P-31 in-vivo NMR investigation on the function of polyphosphates as phosphate-and energysource during the regreening of the green alga Chlorella fusca

The green alga Chlorella fusca accumulates polyphosphates under conditions of nitrogen starvation while deassembling the photosynthetic apparatus. The polyphosphate content of cells regreening after resupply with nitrate under different culture conditions was investigated by P-31 in-vivo NMR spectroscopy. Neither phosphate deficiency nor anaerobiosis during the first hours of regreening inhibited the recovery of the cells. Polyphosphates were degraded during regeening. Differences in the amount of polyphosphates of phosphate supplied and deficient cells occurred only after more then 8 h. After 16 h phosphate deficient cells had still 75% of the polyphosphate content of phosphate suppled cells. In cells kept under anaerobic conditions polyphosphate degradation was much higher than in oxygen supplied cells. After 8 h they contained less than 50% of the polyphosphate content of oxygen supplied cells. These data suggest that polyphosphates serve as obligatory phosphate source during regreening and may be used as an energy source.Non standard abbreviations EDTA Ethylene diamine tetraacetic acid - FID Free induction decay - MOPSO 3-(N-morpholine)-2-hydroxy-propanesulfonic acid - NMR Nuclear magnetic resonance - PP Polyphosphates - PP₄ central phosphate groups of polyphosphates     

In vivo P-31 NMR measurements of phosphate metabolism in Platymonas subcordiformis as related to external pH

The phosphate metabolism of Platymonas subcordiformis was investigated by 31P-NMR spectroscopy with special attention on the effect of external pH. Glycolyzing cells and cells energized by respiration or photosynthesis gave spectra dependent upon their metabolic state. The transition from deenergized to energized states is accompanied by a shift of cytoplasmic pH from 7.1–7.4, an increase of ATP level and-in well energized cells-the appearance of a new signal tentatively assigned to phosphoarginine.The spectra remain stable over a wide range of external pH. Cytoplasmic pH is well regulated in respiring cells for external pH in the range 5.3–12.3. The typical 0.4 units difference of internal pH in energized as compared to deenergized cells is not affected by external pH in the range 6–12. The intensity of a signal attributed to PEP is markedly increased at high external pH. pH regulation is less efficient below external pH of 6 in deenergized cells. Below pH 3.8 oxidative phosphorylation ceases. Upon raising cytoplasmic pH to 7.4 in deenergized cells polyphosphate chains start to disintegrate.Abbreviations PEP Phosphoenolpyruyate - P _i inorganic phosphate - PP _i inorganic pyrophosphate - poly P polyphosphates - PP-1, PP-2, PP-3 terminal, second, and third phosphate residue of polyphosphates - PP-4 core phosphate residues of polyphosphates - pH _i , pH _o internal (cytoplasmic) and external pH - NTP/NDP nucleotide triphosphate/-diphosphate - S/N signal to noise ratio     

The cytoplasmic pH in photosynthesizing cells of the green alga Chlorella fusca,measured by P-31 NMR spectroscopy

P-31 NMR investigations were performed with the green alga Chlorella fusca under anaerobic conditions in the dark and in the light.In spectra of cells in the dark the signal of intracellular, nonvacuolar P_i indicates a pH in its chemical environment of 7.0–7.2. Upon illumination this signal looses intensity and shifts to lower field, corresponding to a pH of 7.7. Further downfield no other signal that could be attributed to a P_i-pool in more alkaline environment was detected. By the use of 2-deoxyglucose-6-phosphate as an indicator of cytoplasmic pH, this P_i-signal was assigned to the cytoplasm. The pH increase in the cytoplasm upon transfer of cells from the dark to the light is the same as that previously observed upon transfer of cells from anaerobic to aerobic conditions.In cells performing only cyclic photophosphorylation the cytoplasmic pH is lower than in photosynthesizing cells but still 0.2 pH units higher than in the cells in the dark. The reasons for the missing of a signal of stromal P_i and for the difference in cytoplasmic pH in photosynthesizing cells and those capable only of cyclic photophosphorylation are discussed.Non-standard abbreviations 2dG 2-Deoxyglucose - dG-6-P 2-deoxyglucose-6-phosphate - DCMU 3,4-dichlorophenyl-dimethylurea - MOPSO 3-(N-morpholino)-2-hydroxypropane sulfonic acid - P-31 NMR P-31 nuclear magnetic resonance     

N-15 in vivo NMR spectroscopic investigation of nitrogen deprived cell suspensions of the green alga Chlorella fusca

