of active NR synthesis is discussed. Losses of NR activity that occurred in nitrate-grown cultures transferred to ammonium medium correlated with decreases in cross-reacting material.

Evidence for Chloroplastic Localization of an Ammonium-Inducible Glutamate Dehydrogenase and Synthesis of Its Subunit from a Cytosolic Precursor-Protein in Chlorella sorokiniana

Chlorella sorokiniana cells, cultured for 12 hours in 30 millimolar ammonium medium, contained an ammonium inducible nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase (NADP-GDH) isoenzyme with subunits having a molecular weight of 53,000. In vitro translation of total cellular poly(A)(+) RNA, isolated from fully induced cells, resulted in synthesis of an NADP-GDH antigen with a molecular weight of 58,500. The 58,500 dalton antigen was processed in vitro, with a 100,000g supernatant prepared from broken fully induced Chlorella cells, to a protein with a molecular weight of 53,000. These data support the inference that the NADP-GDH subunit (M(r) = 53,000) is initially synthesized as a larger precursor protein (M(r) = 58,500). By use of a cytochemical staining procedure, dependent upon NADP-GDH catalytic activity, the holoenzyme was shown to be chloroplast-localized. An immunoelectron microscopy procedure, employing anti-NADP-GDH immunoglobulin G and Protein A-gold complex, showed that NADP-GDH antigen was absent from the nucleus but present in both the chloroplast and cytosol. Since synthesis of the enzyme can be inhibited by cycloheximide, the detection of NADP-GDH antigen in the cytosol was probably due to binding of the NADP-GDH antibody to nascent polypeptide chains of the precursor-protein being synthesized on cytosolic 80S ribosomes.

Properties and activity of zinc chemisorbents on carriers

1. In the preparation of zinc alumocalcium chemisorbents, reactions take place during mixing of the zinc component with the carrier in the aqueous ammonium medium, which lead to the formation of phases of zinc hydroxoaluminate, gibbsite, zinc hydroxide, calcite, an ammonia-carbonate zinc complex, and tricalcium aluminate hexahydrate. 2. The degree of combination of the zinc component into the zinc hydroxoaluminate depends on the degree of hydration of the carrier and decreases in the order: calcium mono-aluminate > calcium dialuminate > aluminum oxide > Galumin. 3. In the thermolysis the zinc hydroxoaluminate is converted to dispersed zinc oxide and disordered zinc-aluminum spinel, the amount of which depends on the nature of the carrier. 4. The sulfur capacity of the zinc chemisorbents depends on the zinc oxide content and on the amount of the free zinc oxide phase; it changes antibatically to the ability of the carrier to bind the zinc component into zinc hydroxoaluminate and zinc-aluminum spinel.

Inorganic nitrogen assimilation and the regulation of glucose 6-phosphate dehydrogenase in unicellular algae

Cultures of unicellular green algae contained higher levels of glucose 6-phosphate dehydrogenase (G6PDH), and 6-phosphogluconate dehydrogenase (6PGDH), activity when grown in nitrate medium compared to those grown in ammonium medium. With Chlamydomonas reinhardii a mutation in the structural gene for nitrate reductase ( nit A) results in a large over production of G6PDH and 6PGDH activity in nitrogen-free and nitrate medium. Increases in G6PDH activity that occur in nit A cultures incubated in nitrate medium under autotrophic conditions depend on protein synthesis. G6PDH was inactivated by reduced thioredoxin. Oxidised glutathione prevented this inactivation and activated the inactive enzyme.

Preparation of ThO2Sols Suitable for Gelation into Microspheres in CCl4-Ammonia Media

Production conditions of ThO2 sols are studied for satisfactorily gelling them into micro- spheres in CCl4-ammonia media, which result gel-spheres with good sphericities but need high-quality source-sols for crackfree gelation. Two-stage pH-setting techniques were added to the previously reported method, which had been developed to produce ThO2 sols suitable for gelation in hexone-ammonia media and was characterized by preneutralization followed by neutralization under pH-control. As a result, colloid fractions of Th in sols were increased without decreasing colloid size. This enabled one to obtain crackfree, large, spherical ThO2 particles by gelation in the CC14-ammonia media.

Regulation by ammonium of glutamate dehydrogenase (NADP+) from Saccharomyces cerevisiae.

The activity of glutamate dehydrogenase (NADP+) (EC 1.4.1.4; NADP-GDH) of Saccharomyces cerevisiae is decreased under conditions in which intracellular ammonia concentrations increases. A high internal ammonia concentration can be obtained (a) by increasing the ammonium sulphate concentration in the culture medium, and (b) by growing the yeast either in acetate + ammonia media, where the pH of the medium rises during growth, or in heavily buffered glucose + ammonia media at pH 7.5. Under these conditions cellular oxoglutarate concentrations do not vary and changes in NADP-GDH activity appear to provide a constant rate of oxoglutarate utilization. The following results suggest that the decrease in NADP-GDH activity in ammonia-accumulating yeast cells is brought about by repression of synthesis: (i) after a shift to high ammonium sulphate concentrations, the number of units of activity per cell decreased as the inverse of cell doubling; and (ii) the rate of degradation of labelled NADP-GDH was essentially the same in ammonia-accumulating yeast cells and in controls, whereas the synthesis constant was much lower in the ammonia-accumulating cells than in the controls.

