Regulation of ammonium and potassium uptake by glutamine and asparagine in Pisum arvense plants

Pisum arvense plants were subjected to 5 days of nitrogen deprivation. Then, in the conditions that increased or decreased the root glutamine and asparagine pools, the uptake rates of 0.5 mM NH₄ ⁺ and 0.5 mM K⁺ were examined. The plants supplied with 1 mM glutamine or asparagine took up ammonium and potassium at rates lower than those for the control plants. The uptake rates of NH₄ ⁺ and K⁺ were not affected by 1 mM glutamate. When the plants were pre-treated with 100 μM methionine sulphoximine, an inhibitor of glutamine synthesis, the efflux of NH₄ ⁺ from roots to ambient solution was enhanced. On the other hand, exposure of plants to methionine sulphoximine led to an increase in potassium uptake rate. The addition of asparagine, glutamine or glutamate into the incubation medium caused a decline in the rate of NH₄ ⁺ uptake by plasma membrane vesicles isolated from roots of Pisum arvense, whereas on addition of methionine sulphoximine increased ammonium uptake. The results indicate that both NH₄ ⁺ and K⁺ uptake appear to be similarly affected by glutamine and asparagine status in root cells. The research was supported by grant of KBN No. 6PO4C 068 08     

Artemisinin production in Artemisia annua hairy root cultures with improved growth by altering the nitrogen source in the medium

Murashige & Skoog medium was modified for enhancing artemisinin production in Artemisia annua hairy root cultures by altering the ratio of NO ₃ ^– /NH ₄ ⁺ and the total amount of initial nitrogen. Increasing ammonium to 60 mM decreased both growth and artemisinin accumulation in hairy root cultures. With NO ₃ ^– /NH ₄ ⁺ at 5:1 (w/w), the optimum concentration of total initial nitrogen for artemisinin production was 20 mM. After 24 days of cultivation with 16.7 mM nitrate and 3.3 mM ammonium, the maximum artemisinin production of hairy roots was about 14 mg l^–1, a 57% increase over that in the standard MS medium.     

Potassium transport in Chlamydomonas reinhardtii: isolation and characterization of transport-deficient mutant strains

Putative potassium-transport-deficient mutant strains of Chlamydomonas reinhardtii Dang. were induced by ultra-violet mutagenesis and were identified by their dependence on abnormally high concentrations of potassium for growth. Potassium transport studies employing ⁸⁶Rb as a tracer were carried out with wild-type cells and with three independently isolated KDP (potassium-dependent phenotype) clones. Wildtype cells exhibit two transport activities. Transport activity A was expressed when cells were grown in medium supplemented with 10 mM KCl. The transporter with type-A activity does not discriminate between either Rb⁺ or K⁺ as a substrate and has a K_m for Rb⁺ equal to 1 mM and a V_max equal to 31 nmol Rb⁺ h^-1 10^-6 cells. Transport activity B was expressed when cells were starved of potassium for 24 h. The transporter with type-B activity prefers K⁺ to Rb⁺ as a substrate; it has a K_m for Rb⁺ equal to 2.5 mM and a V_max equal to 210 nmol Rb⁺ h^-1 10^-6 cells. All three mutant clones exhibit transport activity comparable to type-A when grown in 10 mM KCl. When starved of potassium for 24 h, two KDP clones demonstrate no transport activity and the third clone continues to exhibit only type-A activity.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DES diethylstilbesterol - KDP potassium-dependent phenotype     

