Phosphorus-31 NMR Studies of Cell Wall-Associated Calcium-Phosphates in Ulva lactuca

Phosphate concentrations in the range 0.1 to 2.0 millimolar induced the formation of extracellular amorphous calcium-phosphates in the cell wall of the marine macro algae Ulva lactuca when they were cultivated in light in seawater at 20°C. A broad resonance representing these compounds as well as resonances for extracellular orthophosphate and polyphosphates could be followed by ³¹P-nuclear magnetic resonance spectroscopy. The presence of the calcium-phosphate made the cells brittle and it inhibited the growth of the macro algae and caused mortality within 1 week. The formation of the calcium-phosphates was influenced by the external phosphate concentration, the extracellular pH and the nature and concentration of the external nitrogen source. Furthermore, no formation of these compounds was observed when Ulva lactuca was cultivated in the dark, at low temperatures (5°C) or in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The complex could be removed through washes with ethylenediaminetetraacetate; this treatment did not alter the intracellular pH or the orthophosphate and polyphosphate pools and it restored growth.     

P NMR Observations on the Effect of the External pH on the Intracellular pH Values in Plant Cell Suspension Cultures

³¹P nuclear magnetic resonance (NMR) spectroscopy was used to monitor the response of oil palm (Elaeis guineensis) and carrot (Daucus carota) cell suspensions to changes in the external pH. An airlift system was used to oxygenate the cells during the NMR measurements and a protocol was developed to enable a constant external pH to be maintained in the suspension when required. Phosphonoacetic acid was used as an external pH marker and the intracellular pH values were measured from the chemical shifts of the cytoplasmic and vacuolar orthophosphate resonances. In contrast to earlier studies the cytoplasmic pH was independent of the external pH over the range 5.5 to 8.0 and it was only below pH 5.5 that the cytoplasmic pH varied, falling at a rate of 0.12 pH unit per external unit. Loss of pH control was observed in response to sudden increases in external pH with the response of the cells depending on the conditions imposed. A notable feature of the recovery from these treatments was the transient acidification of the cytoplasm that occurred in a fraction of the cells and overshoot phenomena of this kind provided direct evidence for the time dependence of the regulatory mechanisms.     

Inorganic Carbon Accumulation and Photosynthesis in a Blue-green Alga as a Function of External pH

The blue-green alga Coccochloris peniocystis photosynthesizes optimally over the pH range of 7.0 to 10.0, but the O₂-evolution rate is inhibited below pH 7.0 and ceases below pH 5.25. Measurement of the inorganic carbon pool in this alga in the light, using the silicone-fluid filtration technique demonstrated that the rate of accumulation of dissolved inorganic carbon remained relatively constant over a wide pH range. At external dissolved inorganic carbon concentrations of 0.56 to 0.89 millimolar the internal concentration after 30 seconds illumination was greater than 3.5 millimolar over the entire pH range. Intracellular pH measured in the light using [¹⁴C]5,5-dimethyloxazolidine-2,4-dione and [¹⁴C]methylamine dropped from pH 7.6 at an external pH of 7.0 to pH 6.6 at an external pH of 5.25. Above an external pH of 7.0 the intracellular pH rose gradually to pH 7.9 at an external pH 10.0. Ribulose-1,5-bisphosphate carboxylase activity of cell-free algal extracts exhibited optimal activity at pH 7.5 to 7.8 but was inactive below pH 6.5. It is suggested that the inability of Coccochloris to maintain its intracellular pH when in an acidic environment restricts its photosynthetic capacity by a direct pH effect on the principal CO₂ fixing enzyme.     

The active transport of phosphate into the yeast cell

Phosphate can distribute in the cell wall space, but is not bound to an appreciable extent at the cell surface in non-metabolizing yeast. During metabolism of sugars, phosphate is actively transported into the yeast cell by a mechanism specifically involving glycolysis reactions. The movement of phosphate is in the inward direction only (no appreciable efflux), and it can proceed against a concentration gradient of 100 to 1. It is dependent on external phosphate concentrations in an asymptotic relationship, but is independent of the cellular orthophosphate concentration. The pH optimum for the phosphate uptake of 6.5 is shifted to the acid side by potassium. At certain values of pH a stimulation of 700 per cent by potassium can be observed. The nature of the effects of K⁺ and H⁺ are discussed.     

