Continuous-flow NMR culture system for mammalian cells

A continuous-flow NMR culture system for mammalian cells has been developed on which ³¹P-NMR experiments under complete and strictly physiologic conditions have been performed. Observations on the response of the cellular metabolism to stresses such as starvation, low temperature and changes in environmental pH monitored by ³¹P-NMR are reported. The response of the intracellular pH relative to the external pH of the growth medium is studied. We find that under the experimental conditions used there exists a ΔpH varying between less than 0.2 and more than 0.6 pH units. These results are compatible with those obtained using other techniques.     

Amine Accumulation in Acidic Vacuoles Protects the Halotolerant Alga Dunaliella salina Against Alkaline Stress

Amines at alkaline pH induce in cells of the halotolerant alga Dunaliella a transient stress that is manifested by a drop in ATP and an increase of cytoplasmic pH. As much as 300 millimolar NH₄⁺ are taken up by the cells at pH 9. The uptake is not associated with gross changes in volume and is accompanied by K⁺ efflux. Most of the amine is not metabolized, and can be released by external acidification. Recovery of the cells from the amine-induced stress occurs within 30 to 60 minutes and is accompanied by massive swelling of vacuoles and by release of the fluorescent dye atebrin from these vacuoles, suggesting that amines are compartmentalized into acidic vacuoles. The time course of ammonia uptake into Dunaliella cells is biphasic—a rapid influx, associated with cytoplasmic alkalinization, followed by a temperature-dependent slow uptake phase, which is correlated with recovery of cellular ATP and cytoplasmic pH. The dependence of amine uptake on external pH indicates that it diffuses into the cells in the free amine form. Studies with lysed cell preparations, in which vacuoles become exposed but retain their capacity to accumulate amines, indicate that the permeability of the vacuolar membrane to amines is much higher than that of the plasma membrane. The results can be retionalized by assuming that the initial amine accumulation, which leads to rapid vacuolar alkalinization, activates metabolic reactions that further increase the capacity of the vacuoles to sequester most of the amine from the cytoplasm. The results indicate that acidic vacuoles in Dunaliella serve as a high-capacity buffering system for amines, and as a safeguard against cytoplasmic alkalinization and uncoupling of photosynthesis.     

Charge and acidity compensation during proton-sugar symport in Chlorella: The H+-ATPase does not fully compensate for the sugar-coupled proton influx

This study was undertaken in order to demonstrate the extent to which the activity of the plasmalemma H⁺-ATPase compensates for the charge and acidity flow caused by the sugar-proton symport in cells of chlorella vulgaris Beij.. Detailed analysis of H⁺ and K⁺ fluxes from and into the medium together with measurements of respiration, cytoplasmic pH, and cellular ATP-levels indicate three consecutive phases after the onset of H⁺ symport. Phase 1 occurred immediately after addition of sugar, with an uptake of H⁺ by the hexoseproton symport and charge compensation by K⁺ loss from the cells and, to a smaller degree, by loss of another ion, probably a divalent cation. This phase coincided with strong membrane depolarization. Phase 2 started approximately 5 s after addition of sugar, when the acceleration of the H⁺-ATPase caused a slow-down of the K⁺ efflux, a decrease in the cellular ATP level and an increase in respiration. The increased respiration was most probably responsible for a pronounced net acidification of the medium. This phase was inhibited in deuterium oxide. In phase 3, finally, a slow rate of net H⁺ uptake and K⁺ loss was established for several further minutes, together with a slight depolarization of the membrane. There was hardly any pH change in the cytoplasm, because the cytoplasmic buffering capacity was high enough to stabilize the pH for several minutes despite the net H⁺ fluxes. The quantitative participation of the several phases of H⁺ and K⁺ flow depended on the pH of the medium, the ambient Ca²⁺ concentration, and the metabolic fate of the transported sugar. The results indicate that the activity of the H⁺-ATPase never fully compensated for H⁺ uptake by the sugar-symport system, because at least 10% of symport-caused charge inflow was compensated for by K⁺ efflux. The restoration of pH in the cytoplasm and in the medium was probably achieved by metabolic reactions connected to increased glycolysis and respiration.Abbreviations DMO dimethyloxazolidinedione - EDTA ethylcnediaminetetraacetic acid - p.c. packed cell volume     

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.     

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported¹. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases². Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells³. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis^3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.     