Role of the diaphorase moiety on the reversible inactivation of the Chlamydomonas reinhardii nitrate reductase complex

Nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) of Chlamydomonas reinhardii mutant 305. lacking NAD(P)H-cytochrome c reductase activity, became rapidly and reversibly inactivated in vitro upon incubation with dithionite and reactivated by oxidation with ferricyanide. Nitrate protected against the inactivation. Unlike the native nitrate reductase complex of its parental wild strain, the enzyme of 305 was not inactivated by reduced pyridine nucleotides. In contrast to that of wild-type cells, the in vivo reversible inactivation of mutant 305 nitrate reductase was observed only after complete elimination of nitrate from the media and subsequent transfer to ammonium medium or darkness conditions. The inactive enzyme was reactivated by addition of nitrate to the media without previous removal of ammonium, which indicates that, unlike in the wild-type cells, ammonium does not prevent nitrate uptake by 305 cells. This different regulation pattern is due to the structural modification of 305 nitrate reductase. We conclude that in vitro an active diaphorase moiety is required for the inactivation by reduced pyridine nucleotides of the nitrate reductase of C. reinhardii, and that in vivo the absence of nitrate rather than the presence of ammonium is the triggering event for nitrate reductase inactivation, which can be also achieved by reductants other than NAD(P)H.

Canavanine Resistance and the Mechanism of Arginine Uptake in the Fission Yeast Schizosaccharomyces pombe

The mechanism of resistance to the arginine analogue L-canavanine, and of arginine uptake, were examined in the fission yeast Schizosaccharomyces pombe. Two mutants with increased resistance to canavanine were analysed genetically: both were double mutants, and in each case one mutation conferred resistance to canavanine, while the other enhanced this resistance. Evidence is presented that can 1.1 strains are defective in one system for arginine uptake, which presumably prevents entry of canavanine into the cell. This system operates in the wild-type whether the nitrogen source supplied is ammonium or glutamate. Double mutants carrying can 1.1 and an arginine requirement are unable to grow on ammonium medium even when supplied with exogenous argine, while growth can occur on glutamate plus arginine. This suggested the existence of a second uptake system for arginine which is absent during growth on ammonium, and direct measurements of the rates of arginine uptake under various conditions confirmed this. Our observations closely parallel those made on the budding yeast Saccharomyces cerevisiae. The ability to select for or against function of the can 1 gene should facilitate certain types of genetical analysis in S.pombe.

Role of glutamine synthetase in the uptake and metabolism of methylammonium by Azotobacter vinelandii

Methylammonium is a substrate for the ammonium transport system of Azotobacter vinelandii. During cellular uptake methylammonium is rapidly converted to a less polar metabolite (E. M. Barnes, Jr., and P. Zimniak, J. Bacteriol. 146:512-516, 1981). This metabolite has been isolated from A. vinelandii and identified as gamma-glutamylmethylamide by mass spectroscopy, 1H nuclear magnetic resonance spectroscopy, and cochromatography with the authentic compound. Escherichia coli also accumulated gamma-glutamylmethylamide during methylammonium uptake. The biosynthesis of gamma-glutamylmethylamide in vitro required methylammonium, ATP, L-glutamate, and a soluble cell extract from A. vinelandii. The enzyme responsible for gamma-glutamylmethylamide synthesis was glutamine synthetase. In a crude extract, L-methionine-DL-sulfoximine was equipotent in inhibiting the activities for gamma-glutamyltransferase and for the synthesis of glutamine and gamma-glutamylmethylamide. Likewise, an antiserum against the glutamine synthetase of E. coli precipitated the transferase and both synthetic activities at similar titers. During repression by growth of cells on ammonium medium, the synthesis of glutamine and gamma-glutamylmethylamide in vitro was also inhibited coordinately. A partially purified preparation of glutamine synthetase from A. vinelandii utilized methylammonium as substrate (Km = 78 mM, Vmax = 0.30 mumol/min per mg), although less efficiently than ammonium (Km = 0.089 mM, Vmax = 1.1 mumol/min per mg). The kinetic properties of glutamine synthetase with methylammonium as substrate as well as the insensitivity of this activity to inhibition by T1+ were strikingly different from methylammonium translocation. Thus, methylammonium (ammonium) translocation and intracellular trapping as glutamylamides are experimentally distinguishable processes.