Regulation of NH4 + transport pathways in Pisum arvense roots

The effects of metabolic and protein synthesis inhibitors on NH₄ ⁺ uptake by Pisum arvense plants at low (0.05 mM) and high (1 mM) external ammonium concentration were studied. In short-time experiments cycloheximide decreased the ammonium uptake rate at low level of NH₄ ⁺ and increased the absorption of NH₄ ⁺ from uptake medium containing high ammonium concentration. Arsenate and azide supplied into uptake solutions at low ammonium concentration strongly decreased or completely suppressed the NH₄ ⁺ uptake rate, respectively. When the experiments were carried out at high level of ammonium only azide decreased the uptake rate of NH₄ ⁺ and arsenate stimulated this process. Dinitrophenol very strongly repressed the uptake rate of NH₄ ⁺ at both ammonium concentrations. After removing dinitrophenol from both solutions, neither at low nor high external ammonium level the recovery of NH₄ ⁺ uptake rate was achieved within 150 min or 3 h, respectively. The recovery of NH₄ ⁺ uptake rate after removing azide was observed within 90 min and 3 h at low and high ammonium concentrations, respectively. The regulation of NH₄ ⁺ uptake by some inhibitors at low external ammonium level was investigated using plasma membrane vesicles isolated from roots by two-phase partitioning. Orthovanadate completely suppressed the uptake of NH₄ ⁺ by vesicles and quinacrine decreased the NH₄ ⁺ uptake which 55 suggests that ammonium uptake depends on activities of plasma membrane-bound enzymes. On the other hand, it was found that dinitrophenol completely reduced the NH₄ ⁺ uptake by vesicles. The various effects of inhibitors on ammonium uptake dependent on external ammonium concentration suggest the action of different ammonium transport systems in Pisum arvense roots. The ammonium transport into root cells at low NH₄ ⁺ level requires energy and synthesis of protein in the cytoplasm. The research was supported by grant of KBN No. 6PO4C 068 08     

Arrest of cell division but not protein synthesis in sodium-deficient cells of the marine diatom Phaeodactylum tricornutum Bohlin

Cell division in the marine diatom Phaeodactylum tricornutum was prevented when cultures were maintained in the absence of sodium, regardless of the nitrogen status of the cells or medium. Addition of 10 mM ammonium and 50 mM sodium to cultures preconditioned in nitrogen and sodium-deficient medium for 5 d led to a recovery in cell division and chlorophyll a, and net protein synthesis. Sodium added in the absence of ammonium led to a recovery in cell division, but not net protein synthesis. Ammonium added in the absence of sodium was partially assimilated (as NH₃) and resulted in a small amount of protein synthesis, but without cell division. This effect was enhanced if the cells had lower protein quotas prior to ammonium addition, with total consumption of the added 1 mM ammonium and appreciable net protein synthesis. Respiration was enhanced by 1 or 10 mM ammonium or 10 mM methylammonium addition to nitrogen-deficient cultures maintained in the presence or absence of sodium. In contrast to respiration, photosynthesis was inhibited by these additions in sodium-replete cultures, but was enhanced in sodium-deficient cultures.This research was supported by the Auckland University Research Committee and University Grants Committee of New Zealand.     

Some properties of a Klebsiella pneumoniae ammonium transport negative mutant (Amt−)

The main property of an Amt^- (ammonium transport negative) mutant of Klebsiella pneumoniae is its inability to accumulate NH ₄ ⁺ intracellularly. When growing on nitrogen sources other than NH ₄ ⁺ , the mutant constantly looses NH₃ by diffusion. This loss results in poor growth. The NH₃ excretion suggests the existence of a futile cycle (NH₃ loss/NH ₄ ⁺ reabsorption) in the wild type and possibly other bacterial strains, which do not constantly excrete NH₃.Dedicated to Prof. R. H. Burris     

Ammonium-related metabolic changes affect somatic embryogenesis in pumpkin (Cucurbita pepo L.)