On the measurement of pH in Escherichia coli by 31P nuclear magnetic resonance

The ³¹P high resolution NMR spectra of concentrated suspensions of Escherichia coli cells have been measured at 145.8 MHz. The position of the orthophosphate resonance is used as a measure of internal and external pH. In accord with Paddan, Zilberstein and Rottenberg ((1976) Eur. J. Biochem. 63, 533–541) it is shown that when properly energized the internal pH is 7.5 ± 0.1. By synchronizing the NMR data acquisition with 3-s bursts of O₂ it is possible to measure the internal pH with a time resolution of about 1 s. It is shown that at 20°C the pH remains constant for times longer than 15 s after the oxygen is discontinued and it decays in several minutes.     

Effect of External pH on the Internal pH of Chlorella saccharophila

The overall internal pH of the acid-tolerant green alga, Chlorella saccharophila, was determined in the light and in the dark by the distribution of 5,5-dimethyl-2-[¹⁴C]oxazolidine-2,4-dione ([¹⁴C]DMO) or [¹⁴C]benzoic acid ([¹⁴C]BA) between the cells and the surrounding medium. [¹⁴C]DMO was used at external pH of 5.0 to 7.5 while [¹⁴C]BA was used in the range pH 3.0 to pH 5.5. Neither compound was metabolized by the algal cells and intracellular binding was minimal. The internal pH of the algae obtained with the two compounds at external pH values of 5.0 and 5.5 were in good agreement. The internal pH of C. saccharophila remained relatively constant at pH 7.3 over the external pH range of pH 5.0 to 7.5. Below pH 5.0, however, there was a gradual decrease in the internal pH to 6.4 at an external pH of 3.0. The maintenance of a constant internal pH requires energy and the downward drift of internal pH with a drop in external pH may be a mechanism to conserve energy and allow growth at acid pH.     

Mode of orthophosphate uptake and ATP labeling by mammalian cells

Incubation of HeLa cells with [³²P]orthophosphate results in more rapid labeling of the γ-phosphorus of ATP than of the intracellular pool of orthophosphate. The specific radioactivity of ATP equals that of extracellular orthophosphate after 2h of incubation. A similar pattern of labeling is seen with human erythrocytes when incubated at physiological concentrations of orthophosphate (2 mM) and pH 7.4–7.8. At lower pH, 6.8–7.2, the rate of orthophosphate uptake increases and exceeds the rate of labeling of ATP. These data are explained by the existence of a primary system for ATP uptake which involves the mediation of membrane-bound glyceraldehyde-3-phosphate dehydrogenase. Phosphate first enters the cell as 1,3-diphosphoglyceric acid, is then transferred to ATP, and then enters the intracellular orthophosphate pool. At lower pH monovalent orthophosphate also enters the erythrocyte by a process not involving glyceraldehyde-3-phosphate dehydrogenase.     

Proton Gradients in Intact Cyanobacteria

The internal pH values of two unicellular cyanobacterial strains were determined with electron spin resonance probes, over an external pH range of 6 to 9, in the light and in the dark. The slow growing, thylakoid-lacking Gloeobacter violaceus was found to have a low capacity for maintaining a constant internal pH. The distribution pattern of weak acid and amine nitroxide spin probes across the cell membranes of this organism, in the light and in the dark, was consistent with the assumption that it contains a single intracellular compartment. At an external pH of 7.0, intracellular pH was 6.8 in the dark and 7.2 in the light. The cells of Agmenellum quadruplicatum, a marine species, were found to contain two separate compartments; in the dark, the pH of the cytoplasmic and the intrathylakoid spaces were calculated to be 7.2 and 5.5, respectively. Upon illumination, the former increased and the latter decreased by about 0.5 pH units.     

Proton Electrochemical Gradients in Washed Cells of Nitrosomonas europaea and Nitrobacter agilis

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [³H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, ¹⁴C-benzoic and ¹⁴C-acetyl salicylic, and the weak base [¹⁴C]methylamine. Nitrosomonas europaea accumulated ¹⁴C-benzoic acid and ¹⁴C-acetyl salicylic acid when the external pH was below 7.0 and [¹⁴C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [¹⁴C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Δψ only contributed to Δp. In Nitrosomonas europaea the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.     