The composition of pericarp,cell aging,and changes in water absorption in two tomato genotypes: mechanism,factors, and potential role in fruit cracking

Cracking, a serious problem in many fruits, may cause significant economic losses. It may occur when internal pressure cannot sustain by the epidermis any longer. Water absorption and epidermis are among the most important factors that associated with cracking. To determine whether pericarp composition and its mechanical performance, endogenous cell wall disassembly, and water-absorbing capacity influences tomato fruit cracking, we grew a cracking-resistant genotype ‘LA1698’ and susceptible genotype ‘LA2683’. The results illustrated that the cuticle and subcutaneous layer were thicker in ‘LA1698’ than in ‘LA2683’. Compared with ‘LA2683’, the fruit firmness, consistency, and bursting strength of ‘LA1698’ were all higher. Fruits of ‘LA1698’ had decreased activities of polygalacturonase, β-galactosidase, and cellulose, which can disassemble the polysaccharide network. As a result, it had reduced water-soluble pectin and more covalently and ionically bound pectin that can crosslink with Ca²⁺ and B. These fruits also have a greater abundance of hemicelluloses. In addition, ‘LA1698’ had higher SOD activities and lower relative conductivity, meaning its cells might have a better biological activity to resist changes of the external environment (such as water variation) and to prevent fruit cracking. However, POD in ‘LA2683’ was more abundant than in ‘LA1698’. ‘LA1698’ produced juice with lower total soluble solids, which led to a lower initial water-absorbing ability and difference between the exocarp and mesocarp. In conclusion, a stronger pericarp and cells with a better biological activity in addition to the lower water-absorbing difference between the exocarp and mesocarp made ‘LA1698’ more resistant to cracking.     

Alterations of Intracellular pH in Response to Low Temperature Stresses

+ -ATPase is one of the primary cellular events directly resulting from cold exposure. We demonstrate here that cold-induced inactivation of the proton translocating enzyme is closely linked to the rapid acidification of the cytoplasm and the concomitant alkalization of the vacuoles, suggesting an important role of the enzyme in maintaining homeostasis of the cellular pH in a cold environment. The stability of the vacuolar H⁺-ATPase to cold both in vivo and in vitro is distinctly different between species sensitive and insensitive to cold. These findings provide further insight into the way in which the vacuolar H⁺-ATPase is involved in cold adaptation of plants. In addition, the temperature reduction and the concentration of the cytoplasm as a consequence of freeze-induced dehydration may also result in changes in the cellular pH. In fact, we demonstrate here that the cytoplasm is markedly acidified upon freezing; in particular, in cells of less hardy plants. Freeze-induced acidification is presumably due to changes in the physico-chemical properties of the cytoplasm and the changes in the permeability of the vacuolar membrane both of which result from severe dehydration. The physiological significance of freeze-induced acidification of the cytoplasm is discussed. Received 26 March 1999/ Accepted in revised form 30 March 1999     

The distribution of calmodulin in living mitotic cells

Calmodulin has been labeled with rhodamine isothiocyanate (CaM-RITC) and used as a probe for the location of calmodulin in vivo. CaM-RITC retains its capacity to regulate the activity of brain phosphodiesterase in a Ca²⁺-dependent manner in vitro, indicating that the labeled protein is still active. After injection into living mammalian cells CaM-RITC incorporates rapidly into the mitotic spindle; the details of its localization there mimic closely the distribution of Calmodulin seen by immunofluorescence. In interphase cells the CaM-RITC is excluded from the nucleus, but shows no region of specific concentration within the cytoplasm. Neither a 2-fold increase in cellular CaM nor the injection of anti CaM has any observable effect on the progress of mitosis.     

Ion and water shifts produced by ammonium chloride in high K and low K red cells

The effect of ammonium chloride on the cellular Na⁺, K⁺ and water has been examined in human and horse (high K), cow (medium K) and cat (low K) red cells. It was found that high K red cells, especially those of the horse, gained water an Na⁺, whereas the net movement of K⁺ was negligible. There was a correlation between the increase of cellular Na⁺ concentration and of the packed red cell volume. In contrast, the packed cell volume of low K red cells increased slightly or not at all, and Na⁺ ions leaked out from the cells. The high K cells had a lower Cl^? concentration and higher buffer capacity than the low K cells. The results obtained with the medium K (cow) cells usually lay between those of the other two cell types. In all the cases both the plasma and cell pH decreased resulting from the addition of ammonium chloride. The mechanism of movements of water and Na⁺ ions in high K cells remained unsolved, but the response of low K cells to ammonium chloride was near that of a cation exchange resin.     