Somatic embryogenesis in pumpkin can be induced on auxin-containing medium and also on hormone-free medium containing 1 mM ammonium (NH₄⁺) as the sole source of nitrogen. Growth of NH₄⁺-induced embryogenic tissue was slow and caused considerable acidification of the culture medium. Small spherical cells with dense cytoplasma formed proembryogenic cell clusters that could not develop into late stage embryos. Buffering of NH₄⁺ medium with 25 mM 2-(N-morpholino)-ethane-sulfonic acid enhanced tissue proliferation, but no further differentiation was observed. Later stage embryos developed only after re-supply of nitrogen in form of nitrate or l-glutamine. Effects of nitrogen status and pH of culture media on ammonium assimilation were analyzed by following the activity of glutamine synthetase (GS) in relation to phenylalanine ammonia-lyase (PAL). Increased activity of GS and PAL in NH₄⁺ induced tissue coincided with significantly higher activity of stress-related enzymes superoxide dismutase (SOD) and soluble peroxidase (POD), indicating oxidative stress response of embryogenic tissue to NH₄⁺ as the sole source of nitrogen. In addition, considerable increase was observed in callose accumulation and esterase activity, the early markers of somatic embryogenesis. Activity of stress-related enzymes decreased after the re-supply of nitrate (20 mM) or Gln (10 mM) in combination with NH₄⁺ (1 mM), which subsequently triggered globular embryo development. Together, these results suggest that stress responses, as affected by nitrogen supply, contribute to the regulation of embryogenic competence in pumpkin.     

Influence of Spectral Range and Carbon and Nitrogen Sources on Oxygen Evolution and Emerson Enhancement in Chlamydomonas reinhardtii

Chlamydomonas reinhardtii was grown in medium with different carbon (acetate, CO₂, or both), and nitrogen (ammonium chloride, peptone, urea) sources and under light of different spectral composition. The light-dark cycles were found more suitable for mixotrophic growth than continuous irradiation. Both blue (BR) and red (RR) radiations decreased photosynthetic capacity of mixotrophic cells compared to “white light” (WL). Effect of RR was associated with photon distribution favouring photosystem 1 (PS1) suggesting increased cyclic phosphorylation. Mixotrophic growth in 10 mM NH₄Cl increased photosynthetic oxygen evolution compared to standard concentration of 5 mM NH₄Cl used for growing C. reinhardtii. Autotrophic growth stimulated the photosynthetic capacity compared to mixotrophic one. However, higher photosynthetic capacity was achieved for mixotrophic cells by growing them at high NH₄ ⁺/K⁺ ratio and high phosphate concentration. This revised version was published online in July 2006 with corrections to the Cover Date.     

Simultaneous influx of ammonium and potassium into maize roots: kinetics and interactions

The interaction between ammonium and potassium during influx was examined in roots of dark-grown decapitated corn seedlings (Zea mays L., cv. Pioneer 3369A). Influx was measured during a 10-min exposure to either (¹⁵NH₄)₂SO₄ ranging from 10 to 200 M NH ₄ ⁺ with and without 200 M K(⁸⁶Rb)Cl or to K(⁸⁶Rb)Cl ranging from 10 to 200 M K⁺ with and without 200 M NH ₄ ⁺ as (¹⁵NH₄)₂SO₄. The simple Michaelis-Menten model described the data well only for potassium influx in the presence of ambient ammonium. For the other three instances, the data were improved by assuming that a second influx mechanism became operative as the low-concentration phase approached saturation. Two distinct mechanisms are thus indicated for both ammonium and potassium influx within the range of 10 to 200 M.The influx mechanism operating at low concentrations showed greater affinity for potassium than for ammonium, even though the capacity for ammonium transport was twice as large as that for potassium. It is suggested that this phase involved a common transport system for the two ions and that localized low acidity next to the internal surface, following H⁺ extrusion, favored ammonium deprotonation and dissociation from the transport system-ammonium complex. Parallel decreases in V _max and increases in K_m of the low-concentration saturable phase occurred for ammonium influx when ambient potassium was present and for potassium influx when ambient ammonium was present. The data support a mixed-type inhibition in each case. Simultaneous measurement of potassium and ammonium influx showed that they were highly negatively correlated at the lower concentrations, indicating that the extent to which influx of the inhibited ion was restricted was associated with influx of the inhibitor ion. Presence of ambient ammonium eliminated the second phase of potassium influx. In contrast, the presence of ambient potassium decreased the concentration at which the second phase of ammonium influx was initiated but did not restrict the rate.Paper no. 11131 of the Journal Series of the North Carolina Agricultural Research ServiceThe use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of the products named, nor criticism of similar ones not mentioned     

