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.     

The intracellular pH of isolated,photosynthetically active Asparagus mesophyll cells

The intracellular pH of isolated, photosynthetically active mesophyll cells of Asparagus sprengeri Regel has been determined, in the light and dark, by the distribution of the weak acid 5,5-dimethyl-[2-¹⁴C]oxazolidine-2,4-dione ([¹⁴C]DMO) between the cells and the liquid medium. [¹⁴C]DMO was taken up rapidly, reaching equilibrium in 7–10 min of incubation, but was not metabolized by the cells, and intracellular binding of the compound was minimal. The intracellular pH, measured at saturating light fluence and 1.5 mM sodium bicarbonate, was found to remain relatively constant at 6.95–7.21 over the external pH range of 5.5–7.2. Illumination of the cells increased the intracellular pH compared to dark controls. The pH of the cytoplasm, excluding and including the chloroplasts (cytoplasmic and bulk cytoplasmic, respectively) was calculated from the experimentally derived intracellular [¹⁴C]DMO concentration and estimates of the vacuolar, chloroplastic and cytoplasmic volumes. The calculated cytoplasmic pH was similar in the light and dark, being 7.75 and 7.74, respectively, while the calculated pH of bulk cytoplasm was 7.85 in the light and 7.49 in the dark. Theoretical analysis indicated that intracellular pH is a good indicator of changes in the bulk cytoplasmic pH but insensitive to changes in vacuolar pH. The external pH optimum for photosynthesis (O₂ evolution) of isolated Asparagus cells was pH 7.2. At pH 8.0 photosynthesis was inhibited by 30% and at pH 5.25 by 45%. Inhibition at alkaline pH may be the result of a decrease in the pH gradient between the cells and the medium, causing CO₂ limitation in the cell. At acid pH, decrease in internal pH caused by substantial accumulation of inorganic carbon may account for the loss in photosynthetic activity.Abbreviations [¹⁴C]DMO 5,5-dimethyl[2-¹⁴C]oxazolidine-2,4-dione - pH_i overall intracellular pH - pH_e pH of external medium     

Transmembrane pH gradients and functional heterogeneity in reconstituted vesicle systems

The pH-sensitive, membrane impermeant fluorescence probes 8-hydroxy-1,3,6-pyrenetrisulfonate (pyranine; pK_a = 7.2) and 1-naphthol-3,6-disulfonate (Naps pK_a = 8.2) can be simultaneously entrapped within the intravesicular aqueous compartment of unilamellar vesicles and reconstituted proteoliposomes, where they function as reliable reporters of the intravesicular pH. Because the two probes are sensitive to pH over different but overlapping ranges, the useful monitoring range for the co-trapped probe pair extends from pH 6.5 to 9. In vesicles pre-equilibrated at a given pH and then subjected to a sudden change in external pH, the rate and extent of the subsequent change in internal pH are identical at all times during the re-equilibration, regardless of which probe is used to monitor the change. However, in reconstituted bacteriorhodopsin proteoliposomes, the size of the transmembrane pH gradient generated in the light always appears greater when pyranine is used to monitor internal pH. This discrepancy can most readily be understood in terms of heterogeneity in the vesicle suspension, with at least two populations of vesicles, one active in proton and one inactive. A simple algorithm was developed which generates, from the observed internal pH changes for two probes of different pK_a, the percentage of vesicles which are inactive, as well as the actual internal pH of the active fraction. The applicability of this algorithm was subsequently confirmed using a suspension of vesicles in which the level of heterogeneity was deliberately altered by the addition of various amounts of gramicidin. The apparent transmembrane pH gradient for the vesicle population as a whole decreased with increasing gramicidin, as did the calculated percentage of vesicles able to maintain a pH gradient, while the transmembrane gradient calculated for the active vesicle fraction only was essentially unaffected by gramicidin.     

pKa Values and Partition Coefficients of Nitroxide Spin Probes for Membrane Bioenergetics Measurements

Knowledge of pK_a's is necessary to calculate intracellular/intravesicular pH values from nitroxide accumulation in cells or vesicles as detected with electron spin resonance (ESR) spectroscopy. pK_a values were confirmed in lipid vesicles of known internal pH. To help select probes that do not accumulate in lipid membranes, octanol/buffer partition coefficients of uncharged nitroxides were determined. As an application of selected probes, pH gradients and internal aqueous volumes were analyzed in mitochondria (one internal compartment) and in the cyanobacterium Synechococcus 6311 (two internal compartments). The combination of 3-carboxy-, 3-amino- and 3-aminocarbonyl-2,2,5,5-tetramethylpyrrolidin-1-yloxyl was found to be most satisfactory for determinations of internal pH and volumes.     