The possibility to apply N-15 in vivo NMR spectroscopy to study algal N-metabolism has been investigated. N-15 labelled cells of the green alga Chlorella fusca, subjected to nitrogen starvation and N-14 labelled cells supplied with K¹⁵NO₃ after prolonged nitrogen starvation were monitored by N-15 in vivo NMR spectroscopy at different times after the change in their nitrogen supply. During 20–40 min, necessary for the acquisition of 1 spectrum, the cells were under dark anaerobic conditions, but the relative amounts of the metabolites detected did not change. Signals from 2 acid amides, from the side chain nitrogens of arginine and lysine, from prolin as well as 4 signals from α amino groups of amino acids were detected. Besides two signals not yet reported in the literature were found. They may be due to amino compounds, but not to amino acids. The amount of free amino acids in the cells increases not only upon resupply of nitrogen starved cells with nitrate but also during the first hours after nitrate depletion. The spectra obtained from N-15 labelled autospores show that N-15 in vivo NMR spectroscopy can be applied to the investigation of N metabolism of the cells.     

Ammonia rhythm in Microcystis firma studied by in vivo 15N and 31P NMR spectroscopy

Cultures of the cyanobacterium Microcystis firma show rhythmic uptake and release of ammonia under conditions of carbon limitation. The massive removal of ammonia from the medium during the first light phase has little impact on the intracellular pH: a pH shift of less than 0.2 U towards the alkaline can be measured by in vivo ³¹P NMR. Furthermore, the energy status of the cells remains regulated. In vivo ¹⁵N NMR of M. firma, cultivated either with labelled nitrate or ammonia as the sole nitrogen source, reveals only gradual differences in the pool of free amino acids. Additionally both cultivation types show -aminobutyric acid, acid amides and yet unassigned secondary metabolites as nitrogen storing compounds. Investigating the incorporation of nitrogen under carbon limitation, however, only the amide nitrogen of glutamine is found permanently labelled in situ. While transamination reactions are blocked, nitrate reduction to ammonia can still proceed. Cation exchange processes in the cell wall are considered regarding the ammonia disappearance in the first phase, and the control of ammonia uptake is discussed with respect to the avoidance of intracellular toxification.Abbreviations GABA -aminobutyric acid - GOGAT glutamate synthase - GS glutamine synthetase - MDP methylene diphosphonate - MOPSO 3-(N-morpholino)-2-hydroxy-propanesulfonic acid - NDPS nucieoside diphosphosugars - NOE nuclear Overhauser effect - NMR nuclear magnetic resonance For convenience, the term ammonia is used throughout to denote ammonia or ammonium ion when there is no good evidence as to which chemical species is involved     

Ammonium rhythm in cultures of the cyanobacterium Microcystis firma

Over a period of several days, rhythmic changes in extracellular NH⁺₄ concentration take place in cultures of the cyanobacterium Microcystis firma (Bré et Lenorm.) Schmidle, strain Gromov/St. Petersb. 398, under conditions of restricted CO₂ supply and light/dark alternation. The changes are enhanced by nitrate supply. Among the various processes generating intracellular NH⁺₄ (NH⁴₄ uptake, NO⁻₃ reduction, protein and amino acid degradation, photorespiration), NO⁻₃ reduction appears as the one most important. This can be concluded from experiments with and without nitrate and/or ammonium in the medium. In the presence of saturating CO₂, continuous light, or continuous darkness, rhythmic NH⁺⁴₄ oscillations are not induced. Studies of the incorporation of NH⁺₄ nitrogen by in vivo ¹⁵N-NMR show that if CO₂ is supplied, ¹⁵N is accumulated in several components with the following time course: in the first hour in Gln (δ), in the second hour in the α-amino groups of most nonbranched amino acids, in the third hour in γ-aminobutyric acid (GABA), Orn (δ) and Lys (ε), and in the sixth hour in Ala. Carbon limitation, however, results in accumulation of label in the amide nitrogen of glutamine only.     