Polyphosphate Hydrolysis within Acidic Vacuoles in Response to Amine-Induced Alkaline Stress in the Halotolerant Alga Dunaliella salina

The location and mobilization of polyphosphates in response to an amine-induced alkaline stress were studied in the halotolerant alga Dunaliella salina. The following observations suggest that polyphosphates accumulate in acidic vacuoles: (a) Accumulation of large amounts of polyphosphates is manifested as intravacuolar dense osmiophilic bodies in electron micrographs. (b) Uptake of amines into the vacuoles induces massive hydrolysis of polyphosphates, demonstrated by in vivo ³¹P-nuclear magnetic resonance, and by analysis of hydrolytic products on thin layer chromatograms. The analysis indicates that: (a) Polyphosphate hydrolysis is kinetically correlated with amine accumulation and with the recovery of cytoplasmic pH. (b) The major hydrolytic product is tripolyphosphate. (c) The peak position of the tripolyphosphate terminal phosphate in nuclear magnetic resonance spectra is progressively shifted as the cells recover, indicating that the pH inside the vacuoles increases while the pH in the cytoplasm decreases. (d) In lysed cell preparations, in which vacuoles become exposed to the external pH, mild alkalinization in the absence of amines induces polyphosphate hydrolysis to tripolyphosphates. It is suggested that amine accumulation within vacuoles activates a specific phosphatase, which hydrolyzes long-chain polyphosphates to tripolyphosphates. The hydrolysis increases the capacity of the vacuoles to sequester amines from the cytoplasm probably by releasing protons required to buffer the amine, and leads to recovery of cytoplasmic pH. Thus, polyphosphate hydrolysis provides a high-capacity buffering system that sustains amine compartmentation into vacuoles and protects cytoplasmic pH.     

Cyto B dependent and ouabain insensitive Regulatory Volume Decrease in bicellular mouse embryo

Mouse single-cell embryos exhibit robust Regulatory Volume Decrease (RVD). In what manner the very early mammalian embryo following zygote stage is appreciably altered by the anisotonic extracellular solution is, as yet, totally unclear. Little attention was paid to this direction since there was no way to determine the blastomere volume. This work has served to quantitatively investigate the osmotic response of bicellular mouse embryos employing Laser Scanning Microtomography (LSM) followed with three-dimensional reconstruction (3 DR). We have shown that bicellular mouse embryos in hypotonic Dulbecco’s experience RVD. Embryonic cells subjected to hyposmolar exhibit rapid osmotic swelling followed by gradual shrinking back toward their original volume. The van’ t Hoff law defines swelling phase with the effective hydraulic conductivity of 0.3 micron · min⁻¹ · atm⁻¹. Water release during RVD in bicellular mouse embryos is abolished by Cytochalasin B (Cyto B) and the volume recovery is insensitive to ouabain treatment.     

Volume regulation in osmotically-stressed coelomic erythrocytes from Priapulus caudatus Lamarck (phylum Priapuloidea)

Volume regulation in the haemerythrin-rich coelomic erythrocytes from the benthic burrower Priapulus caudatus Lamarck (phylum Priapuloidea) in response to increased and decreased plasma osmolarity was investigated.In contrast to previously-investigated erythrocytes, Priapulus erythrocytes exhibited only slight capacity for volume control. Measurement of ninhydrin positive substances indicates that an efflux of cellular amino acids may be implicated in the slight volume regulatory decrease observed under strongly hypo-osmotic stress, bin no evidence was found for changes in amino acid-protein equilibrium involving the haemerythrin.The findings are described in relation to their possible adaptive and phylogenetic significance and the alleged ubiquity of pronounced volume regulatory capabilities in animal cells.     

Cellular acidosis inhibits assembly, disassembly, and motility of stress granules

Stress granules (SGs) are large ribonucleoprotein (RNP)-containing particles that form in cytoplasm in response to a variety of acute changes in the cellular environment. One of the general parameters of the cell environment is pH. In some diseases, as well as in muscle fatigue, tissue acidosis occurs, leading to decrease in intracellular pH. Here we studied whether decrease in pH causes the formation of SGs in cultured animal cells, whether it affects the formation of the SGs under the action of arsenite and, if such effects occur, what are the mechanisms of the influence of acidosis. Acidosis was simulated by decreasing the pH of the culture medium, which acidified the cytoplasm. We found that medium acidification to pH 6.0 in itself did not cause formation of SGs in cells. Moreover, acidification prevented the formation of SGs under treatment with sodium arsenite or sodium arsenite together with the proteasome inhibitor MG132, and it inhibited the dissociation of preformed SGs under the influence of cycloheximide. We established that pH decrease did not affect the phosphorylation of eIF2α that occurs under the action of sodium arsenite, and even caused such phosphorylation by itself. We also found that the velocity of SG motion in cytoplasm at acidic pH was very low, and the mobile fraction of SG-incorporated PABP protein revealed by FRAP was decreased. We suppose that acidic pH impairs biochemical processes favoring assembly of RNPs in stress conditions and RNP dissociation on the termination of stress. Thus, in acidosis the reaction of the cellular translation apparatus to stress is modified.     