Effects of increased intracellular pH-buffering capacity on the light response of Limulus ventral photoreceptor

Aspects of a possible involvement of hydrogen ions in the electrophysiological responses to light of Limulus ventral photoreceptors were investigated. A I M solution of either a zwitter-ionic pH buffer or a weakly-buffering control substance was pressure injected through a micropipette into a ventral photoreceptor cell. To estimate the amount injected, ³⁵SO₄ was included in the solution. Membrane currents induced by light flashes were measured by a voltage-clamp technique. The buffer-filled micropipette passed current and a 3 M KCl filled micropipette monitored membrane voltage. The sensitivity (peak light-induced current/stimulus energy) was measured, after dark adaptation, before and after injection. Injections of buffers, pH 6.3–7.2, to intracellular concentrations of at least 40–200 mM produced only a small mean decrease in sensitivity, approximately equal to that caused by injections of control substances. Excitation, therefore, apparently is not mediated by a change in intracellular pH. Buffers with pH values 5.4–8.4 were also injected. The time to peak of the response depended on pH, being shortened by up to 20% at pH values below 7.7 and lengthened at higher pH values. The time to peak of the response appeared to be shortened by an increase in intracellular pH-buffering capacity even when there was no change in intracellular pH.     

Errors in measurements of aquatic macrophyte gas exchange due to oxygen storage in internal airspaces

Dissolved oxygen measurements of respiratory and photosynthetic gas exchanges of the aquatic angiosperm Egeria densa Planch. and vesicles of the macroalga Carpophyllum maschalocarpum (Turn.) Grev. were made using a Clark-type oxygen electrode in a well-stirred closed chamber. Comparisons were made of dark—light and light—dark transients and light and dark oxygen exchanges for intact material and the same material with the internal airspaces flooded.For Egeria, there was an apparently greater gas exchange (up to 17% for photosynthesis and 50% for respiration) for infiltrated material. The duration of transient lags following light and dark treatments remained unchanged by infiltration, indicating that the storage error could not be detected from lag duration. The storage error during photosynthesis agreed closely with theoretical predictions based on oxygen solubility, but additional factors contribute to the dark error. Carpophyllum results were unpredictable from solubility calculations as the thick vesicle walls restricted exchange between the internal and external phases, allowing internal oxygen storage only at high external partial pressures.The widespread assumption that short lag phases indicate negligible internal oxygen storage in the gas spaces is questioned on the basis of these results. Solutions to the storage error are proposed.     

Maintenance of Intracellular pH and Acid Tolerance in Rhizobium meliloti

The development and function of the Rhizobium meliloti-Medicago sp. symbiosis are sensitive to soil acidity. Physiological criteria that can be measured in culture which serve to predict acid tolerance in soil would be valuable. The intracellular pH of R. meliloti was measured using either radioactively labeled weak acids (5,5-dimethyloxazolidine-2,4-dione and butyric acid) or pH-sensitive fluorescent compounds; both methods gave similar values. Six acid-tolerant strains (WSM419, WSM533, WSM539, WSM540, WSM852, and WSM870) maintained an alkaline intracellular pH when the external pH was between 5.6 and 7.2. In contrast, two Australian commercial inoculant strains (CC169 and U45) and four acid-sensitive strains from alkaline soils in Iraq (WSM244, WSM301, WSM365, and WSM367) maintained an alkaline intracellular pH when the external pH was ≥6.5, but had intracellular pH values of ≤6.8 when the external pH was ≤6.0. Four transposon Tn5-induced mutants of acid-tolerant strain WSM419, impaired in their ability to grow at pH 5.6, showed limited control over the intracellular pH. The ability to generate a large pH gradient under acid conditions may be a better indicator of acid tolerance in R. meliloti under field conditions than is growth on acidic agar plates.     

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.     