P-31 NMR saturation transfer experiments in Chlamydomonas reinhardtii : evidence for the NMR visibility of chloroplastidic Pi

ATP synthesis and consumption in respiring cells of the green alga Chlamydomonas reinhardtii were measured with ³¹P in vivo NMR saturation transfer experiments to determine the intracellular compartmentation of inorganic phosphate. Most of the observed flux towards ATP synthesis was catalyzed by the coupled enzymes glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase (GAPDH/PGK). The attribution of the measured flux to these enzymes is supported by the observation, that (i) the magnetization transfer was strongly reduced by iodoacetate, an irreversible inhibitor of GAPDH and that (ii) the unidirectional flux was much greater than the net flux through the mitochondrial F₀F₁-ATPase as determined by oxygen consumption measurements. In Chlamydomonas, glycolysis is divided into a chloroplastidic and a cytosolic part with the enzymes GAPDH/PGK being located in the chloroplast stroma (Klein 1986). The ³¹P-NMR signal of inorganic phosphate must, therefore, originate from the chloroplast. The life time of the magnetic label transferred to P_i by these enzymes is too short for it to be transported to the cytosol via the phosphate translocator of the chloroplast envelope. When the intracellular compartmentation of P_i was taken into consideration the calculated unidirectional ATP synthesis rate was equal to the consumption rate, indicating operation of GAPDH/PGK near equilibrium. The assignment of most of the intracellular P_i to the chloroplast is in contradiction to earlier reports, which attributed the Pi signal to the cytosol. This is of special interest for the use of the chemical shift of the P_i signal as an intracellular pH-marker in plant cells.Abbreviations 3-PGA 3-phosphoglycerate - CW continuous wave - dG6P 2-deoxyglucose-6-phosphate - GAPDH glyceraldehyde-3-phosphate dehydrogenase - MO equilibrium z-magnetization - M₀ instantaneous z-magnetization after selective saturation for time t - MDP methylene-diphosphonic acid - PDE phosphodiester - PGK phosphoglycerate kinase - P_i inorganic orthophosphate - polyP polyphosphate - T₁ longitudinal relaxation time - ₁ longitudinal relaxation time with chemical exchange - TCA cycle tricarboxylic acid cycle Correspondence to: A. Mayer     

Changes in organic solutes,volume, energy state,and metabolism associated with osmotic stress in a glial cell line: A multinuclear NMR study

Diffusion-weighted in vivo¹H-NMR spectroscopy of F98 glioma cells embedded in basement membrane gel threads showed that the initial cell swelling to about 180% of the original volume induced under hypotonic stress was followed by a regulatory volume decrease to nearly 100% of the control volume in Dulbecco's modified Eagle's medium (DMEM) but only to 130% in Krebs-Henseleit buffer (KHB, containing only glucose as a substrate) after 7 h. The initial cell shrinkage to approx. 70% induced by the hypertonic stress was compensated by a regulatory volume increase which after 7 h reached almost 100% of the control value in KHB and 75% in DMEM.¹H-,¹³C-and³¹P-NMR spectroscopy of perchloric acid extracts showed that these volume regulatory processes were accompanied by pronounced changes in the content of organic osmolytes. Adaptation of intra- to extracellular osmolarity was preferentially mediated by a decrease in the cytosolic taurine level under hypotonic stress and by an intracellular accumulation of amino acids under hypertonic stress. If these solutes were not available in sufficient quantities (as in KHB), the osmolarity of the cytosol was increasingly modified by biosynthesis of products and intermediates of essential metabolic pathways, such as alanine, glutamate and glycerophosphocholine in addition to ethanolamine. The cellular nucleoside triphosphate level measured by in vivo³¹P-NMR spectroscopy indicated that the energy state of the cells was more easily sustained under hypotonic than hypertonic conditions.To whom to address reprint requests.     

Structural and membrane modifying porperties of suzukacillin, a peptide antibiotic related to alamethicin. Part B. Pore formation in black lipid films.

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete condustance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger alpha-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of "inactivation".     

Structural and membrane modifying properties of suzukacillin,a peptide antibiotic related to alamethicin: Part B. Pore formation in black lipid films

Suzukacillin, a polypeptide consisting of presumably 23 amino acids and 1 phenylalaninol, is produced by a Trichoderma viride strain No. 1037 and it can be isolated from the culture medium. It shows membrane-modifying properties similar to those of alamethicin. Discrete conductance fluctuations indicate the formation of oligomer pores of varying diameter. On the basis of voltage jump relaxation experiments evidence is given that the dimer is the nucleation state from which pore formation tion starts and the oligomer disappears. According to the voltage-current characteristics, voltage-dependent and voltage-independent conductances are observed. A slow process is involved, which can be interpreted as a change in the equilibrium distribution between different conformations of the suzukacillin monomer at the membrane interphase. This change results from its interaction with the lipid matrix. Differences in experimental observations between suzukacillin and alamethicin are attributed to the relatively larger α-helix and higher number of aliphatic side chains of the suzukacillin monomer and to a more intense interaction with the lipid membrane. This leads to a higher probability of forming dimers from monomers and to the occurrence of “inactivation”.