Use of Na-Nuclear Magnetic Resonance To Follow Sodium Uptake and Efflux in NaCl-Adapted and Nonadapted Millet (Panicum miliaceum) Suspensions

Cellular Na⁺ transport was followed in vivo by ²³Na nuclear magnetic resonance (NMR) using anionic dysprosium-based shift reagents to resolve internal and external ²³Na⁺ resonances. Proso millet (Panicum miliaceum) cell suspensions adapted for rapid growth on 130 mm NaCl had biphasic ²³Na efflux kinetics when shifted to low Na⁺ medium, while nonadapted cells had little measurable Na⁺ efflux after preloading with ²³NaCl. Uptake of ²³Na was also observed using ²³Na NMR. The resonance frequency of the external Na⁺-dysprosium (III) triphosphate, relative to that of the ²³Na in the cells, was sensitive to pH, permitting the pH of the external medium to be followed during the course of in vivo experiments.     

Regulatory properties of rabbit red blood cell hexokinase at conditions close to physiological

The true level of hexokinase in rabbit erythrocytes was determined by three different methods, including the spectrophotometric glucose-6-phosphate dehydrogenase coupled assay and a new radioisotopic assay. The value found at 37°C (pH 7.2) was 10.23±1.90 μmol/h per ml red blood cells, which is lower than previously reported values. More than 40 cellular components of the rabbit erythrocytes were tested for their effects on the enzyme. Their intracellular concentrations were also determined. Several of these compounds were found to be competitive inhibitors of the enzyme with respect to Mg·ATP^2?. Furthermore, reduced glutathione at a concentration of 1 mM was able to maintain hexokinase in the reduced state with full catalytic activity. The ability of orthophosphate to remove the inhibition of some phosphorylated compounds was examined under conditions similar to cellular (pH 7.2 and 50 μM of orthophosphate) and found to be of no practical interest. In contrast, the binding of ATP^4? and 2,3-diphosphoglycerate to the rabbit hemoglobin significantly modifies their intracellular concentrations and the formation of the respective Mg complexes. The pH-dependence of the reaction velocity and of the kinetic properties of the enzyme in different buffer systems were also considered. This information was computerized, and the rate of glucose phosphorylation in the presence of the mentioned compounds was determined. The value obtained, 1.94±0.02 μmol/h per ml red blood cells, is practically identical to the measured rate of glucose utilization by intact rabbit erythrocytes (1.92±0.3 μmol/h per ml red blood cells). These results provide further evidence for the central role of hexokinase in the regulation of red blood cell glycolysis.     

Intracellular Accumulation of Glycine in Polyphosphate-Accumulating Organisms in Activated Sludge,a Novel Storage Mechanism under Dynamic Anaerobic-Aerobic Conditions

Dynamic anaerobic-aerobic feast-famine conditions are applied to wastewater treatment plants to select polyphosphate-accumulating organisms to carry out enhanced biological phosphorus removal. Acetate is a well-known substrate to stimulate this process, and here we show that different amino acids also are suitable substrates, with glycine as the most promising. ¹³C-labeled glycine and nuclear magnetic resonance (NMR) were applied to investigate uptake and potential storage products when activated sludge was fed with glycine under anaerobic conditions. Glycine was consumed by the biomass, and the majority was stored intracellularly as free glycine and fermentation products. Subsequently, in the aerobic phase without addition of external substrate, the stored glycine was consumed. The uptake of glycine and oxidation of intracellular metabolites took place along with a release and uptake of orthophosphate, respectively. Fluorescence in situ hybridization combined with microautoradiography using ³H-labeled glycine revealed uncultured actinobacterial Tetrasphaera as a dominant glycine consumer. Experiments with Tetrasphaera elongata as representative of uncultured Tetrasphaera showed that under anaerobic conditions it was able to take up labeled glycine and accumulate this and other labeled metabolites to an intracellular concentration of approximately 4 mM. All components were consumed under subsequent aerobic conditions. Intracellular accumulation of amino acids seems to be a novel storage strategy for polyphosphate-accumulating bacteria under dynamic anaerobic-aerobic feast-famine conditions.     