Characteristics of light-dependent inorganic carbon uptake by isolated spinach chloroplasts

The light-dependent accumulation of radioactively labeled inorganic carbon in isolated spinach (Spinacia oleracea L.) chloroplasts was determined by silicone oil filtering centrifugation. Intact chloroplasts, dark-incubated 60 seconds at pH 7.6 and 23°C with 0.5 millimolar sodium bicarbonate, contained 0.5 to 1.0 millimolar internal inorganic carbon. The stromal pool of inorganic carbon increased 5- to 7-fold after 2 to 3 minutes of light. The saturated internal bicarbonate concentration of illuminated spinach chloroplasts was 10- to 20-fold greater than that of the external medium. This ratio decreased at lower temperatures and with increasing external bicarbonate. Over one-half the inorganic carbon found in intact spinach chloroplasts after 2 minutes of light was retained during a subsequent 3-minute dark incubation at 5°C. Calculations of light-induced stromal alkalization based on the uptake of radioactively labeled bicarbonate were 0.4 to 0.5 pH units less than measurements performed with [¹⁴C]dimethyloxazolidine-dione. About one-third of the binding sites on the enzyme ribulose 1,5-bisphosphate carboxylase were radiolabeled when the enzyme was activated in situ and ¹⁴CO₂ bound to the activator site was trapped in the presence of carboxypentitol bisphosphates. Deleting orthophosphate from the incubation medium eliminated inorganic carbon accumulation in the stroma. Thus, bicarbonate ion distribution across the chloroplast envelope was not strictly pH dependent as predicted by the Henderson-Hasselbach formula. This finding is potentially explained by the presence of bound CO₂ in the chloroplast.     

Phosphorus metabolism and intracellular pH in the halotolerant alga Dunaliella parva studied by 31P-NMR

The metabolism of the green unicellular halotolerant alga Dunaliella parva was studied by means of ³¹P nuclear magnetic resonance spectroscopy. The major soluble phosphate compounds were found to be similar to those in other organisms but two phosphodiesters, glycerophosphorylglycerol and glycerophosphorylcholine, were identified in algal tissue for the first time. Only a single pool of intracellular orthophosphate was observed and the chemical shift of the corresponding resonance was used to monitor the intracellular pH. The cell pH and the orthophosphate content were sensitive both to the oxygenation of the cells and to the illumination of the cell suspension. The intracellular pH was controlled over an external pH range of 6–9, but at pH 5 the cell contents became acidic. Carbonyl cyanide p-trifluoromethoxyphenylhydrazone was observed to uncouple oxidative phosphorylation but it did not equilibrate the pH difference across the cell membrane in experiments conducted at an external pH of 7.8.     

Photosynthesis in Ulva fasciata: V. Evidence for an Inorganic Carbon Concentrating System, and Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase CO(2) Kinetics

Evidence of an inorganic carbon concentrating system in a marine macroalga is provided here. Based on an O₂ technique, supported by determinations of inorganic carbon concentrations, of experimental media (as well as compensation points) using infrared gas analysis, it was found that Ulva fasciata maintained intracellular inorganic carbon levels of 2.3 to 6.0 millimolar at bulk medium concentrations ranging from 0.02 to 1.5 millimolar. Bicarbonate seemed to be the preferred carbon form taken up at all inorganic carbon levels. It was found that ribulose-1,5-bisphosphate carboxylase/oxygenase from Ulva had a K_m(CO₂) of 70 micromolar and saturated at about 250 micromolar CO₂. Assuming a cytoplasmic pH of 7.2 (as measured for another Ulva species, P Lundberg et al. [1988] Plant Physiol 89: 1380-1387), and given the high activity of internal carbonic anhydrase (S Beer, A Israel [1990] Plant Cell Environ [in press]) and the here measured internal inorganic carbon level, it was concluded that internal CO₂ in Ulva could, at ambient external inorganic carbon concentrations, be maintained at a high enough level to saturate ribulose-1,5-bisphosphate carboxylase/oxygenase carboxylation. It is suggested that this suppresses photorespiration and optimizes net photosynthetic production in an alga representing a large group of marine plants faced with limiting external CO₂ concentrations in nature.     