Seasonal variability of apoplastic NH4 + and pH in an intensively managed grassland

The stomatal compensation point of ammonia (_s) is a major factor controlling the exchange of atmospheric ammonia (NH₃) with vegetation. It is known to depend on the supply of nitrogen and to vary among plant species, but its seasonal variation has not yet been reported for grassland. In this study, we present the temporal variation of apoplastic NH₄ ⁺ concentration ([NH₄ ⁺]_apo) and pH (pH_apo) measured in leaves of Lolium perenne L. in a grassland, through two periods of cutting / fertilisation, followed by a livestock grazing period. The total free NH₄ ⁺ concentration measured in foliage ([NH₄ ⁺]_fol), and soil mineral NH₄ ⁺ and NO₃ ^– concentration are also presented. The value of [NH₄ ⁺]_apo varied from less than 0.01 mM to a maximum of 0.5 mM occurring just after fertilisation, whereas the apoplastic pH ranged from pH 6 to 6.5 for most of the time and increased up to pH 7.8, 9 days after the second fertilisation, when grazing started. [NH₄ ⁺]_fol varied between 20 and 50 g N-NH₄ ⁺ g^–1 f.w. The compensation point at 20°C, ranged from 0.02 g NH₃ m^–3 between the fertilisations to 10 g NH₃ m^–3 just after the second fertilisation. The reasons for these seasonal changes are discussed, with respect to plant metabolism and the concentration of ammonium and nitrate in the soil.     

Alanine dehydrogenase of the N2-fixing blue-green alga,Anabaena cylindrica

The l-alanine dehydrogenase (ADH) of Anabaena cylindrica has been purified 700-fold. It has a molecular weight of approximately 270000, has 6 sub-units, each of molecular weight approximately 43000, and shows activity both in the aminating and deaminating directions. The enzyme is NADH/NAD⁺ specific and oxaloacetate can partially substitute for pyruvate. The K _m ^app for NAD⁺ is 14 M and 60 M at low and high NAD⁺ concentrations, respectively. The K _m ^app for l-alanine is 0.4 mM, that for pyruvate is 0.11 mM, and that for oxaloacetate is 3.0 mM. The K _m ^app for NH ₄ ⁺ varies from 8–133 mM depending on the pH, being lowest at high pH levels (pH 8.7 or above). Alanine, serine and glycine inhibit ADH activity in the aminating direction. The enzyme is active both in heterocysts and vegetative cells and activity is higher in nitrogen-starved cultures than in N₂-fixing cultures. The data suggest that although alanine is formed by the aminating activity of ADH, entry of newly fixed ammonia into organic combination does not occur primarily via ADH in N₂-fixing cultures of A. cylindrica. Ammonia assimilation via ADH may be important in cultures with an excess of available nitrogen. The deaminating activity of the enzyme may be important under conditions of nitrogen-deficiency.Abbreviations ADH alanine dehydrogenase - DEAE diethylamino ethyl cellulose - EDTA ethylenediamine tetraacetic acid - GDH glutamic dehydrogenase - GS glutamine synthetase - GOT aspartate-glutamate aminotransferase - NAD⁺ nicotinamide adenine dinucleotide - NADH reduced nicotinamide adenine dinucleotide - NADP⁺ nicotinamide adenine dinucleotide phosphate - NADPH reduced nicotinamide adenine dinucleotide phosphate - SDS sodium dodecyl sulphate - Tris tris(hydroxymethyl) aminomethane     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+/K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM–500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na⁺/K⁺-ATPase activity was studied. The response of enzymatic activity in the presence of various K⁺ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na⁺/K⁺-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K⁺ ions below 20 mM in the medium assay. The IC₅₀ values for high/low isoforms 2.77 × 10^{? 6} M / 8.56 × 10^{? 5} M and 7.06 × 10^{? 7} M /1.87 × 10^{? 5} M were obtained in the presence of optimal (20 mM) and 2 mM K⁺, respectively. However, preincubation in the presence of elevated K⁺ concentration (50 – 500 mM) in the medium assay prior to Na⁺/K⁺-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC₅₀ values for both isoforms was the same as in the presence of the optimal K⁺ concentration. On the contrary, addition of 200 mM K⁺ into the medium assay after 10 minutes exposure of Na⁺/K⁺-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na⁺/K⁺-ATPase by reducing maximum enzymatic velocity (V_max) and K_m, implying an uncompetitive mode of interaction.     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Mutants of Azospirillum brasilense resistant to methylammonium