Effect of Elicitation and Changes in Extracellular pH on the Cytoplasmic and Vacuolar pH of Suspension-Cultured Soybean Cells

We have employed both ³¹P nuclear magnetic resonance spectroscopy and two intracellular fluorescent pH indicator dyes to monitor the pH of the vacuole and cytoplasm of suspension-cultured soybean cells (Glycine max Merr cv Kent). For the ³¹P nuclear magnetic resonance studies, a flow cell was constructed that allowed perfusion of the cells in oxygenated growth medium throughout the experiment. When the perfusion medium was transiently adjusted to a pH higher than that of the ambient growth medium, a rapid elevation of vacuolar pH was observed followed by a slow (approximately 30 minute) return to near resting pH. In contrast, the concurrent pH changes in the cytoplasm were usually fourfold smaller. These data indicate that extracellular pH changes are rapidly communicated to the vacuole in soybean cells without significantly perturbing cytoplasmic pH. When elicitors were dissolved in a medium of altered pH and introduced into the cell suspension, the pH of the vacuole, as above, quickly reflected the pH of the added elicitor solution. In contrast, when the pH of either a polygalacturonic acid or Verticillium dahliae elicitor preparation was adjusted to the same pH as the ambient medium, no significant change in either vacuolar or cytoplasmic pH was observed during the 35 minute experiment. These results were confirmed in experiments with pH-sensitive fluorescent dyes. We conclude that suspension-cultured soybean cells do not respond to elicitation by significantly changing the pH of their vacuolar or cytoplasmic compartments.     

Kinetics of phosphorus absorption byCorynebacterium bovis

The initial rate of phosphorus uptake by phosphorus-limited cells ofCorynebacterium bovis grown in batch culture and in a chemostat was measured with [³²P] orthophosphate. It was dependent on the external phosphorus concentration and was inversely related to the amount of intracellular phosphorus. The relationship between the initial rate of uptake, intracellular phosphorus, and phosphorus concentration in the medium can be expressed in terms of Haldane's modification of the Michaelis-Menten equation.     

Effects of ethanol on Saccharomyces cerevisiae as monitored by in vivo 31P and 13C nuclear magnetic resonance

Cell suspensions of a respiratory deficient mutant of Saccharomyces cerevisiae were monitored by in vivo ³¹P and ¹³C Nuclear Magnetic Resonance in order to evaluate the effect of ethanol in intracellular pH and metabolism. In the absence of an added energy source, ethanol caused acidification of the cytoplasm, as indicated by the shift to higher field of the resonance assigned to the cytoplasmic orthophosphate. Under the experimental conditions used this acidification was not a consequence of an increase in the passive influx of H⁺. With cells energized with glucose, a lower value for the cytoplasmic pH was also observed, when ethanol was added. Furthermore, lower levels of phosphomonoesters were detected in the presence of ethanol, indicating that an early event in glycolysis is an important target of the ethanol action. Acetic acid was identified as responsible for the acidification of the cytoplasm, in experiments where [¹³C]ethanol was added and formation of labeled acetic acid was detected. The intracellular and the extracellular concentrations of acetic acid were respectively, 30 mM and 2 mM when 0.5% (120 mM) [¹³C]ethanol was added.Abbreviations P_i inorganic phosphate - P_ic inorganic phosphate in the cytoplasm - P_iv inorganic phosphate in the vacuole - tP terminal phosphate in polyphosphate     

In Vivo pH Regulation by a Na/H Antiporter in the Halotolerant Alga Dunaliella salina

Na⁺/H⁺ exchange activity in whole cells of the halotolerant alga Dunaliella salina can be elicited by intracellular acidification due to addition of weak acids at appropriate external pH. The changes in both intracellular pH and Na⁺ were followed. Following a mild intracellular acidification, intracellular Na⁺ content increased dramatically and then decreased. We interpret the phase of Na⁺ influx as due to the activation of the plasma membrane Na⁺/H⁺ antiporter and the phase of Na⁺ efflux as due to an active Na⁺ extrusion process. The following observations are in agreement with this interpretation: (a) the Na⁺ influx phase was sensitive to Li⁺, which is an inhibitor of the Na⁺/H⁺ antiporter, did not require energy, and was insensitive to vanadate; (b) the Na⁺ efflux phase is energy-dependent and sensitive to the plasma membrane ATPase inhibitor, vanadate. Following intracellular acidification, a drastic decrease in the intracellular ATP content is observed that is reversed when the cells regain their neutral pH value. We suggest that the intracellular acidification-induced change in the internal Na⁺ concentration is due to a combination of Na⁺ uptake via the Na⁺/H⁺ antiporter and an active, ATPase-dependent, Na⁺ extrusion. The Na⁺/H⁺ antiporter seems, therefore, to play a principal role in internal pH regulation in Dunaliella.     