Intracellular Conditions Required for Initiation of Solvent Production by Clostridium acetobutylicum

We investigated the intracellular physiological conditions associated with the induction of butanol-producing enzymes in Clostridium acetobutylicum. During the acidogenic phase of growth, the internal pH decreased in parallel with the decrease in the external pH, but the internal pH did not go below 5.5 throughout batch growth. Butanol was found to dissipate the proton motive force of fermenting C. acetobutylicum cells by decreasing the transmembrane pH gradient, whereas the membrane potential was affected only slightly. In growing cells, the switch from acid to solvent production occurred when the internal undissociated butyric acid concentration reached 13 mM and the total intracellular undissociated acid concentration (acetic plus butyric acids) was at least 40 to 45 mM. Similar values were obtained when cultures were supplemented with 50 mM butyric acid initially or when a phosphate-buffered medium was used instead of an acetate-buffered medium. To measure the induction of the enzymes involved in solvent synthesis, we determined the rates of conversion of butyrate to butanol in growing cells. The rate of butanol formation reached a maximum in the mid-solvent phase, when the butanol concentration was 50 mM. Although more solvent accumulated later, de novo enzyme synthesis decreased and then ceased.     

Properties of the Isolated Intact Chloroplast at Cytoplasmic K Concentrations : I. Light-Induced Cation Uptake into Intact Chloroplasts is Driven by an Electrical Potential Difference

Photosynthesis, stroma-pH, and internal K⁺ and Cl⁻ concentrations of isolated intact chloroplasts from Spinacia oleracea, as well as ion (K⁺, H⁺, Cl⁻) movements across the envelope, were measured over a wide range of external KCl concentrations (1-100 millimolar).

Isolated intact chloroplasts are a Donnan system which accumulates cations (K⁺ or added Tetraphenylphosphonium⁺) and excludes anions (Cl⁻) at low ionic strength of the medium. The internally negative dark potential becomes still more negative in the light as estimated by Tetraphenylphosphonium⁺ distribution. At 100 millimolar external KCl, potentials both in the light and in the dark and also the light-induced uptake of K⁺ or Na⁺ and the release of protons all become very small. Light-induced K⁺ uptake is not abolished by valinomycin suggesting that the K⁺ uptake is not primarily active. Intact chloroplasts contain higher K⁺ concentrations (112-157 millimolar) than chloroplasts isolated in standard media. Photosynthetic activity of intact chloroplasts is higher at 100 millimolar external KCl than at 5 to 25 millimolar. The pH optimum of CO₂ fixation at high K⁺ concentrations is broadened towards low pH values. This can be correlated with the observation that high external KCl concentrations at a constant pH of the suspending medium produce an increase of stroma-pH both in the light and in the dark. These results demonstrate a requirement of high external concentrations of monovalent cations for CO₂ fixation in intact chloroplasts.

     

Phosphorus-31 and Nitrogen- 14 NMR Studies of the Uptake of Phosphorus and Nitrogen Compounds in the Marine Macroalgae Ulva lactuca

Cytoplasmic phosphomonoesters and inorganic phosphate, as well as vacuolar inorganic phosphate and polyphosphates, gave rise to the major peaks in ³¹P nuclear magnetic resonance (NMR) spectra of the marine macroalgae Enteromorpha sp., Ceramium sp., and Ulva lactuca which were collected from the sea. In contrast, NMR-visible polyphosphates were lacking in Pylaiella sp. and intracellular vacuolar phosphate seemed to act as the main phosphorus store in this organism. In laboratory experiments, polyphosphates decreased in growing U. lactuca which was cultivated in continuous light under phosphate-deficient conditions. In contrast, the same organism cultivated in seawater with added phosphate and ammonium, accumulated phosphate mainly in the form of polyphosphates. When nitrate was provided as the only nitrogen source, accumulation of polyphosphates in the algae decreased with increasing external nitrate concentration. From the chemical shift of the cytoplasmic Pi peak, the cytoplasmic pH of superfused preparations of Ulva was estimated at 7.2. The vacuolar pH, determined from the chemical shifts of the vacuolar Pi and the terminal polyphosphate peaks, was between 5.5 and 6.0. The intracellular nitrate and ammonium levels in U. lactuca were determined by ¹⁴N NMR. Both nitrogen sources were taken up and stored intracellularly; however, the uptake of ammonium was much faster than that of nitrate.     