One hundred and twenty-nine mutants of Azospirillum brasilense strain Sp6, resistant to methylammonium, were isolated. Three of the mutants were found to be able to reduce acetylene in the presence of 4 mM ammonium or 120mM methylammonium, concentrations which strongly reduced the nitrogenase activity of the parental strain. Under N₂-fixing conditions, two mutants failed to switch off nitrogenase when NH₄Cl was added. Moreover, the three mutants showed a reduced capacity to incorporate [¹⁴C]methylammonium. The level of glutamine synthetase activity found in the mutants was not reduced as compared to that of the parental strain. All of the data indicate an impairement in the mechanism of ammonium uptake by the bacterial cell.Abbreviations MEA Methylammonium - MSP minimal medium (ammonium free) - PY complete medium - GS glutamine synthetase     

Peptidergic neurons of the crab,Cardisoma carnifex,in defined culture maintain characteristic morphologies under a variety of conditions

Summary Peptidergic neurons dissociated from the neurosecretory cell group, the X-organ, of adult crabs (Cardisoma carnifex) show immediate outgrowth on unconditioned plastic dishes in defined medium. Most of the neurons can be categorized as small cells, branchers or veilers. A fourth type, superlarge, found occasionally, has a soma diameter greater than 40 m and multipolar outgrowth. We report here the effects on morphology that follow alterations of the standard defined culturing conditions. The three common types of neurons are present when cells are grown in crab saline or saline with l-glutamine and glucose (saline medium). Changes of pH between 7.0 to 7.9 have no effect. Osmolarity changes cause transient varicosities in small cells. In some veilers, pits rapidly appear in the veil and then disappear within 35 min. In cultures at 26° C instead of 22° C, veilers extend processes from the initial veil in a pattern similar to branchers, and the processes of adjacent veilers sometimes form appositions. Culturing in higher [K⁺]_o medium ([K⁺]_o=15–110 mM; standard=11 mM) has no long-term effect, but growth is arrested by [K⁺]_o greater than 30 mM. Cultures were also grown in media in which [Ca²⁺]_o ranged from 0.1 M to 26 mM (standard=13 mM). Outgrowth occured from all neuronal types in all [Ca²⁺]_o tested. Thus, the expression of different outgrowth morphologies occurs under a wide variety of culturing conditions.     

Relationship of the Escherichia coli TrkA System of Potassium Ion Uptake with the F0F1-ATPase Under Growth Conditions Without Anaerobic or Aerobic Respiration