Derepression of asparaginase II during exponential growth of Saccharomyces cerevisiae on ammonium ion

The biosynthesis of asparaginase II in Saccharomyces cerevisiae is sensitive to nitrogen catabolite repression. In cell cultures growing in complete ammonia medium, asparaginase II synthesis is repressed in the early exponential phase but becomes derepressed in the midexponential phase. When amino acids such as glutamine or asparagine replace ammonium ion in the growth medium, the enzyme remains repressed into the late exponential phase. The three nitrogen compounds permit a similar rate of cell growth and are assimilated at nearly the same rate. In the early exponential phase the internal amino acid pool is larger in cells growing with glutamine or asparagine than in cells growing with ammonium sulfate as the sole source of nitrogen.

Expression of the ROAM mutations in Saccharomyces cerevisiae: involvement of trans-acting regulatory elements and relation with the Ty1 transcription.

The regulatory mutations in Saccharomyces cerevisiae designated cargA + Oh, cargB + Oh, and durOh are alterations in the control regions of the respective structural genes. The alteration causing the cargA + Oh mutation has been shown to be an insertion of a Ty1 element in the 5' noncoding region of the CAR1 ( cargA ) locus. All three mutations cause overproduction of their corresponding gene products and belong to the ROAM family of mutations (Regulated Overproducing Allele responding to Mating signals) in yeast. The amount of overproduction in ROAM mutants is determined, at least in part, by signals that control mating functions in yeast. We report the identification of two genetic loci that regulate Oh mutant gene expression but that do not affect mating ability. These loci are defined by the recessive roc mutations ( ROAM mutation control) that reduce the amount of overproduction caused by the cargA + Oh, cargB + Oh, and durOh mutations. RNAs homologous to CAR1 ( cargA ), DUR1 ,2 and Ty1 DNA probes were analyzed by the Northern hybridization technique. In comparison with wild-type strains, cargA + Oh and durOh mutant strains grown on ammonia medium contain increased amounts of CAR1 and DUR1 ,2 RNA. This RNA overproduction is diminished in MATa/MAT alpha diploid strains as well as in haploid strains that also carry the ste7 mutation which prevents mating or that carry either of the roc1 or roc2 mutant alleles. The amount of RNA homologous to Ty1 DNA is also reduced in ste7 , roc1 , and roc2 mutant strains. This reduction is not observed in a strain with the ste5 mutation, which prevents mating but has no effect on overproduction of ROAM mutant gene products.(ABSTRACT TRUNCATED AT 250 WORDS)

Light Requirement for Induction and Continuous Accumulation of an Ammonium-Inducible NADP-Specific Glutamate Dehydrogenase in Chlorella

The ammonium-inducible NADP-specific glutamate dehydrogenase of Chlorella sorokiniana was shown to require light for both its induction by ammonia in uninduced cells, and its continuous accumulation in fully induced cells. Addition of ammonia to uninduced cells in the light resulted in a 35-minute induction lag followed by linear and coincident increases in enzyme activity and antigen. Enzyme activity was not induced in the dark; however, transfer of these cells to the light resulted in an immediate increase in enzyme activity and antigen. The absence of an induction lag suggested that mRNA sequences and/or an enzyme precursor with different antigenic properties than the active holoenzyme accumulated in cells in the dark in ammonium medium. When fully induced cells were transferred to the dark, the activity of the enzyme quickly ceased to accumulate. In contrast to the NADP-specific isozyme, the cells also contain a constitutive NAD-specific isozyme which was shown to accumulate in cells in the dark in either ammonium or nitrate medium.

The effect of changes in pH on phosphate and potassium uptake by Monascus rubiginosus ATCC 16367 in submerged shaken culture.

Monascus rubiginosus ATCC 16367 was cultivated aerobically in media containing ammonium and nitrate as nitrogen source. The pH of the medium was adjusted at different times, the pH of the nitrate medium being lowered to the pH of the ammonium medium and the pH of the ammonium medium raised to that of the nitrate. More phosphate was taken up on the nitrate medium, but potassium uptake did not start until 24h. On the ammonium medium, both were taken up in parallel from the beginning, but the amount of phosphate taken up never reached the same level as on nitrate medium. When the pH was adjusted, the uptakes changed, especially on the ammonium medium where a great increase in phosphate uptake was observed. More conidia were formed on the nitrate medium and more pigment on the ammonium medium. When the pH of either media was adjusted, the development of conidia and pigment production changed to that of the other control medium where the pH evolved normally in the direction of the change, regardless of the source of nitrogen. The reasons for the development of conidia on nitrate medium or where the pH is high, and the production of pigment on ammonium medium or at low pH is discussed.