Regulation of endogenous and heterologous Ca release-activated Ca currents by pH

Deviations from physiological pH (∼pH 7.2) as well as altered Ca²⁺ signaling play important roles in immune disease and cancer. One of the most ubiquitous pathways for cellular Ca²⁺ influx is the store-operated Ca²⁺ entry (SOCE) or Ca²⁺ release-activated Ca²⁺ current (I_CRAC), which is activated upon depletion of intracellular Ca²⁺ stores. We here show that extracellular and intracellular changes in pH regulate both endogenous I_CRAC in Jurkat T lymphocytes and RBL2H3 cells, and heterologous I_CRAC in HEK293 cells expressing the molecular components STIM1/2 and Orai1/2/3 (CRACM1/2/3). We find that external acidification suppresses, and alkalization facilitates IP₃-induced I_CRAC. In the absence of IP₃, external alkalization did not elicit endogenous I_CRAC but was able to activate heterologous I_CRAC in HEK293 cells expressing Orai1/2/3 and STIM1 or STIM2. Similarly, internal acidification reduced IP₃-induced activation of endogenous and heterologous I_CRAC, while alkalization accelerated its activation kinetics without affecting overall current amplitudes. Mutation of two aspartate residues to uncharged alanine amino acids (D110/112A) in the first extracellular loop of Orai1 significantly attenuated both the inhibition of I_CRAC by external acidic pH as well as its facilitation by alkaline conditions. We conclude that intra- and extracellular pH differentially regulates I_CRAC. While intracellular pH might affect aggregation and/or binding of STIM to Orai, external pH seems to modulate I_CRAC through its channel pore, which in Orai1 is partially mediated by residues D110 and D112.     

Composition and Toxicity of the Inclusion of Bacillus thuringiensis subsp. israelensis

The multisegmented ovoidal inclusion of Bacillus thuringiensis subsp. israelensis was found to be composed of two structurally and biochemically distinct components. Electron microscopy of the inclusion revealed it to be composed mainly of osmiophobic or lightly stained segments crystallized in a lattice showing a repeat of approximately 4.3 nm. These light segments of the inclusions were shared by osmiophylic darkly stained segments with a crystal lattice repeat of approximately 7.8 nm. The lightly stained segments were soluble at pH 9.2 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The extracts of lightly stained segments were lytic to mammalian erythrocytes, and the precipitate obtained by lowering the pH to 5.2 was toxic to the larvae of Aedes egypti. The dark inclusion segments remaining, besides being much less toxic to larvae, were nonlytic to erythrocytes and were soluble at pH 10.5 in sodium dodecyl sulfate-dithiothreitol-Tris-hydrochloride. The light segment was composed of two major polypeptide doublets with molecular weights of 145,000 and 135,000, and 27,000 and 26,500, and the dark segments were composed of a single major polypeptide with a molecular-weight of 70,000. Hence, the inclusion of B. thuringiensis subsp. israelensis is more complex than previously reported, and we conclude that the toxin may be the polypeptide with a molecular weight of 27,000 and 26,500.     

Fluorimetric pH measurement in whole cells of dark aerobic and anaerobic cyanobacteria

9-Aminoacridine and atebrin fluorescence quenching by dark aerobic and anaerobic suspensions of Anacystis nidulans, Plectonema boryanum and Gloeobacter violaceus was determined at external pH 7–9. Individual pH values in cytosol and thylakoid compartments were calculated from the simultaneously different intracellular enrichment factors of the monovalent and the divalent base. Concomitantly, at external pH 4–7 the cytosolic pH was measured with fluorescein diacetate which is taken up and decomposed in the cell by cytosolic hydrolases. The pH-dependent fluorescence of the free fluorescein, which remains trapped in the cell, monitors the cytosolic pH. The fact that the latter was higher in aerobic than in anaerobic cells, and insensitive to saturating concentrations of dicyclohexylcarbodiimide aerobically, was taken to support the concept of a proton-translocating respiratory chain in the plasma membrane.     

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     

The effects of extracellular-and intracellular-pH on transmitter mobilization and fractional release in Helix pomatia

• 1.1. Transmitter mobilization and fractional release were studied in Helix pomatia. The right palliai nerve was stimulated and a synaptic potential was recorded in cell F76 in the right parietal ganglion.
• 2.2. The extra- and intra-cellular pH were changed with Tris-maleate, CO₂ or (NH₄)₂SO₄.
• 3.3. The time constant for the monoexponential part of mobilization decreased with reduced intracellular pH. Only a fraction of this effect could be related to an increase in the intracellular Ca-activity.
• 4.4. Fractional release was reduced in low external pH, but was increased in low intracellular pH. Fractional release is affected more by changes in internal pH than external pH.

     

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.