K⁺ uptake by the Escherichia coli TrkA system is unusual in that it requires both ATP and ; a relation withH⁺ circulation through the membrane is thereforesuggested. The relationship of this system with theF₀F₁-ATPase was studied in intact cells grownunder different conditions. A significant increase of theN,N-dicyclohexylcarbodiimide(DCCD)-inhibitedH⁺ efflux through the F₀F₁ by 5 mMK⁺, but not by Na⁺ added into thepotassium-free medium was revealed only in fermenting wild-type orparent cells, that were grown under anaerobic conditions withoutanaerobic or aerobic respiration and with the production ofH₂. Such an increase disappeared in the unc or the trkA mutants that have alteredF₀F₁ or defective TrkA, respectively.This finding indicates a closed relationship between TrkA andF₀F₁, with these transport systems beingassociated in a single mechanism that functions as an ATP-drivenH⁺–K⁺-exchanging pump. ADCCD-inhibited H⁺–K⁺-exchangethrough these systems with the fixed stoichiometry of H⁺and K⁺ fluxes(2H⁺/K⁺) and a higherK⁺ gradient between the cytoplasm and the externalmedium were also found in these bacteria. They were not observed incells cultured under anaerobic conditions in the presence of nitrate orunder aerobic conditions with respiration and without production ofH₂. The role of anaerobic or aerobic respiration as adeterminant of the relationship of the TrkA with theF₀F₁ is postulated. Moreover, an increase ofDCCD-inhibited H⁺ efflux by added K⁺, aswell as the characteristics of DCCD-sensitiveH⁺–K⁺-exchange found in a parentstrain, were lost in the arcA mutant with a defectiveArc system, suggesting a repression of enzymes in respiratorypathways. In addition, K⁺ influx in the latest mutantwas not markedly changed by valinomycin or with temperature. ThearcA gene product or the Arc system is proposed to beimplicated in the regulation of the relationship between TrkAand F₀F₁.     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Fixed NH4-N in relation to EUF-extractable K

Summary Representative arable soils from Hesse were investigated for their contents of fixed NH₄ ⁺ and EUF-extractable potassium in the rooting zone. Alluvial soils were found to be rich in fixed ammonium and low in EUF-extractable potassium, while soils of basaltic origin were low in fixed ammonium and rich in EUF-extractable potassium. A negative correlation (r=0.79^*) was found between fixed NH₄ ⁺ and EUF-extractable soil K⁺. The content of fixed NH₄ ⁺ in the soil profile showed considerable and significant changes during the growing season, which finding is supposed to be due to NH₄ ⁺ uptake by the crop.     

Na<Superscript>+</Superscript>-mediated piezoprotection in<Emphasis Type="Italic"> Rhodotorula rubra</Emphasis>

Sodium concentrations as low as 2 mM exerted a significant protective effect on the high-pressure inactivation (160–210 MPa) of Rhodotorula rubra at pH 6.5, but not on two other yeasts tested (Shizosaccharomyces pombe and Saccharomyces cerevisiae). A piezoprotective effect of similar magnitude was observed with Li⁺ (2 and 10 mM), and at elevated pH (8.0–9.0), but no effect was seen with K⁺, Ca²⁺, Mg²⁺, Mn²⁺, or NH₄ ⁺. Intracellular Na⁺ levels in cells exposed to low concentrations of Na⁺ or to pH 8.0–9.0 provided evidence for the involvement of a plasma membrane Na⁺/H⁺ antiporter and a correlation between intracellular Na⁺ levels and pressure resistance. The results support the hypothesis that moderate high pressure causes indirect cell death in R. rubra by inducing cytosolic acidification.Communicated by K. Horikoshi     

Cyclic 2,3-diphosphoglycerate metabolism in Methanobacterium thermoautotrophicum (strain ΔH): characterization of the synthetase reaction

The levels of cyclic 2,3-diphosphoglycerate (cDPG) in methanogenic bacteria are governed by the antagonistic activities of cDPG synthetase and cDPG hydrolase. In this paper we focus on the synthetase from Methanobacterium thermoautotrophicum. The cytoplasmic 150 kDa enzyme catalyzed cDPG synthesis from 2,3-diphosphoglycerate (apparent K_m=21 mM), Mg²⁺ (K_m=3.1 mM) and ATP (K_m=1–2 mM). In batch-fed cultures, the enzyme was constitutively present (6–6.5 nmol per min per mg protein) during the different growth phases. In continuous cultures, activity decreased in response to phosphate limitation. The synthetase reaction proceeded with maximal rate at pH 6 and at 65° C and was specifically dependent on high (>0.3M) K⁺ concentrations. The reaction conditions remarkably contrasted to those of cDPG degradation catalyzed by the previously described membrane-bound cDPG hydrolase.Abbreviations cDPG Cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-Diphosphoglycerate - 2-PG 2-Phosphoglycerate - 3-PG 3-Phosphoglycerate