Glutamate Dehydrogenase from Tea Rootlet

Glutamate dehydrogenase (GDH) (1.4.1.2) was prepared from tea rootlet. The GDH from tea rootlet used NADH and NADPH but NADH was favored. Opt pH of the reductive amination of GDH was ca. pH 8.0. The Km values for 2-oxoglutarate, NH4+ and NADH were 0.42 mM, 24 mm and 0.042 mm, respectively. The GDH was inhibited by high concentrations of both 2-oxoglutarate and NH4+. The activity of GDH from tea rootlet was slightly stimulated by Ca2+ and Mg2+. However, Co2+ and Mn2+ were not effective. On the contrary, Zn2+ showed clearly an inhibitory effect. EDTA or p-chloromercuribenzoate inhibited the activity of GDH. The inhibitory effect of EDTA on the GDH was restored by adding Ca2+ and Mg2+. However, Zn2+ increased the inhibitory effect of EDTA. The activity of GDH was inhibited by the high concentration of glutamate. Glutamine, theanine and alanine stimulated the activity of GDH. The activity of GDH in tea rootlets cultured upon glutamate medium was remarkably repressed. The activity of glutamate treated GDH was reactivated when tea rootlets were transferred onto ammonia medium. The GDH enzyme protein from tea rootlet was separated into two fractions on the DEAE chromatogram. And the both fractions increased when the rootlets were supplied with ammonia.

Activation, synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Rates of synthesis and turnover of nitrate reductase in Chlorella vulgaris have been estimated from density-labelling experiments. It is demonstrated that nitrate stimulates the rate of turnover of the enzyme. Upon transfer of Chlorella from ammonium to nitrate medium there is a rapid increase in nitrate reductase activity, only part of which is a consequence of increased de novo synthesis: there must also be considerable activation of existing enzyme. It is shown that, in the absence of nitrate, the enzyme is very stable in vivo. Upon transfer from nitrate to ammonium medium, synthesis of nitrate reductase is reduced below the limits of defection. These effects are separate from the reversible (ferricyanide-reactivatable) inactivation of the enzyme which is also observed.

Temperature Sensitivity of Daily Respiratory Patterns Entrained through Phytochrome Action in Lemna paucicostata Strain 6746

AbstractThe respiration rate of heterotrophic Lemna paucicostata Hegelm. 6746 cultures at 26–27°C and given a brief pulse of red light, assumes patterns with properties dependent on the nitrogen source supplied. In a search for conditions specifically affecting features of those patterns expressing photperiodic timing, their amplitudes have been measured as relative peak height (RPH) — the increased height at the daily peak as a proportion of the average daily minimum — at both 21°C and 28°C. On nitrate, ammonium or aspartate medium, RPH is reduced roughly 70% at 21°C as compared to 28°C, but on nitrogen‐deficient medium the reduction is only 20–30%. Yet growth and the actual intensity of respiration are not differently temperature sensitive on NO3 and N‐deficient media. Nor does RPH itself correlate with growth rate.Previous evidence indicates that patterns on NO3 and NH4 media reflect photoperiodic timing and that those on aspartale and N‐deficient media do not; hence the temperature sensitivity of RPH does not correlate with whether or not a pattern reflects photo‐periodic timing. However, different daily patterns arc elicited by pulses of red or far‐red on NO3. NH4 and aspartale media but not on the N‐deficient. Hence the temperature sensitivity of RPH does correlate with the degree to which the patterns distinguish between red and far‐red, which is to say between high anti low levels of Pfr‐phytochrome. This suggests that high temperature sensitivity in RPH reflects a reaction limited by N assimilation and saturating only at relatively high levels of Pfr.

The Reaction of Wood Pulp with α,β-Amic Acids

In this study, liquid anhydrous ammonia has been used as a pre-swelling treatment for wood pulp and as a solvent for the reactants. With the single-step reactions, slightly more crosslinking resulted when the reactant was applied from liquid ammonia than from aqueous media. The multiple bake reactions, however, resulted in higher total degree of substitution (DS) values on the order of 1.0 and greater, but also in disproportionately more crosslinking. Degradation was minimal by the use of aqueous ammonium succinamate from single pad bake reactions, resulting in an average DP of 331. This is compared to the multiple pad-bake reactions with aqueous ammonium succinamate and with suc cinic anhydride in liquid ammonia media, which resulted in very low DP values ranging from 155 to 200. Discoloration of the derivatives accompanied the increases in DS and reductions in DP. Some cellulose derivatives appear to have been dissolved in concentrations to 4% (w/v) in 10 to 13% aqueous sodium hydroxide at 5.5°C and up to 0.7% in DMSO by refluxing. Infrared analyses have shown the material coagulated from the aqueous sodium hydroxide solutions to be pure cellulose, and the coagulant from DMSO solution to be cellulose hemisuccinate. Both infrared crystallinity index determinations and x-ray diffraction patterns indicated that the cellulose regenerated from aqueous sodium hydroxide solution was suhstantially crystalline.