A. Mayer

Distribution of polyphosphates in cell-compartments of Chlorella fusca as measured by 31P-NMR-spectroscopy

In suspensions of the green alga Chlorella fusca the influence of high pH and high ethylene-diamine-tetraacetic acid concentrations in the external medium, of French-press and perchloric acid extraction of the cells and of alkalization of the intracellular pH on the polyphosphate signal in 31P-nuclear magnetic resonance (31P NMR) spectra was investigated.The results show that part of the polyphosphates of asynchronous Chlorella cells are located outside the cytoplasmic membrane and complexed with divalent metal-ions. These polyphosphates are tightly bound to the cell wall and/or the cytoplasmic membrane and are not susceptible to hydrolyzation by strong acid at room temperature, in contrast to the intracytoplasmic polyphosphates.Upon alkalization of the internal pH of Chlorella cells, polyphosphates, previously not visible in the spectra become detectable by 31P-NMR-spectroscopy. 31P-NMR spectroscopic monitoring of polyphosphates during gradual alkalization of the extra-and intracellular space is proposed as a quick method for the estimation of the cellular polyphosphate content and distribution.

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.

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 Pi 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 Pi-pool in more alkaline environment was detected. By the use of 2-deoxyglucose-6-phosphate as an indicator of cytoplasmic pH, this Pi-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 Pi and for the difference in cytoplasmic pH in photosynthesizing cells and those capable only of cyclic photophosphorylation are discussed.

The dependence of the cytoplasmic pH in aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris on the pH of the medium as determined by 31P in vivo NMR spectroscopy

The pH in the cytoplasm of aerobic and anaerobic cells of the green algae Chlorella fusca and Chlorella vulgaris was determined in dependence on the pH of the external medium, which was varied between pH 3 and pH 10. In aerobic cells of both species the cytoplasmic pH is maintained at a value above 7.2 even at an external pH of 3 and below 7.8 at an external pH of 10. In anaerobic cells the cytoplasmic pH shows linear dependence on external pH in the range of pH 6 to 9 (cytoplasmic pH 6.9 to 7.2), while below an external pH of 6 cytoplasmic pH is maintained at about 6.5.

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.

Heat shock- and ethanol-induced ionic changes in C6 rat glioma cells determined by NMR and fluorescence spectroscopy

The effects of two different stressors, heat shock (HS; 44 degrees C, 20 min) and ethanol (1.2 M, 60 min), on ion content and membrane potential were investigated in C6 rat glioma cells. Both treatments were previously shown to induce the HS response [26]. Intracellular pH (pH(i)), sodium ion concentration ([NA+]i), potassium ion concentration ([K+]i) and membrane potential were determined by means of continuous 31P and 23Na nuclear magnetic resonance (NMR), continuous fluorescence spectroscopy and 86Rb uptake. Lactate extrusion was determined in addition with respect to pH(i) regulation. The aim of this study was a detailed picture of HS and ethanol-induced ion changes in a single cell type, because stress-induced changes in the intracellular ionic balance may be important factors for determining proliferation, stress response and apoptosis. HS lowered the pH(i) from 7.38 +/- 0.04 to about 7.05 +/- 0.04. [Na+]i decreased during HS to 50% of the control and recovered to normal level 95 min after HS treatment. During HS, [K+]i remained constant but increased after HS. The membrane potential hyperpolarized from -83 mV to -125 mV and returned to initial values during HS treatment. Lactate extrusion increased 3-fold after HS. Ethanol (1.2 M) lowered the pH(i) from pH 7.38 +/- 0.04 to pH 7.0 +/- 0.04, but in contrast to heat strongly increased [Na]i. It hyperpolarized the membrane potential from -83 to -125 mV. Ethanol also increased lactate extrusion similar to HS. Also in contrast to the effect of HS, the potassium concentration decreased during ethanol treatment. The Na(+)-H+ exchanger monensin was used to overcome the apparent inhibition of the cellular Na(+)-H+ exchanger by HS. At normal pH(e) (7.4) monensin increased [Na+]i and pH(i) considerably. A subsequent HS reduced [Na+]i only minimally. Acidification of the cells by low pH(e) (6.2) prior to HS did not abolish the HS-induced drop of pH(i), indicating that the Na(+)-H+ exchanger was also inhibited at low pH(i). At low pH(e), monensin transports H+ into the cell. A subsequent HS decreased pH(i) only little, showing the importance of inhibition of the Na(+)-H+ exchanger for the HS-induced pH(i) decrease. 100 microM amiloride reduced pH(i) and [Na+]i in a similar way as HS, but did not change pH(i) and [Na+]i much during a HS. These results indicate that some of the HS-induced ionic changes are mediated by inhibition of the Na(+)-H+ exchanger, activation of Na(+)-K(+)-ATPase and changes of membrane conductance for ions.

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 γ-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.

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.

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 Pi 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 Pi 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 Pi 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 Pi signal as an intracellular pH-marker in plant cells.

Sodium binding to and protonation of ATP: a multinuclear magnetic double resonance study at 8.46 tesla

We show that the interaction of ATP with Na+ and H+, whether binding or dissociation, gives rise to exchange broadened 31P-NMR spectra at 8.4 T, pH 6.7 and 310 K. We interpret the effect as being due to a two-step conversion between two NMR-differentiated ATP pools. A quantitative analysis yields all involved equilibrium constants and some of the dynamic parameters. Our results help to understand previous studies of magnesium binding to ATP and the appearance of high-field in vivo 31P-NMR spectra.