Intracellular pH Regulation in an Acidophilic Unicellular Alga, Cyanidium caldarium: 31P-NMR Determination of Intracellular pH

The intracellular pH of an acidophilic unicellular alga, Cyanidiumcaldarium, was determined as a function of external pH by ³¹Pnuclear magnetic resonance. The algal cells incubated underaerobic conditions or under anaerobic and illuminated conditionsmaintained the intracellular pH in the range from 6.8 to 7.0even when the external pH was changed from 1.2 to 8.4. Underanaerobic and dark conditions, however, the intracellular pHacidified at the acidic pH region of the external medium. Theacidified intracellular pH reversibly returned to neutral eitheron aeration or illumination. The results indicate that, in Cyanidiumcells growing in extremely acidic environments, an active H⁺efflux (H⁺ pump) which depends on metabolic activity (respirationor photosynthesis) is essential to maintain the intracellularpH at a constant physiological level against the passive H⁺leakage due to the steep pH gradient across the cell membrane. (Received March 19, 1986; Accepted July 17, 1986)     

The Influence of H+ on the Membrane Potential and Ion Fluxes of Nitella

The resting membrane potential of the Nitella cell is relatively insensitive to [K]_o, but behaves like a hydrogen electrode. K⁺ and Cl^- effluxes from the cell were measured continuously, while the membrane potential was changed either by means of a negative feedback circuit or by external pH changes. The experiments indicate that P_K and P_Cl are independent of pH but are a function of membrane potential. Slope ion conductances, G_K, G_Cl, and G_Na were calculated from efflux measurements, and their sum was found to be negligible compared to membrane conductance. The possibility that a boundary potential change might be responsible for the membrane potential change was considered but was ruled out by the fact that the peak of the action potential remained at a constant level regardless of pH changes in the external solution. The conductance for H⁺ was estimated by measuring the membrane current change during an external pH change while the membrane potential was clamped at K⁺ equilibrium potential. In the range of external pH 5 to 6, H⁺ chord conductance was substantially equal to the membrane conductance. However, the [H]_i measured by various methods was not such as would be predicted from the [H]_o and the membrane potential using the Nernst equation. In artificial pond water containing DNP, the resting membrane potential decreased; this suggested that some energy-consuming mechanism maintains the membrane potential at the resting level. It is probable that there is a H⁺ extrusion mechanism in the Nitella cell, because the potential difference between the resting potential and the H⁺ equilibrium potential is always maintained notwithstanding a continuous H⁺ inward current which should result from the potential difference.     

Change in Intracellular pH Causes the Toxic Ca<Superscript>2+</Superscript> Entry via NCX1 in Neuron- and Glia-Derived Cells

Brain hypoxia or ischemia causes acidosis and the intracellular accumulation of Ca²⁺ in neuron. The aims of the present study were to elucidate the interaction between intracellular pH and Ca²⁺ during transient acidosis and its effects on the viability of neuronal and glial cells. Intracellular Ca²⁺ and pH were measured using the fluorescence of fura-2 and 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester in neuroblastoma (IMR-32), glioblastoma (T98G), and astrocytoma (CCF-STTG1) cell lines. The administration of 5 mM propionate caused intracellular acidification in IMR-32 and T98G cells but not in CCF-STTG1 cells. After the removal of propionate, the intracellular pH recovered to the resting level. The intracellular Ca²⁺ transiently increased upon the removal of propionate in IMR-32 and T98G cells but not in CCF-STTG1 cells. The transient Ca²⁺ increase caused by the withdrawal of intracellular acidification was abolished by the removal of external Ca²⁺, diminished by a reduction of external Na⁺, and inhibited by benzamil. Transient acidosis caused cell death, whereas the cells were more viable in the absence of external Ca²⁺. Benzamil alleviated cell death caused by transient acidosis in IMR-32 and T98G cells but not in CCF-STTG1 cells. These results suggest that recovery from intracellular acidosis causes a transient increase in cytosolic Ca²⁺ due to reversal of Ca²⁺ transport via Na⁺/Ca²⁺ exchanger coactivated with Na⁺/H⁺ exchanger, which can cause cell death.     

The Nature and Origin of Chemical Shift for Intracellular Water Nuclei in Artemia Cysts

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and ¹⁷O nuclei.

We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei (¹H, ²H, and ¹⁷O) is due to significant water-macromolecular interactions.