Acid-base regulation during ammonium assimilation in Hydrodictyon africanum

Abstract The acid-base balance during ammonium (used to mean NH ₄⁺ and/or NH₃) assimilation in Hydrodictyon africanum has been measured on cells growing with about 1 mol m^?3 ammonium at an external pH of about 6.5. Measurements made included (1) ash alkalinity (corrected for intracellular ammonium) which yields net organic negative charge, (2) the accumulation of organic N in the cells and (3) the change in extracellular H⁺ (from the pH change and the buffer capacity). These measurements showed that some 0.25 excess organic negative charge (half in the cell wall, half inside the plasmalemma) accumulates per organic N synthesized, while some 1.25H⁺ accumulate in the medium per organic N synthesized. Granted a permeability (PNH₃) of some 10^?3 cm s^?1, and a finite [NH₃] in the cytoplasm of these N-assimilating cells it is likely that most of the ammonium entering these growing cells is as NH ₄⁺. This means that most of the H ⁺ appearing in the medium must have originated from inside the cell and have been subjected to active efflux at the plasmalemma: H⁺ accumulates in the medium equivalent to any NH₃ entry by requilibration from exogenous NH ₄⁺. The cell composition (net organic negative charge, organic N content) is very similar in these ammonium-grown cells to that of NO₃⁺grown cells, suggesting that there is no action of a ‘biochemical pH stat’ during longterm assimilation of NO₃⁺in H. africanum. Short-term experiments were carried out at an external pH of 7.2 in which ammonium at various concentrations were supplied to NO₃⁺-grown cells. There was in all cases a rapid influx followed by a slower uptake; at least at the lower concentrations (less than 100 μmol dm^?3) the net influx was all attributable to NH₄⁺influx via a uniporter, probably partly short-circuited by a passive NH₃ efflux due to intrinsic membrane permeability to NH₃. The net ammonium influx was in all cases associated with H⁺ accumulation in the medium. (1.3-1.7 H ⁺ per ammonium taken up); as in the growth experiments, most of the ammonium taken up was assimilated. Determinations of cytoplasmic pH showed either no effect on, or a slight decrease in, pH during ammonium assimilation; the changes that occurred were in the direction expected for actuating a ‘pH-regulating’ change in H⁺ fluxes.     

Intracellular pH modulates quinary structure

NMR spectroscopy can provide information about proteins in living cells. pH is an important characteristic of the intracellular environment because it modulates key protein properties such as net charge and stability. Here, we show that pH modulates quinary interactions, the weak, ubiquitous interactions between proteins and other cellular macromolecules. We use the K10H variant of the B domain of protein G (GB1, 6.2 kDa) as a pH reporter in Escherichia coli cells. By controlling the intracellular pH, we show that quinary interactions influence the quality of in‐cell ¹⁵N–¹H HSQC NMR spectra. At low pH, the quality is degraded because the increase in attractive interactions between E. coli proteins and GB1 slows GB1 tumbling and broadens its crosspeaks. The results demonstrate the importance of quinary interactions for furthering our understanding of protein chemistry in living cells.     

"Nanosized voltmeter" enables cellular-wide electric field mapping

Previously, all biological measurements of intracellular electric fields (E fields), using voltage dyes or patch/voltage clamps, were confined to cellular membranes, which account for <0.1% of the total cellular volume. These membrane-dependent techniques also frequently require lengthy calibration steps for each cell or cell type measured. A new 30-nm "photonic voltmeter", 1000-fold smaller than existing voltmeters, enables, to our knowledge, the first complete three-dimensional E field profiling throughout the entire volume of living cells. These nanodevices are calibrated externally and then applied for E field determinations inside any live cell or cellular compartment, with no further calibration steps. The results indicate that the E fields from the mitochondrial membranes penetrate much deeper into the cytosol than previously estimated, indicating that, electrically, the cytoplasm cannot be described as a simple homogeneous solution, as often approximated, but should rather be thought of as a complex, heterogeneous hydrogel, with distinct microdomains.     

Cation exchange in isolated xylem cell walls of tomato. I. Cd2+ and Rb+ exchange in adsorption experiments

Abstract Purified xylem cell walls were prepared from isolated xylem bundles of tomato (an inbred line of Lycopersicon esculentum Mill, cv. Tiny Tim). Adsorption and exchange experiments were carried out with ¹¹⁵Cd²⁺, ⁸²Rb⁺ and ⁸²Br^?. The application of γ-ray spectroscopy permitted the simultaneous measurement of several ions applied together. The cell-wall water volume was shown to be independent of the external pH and solution ionic strength, possibly due to the presence of lignin. The Donnan Free Space (DFS) volume could be determined as a constant 0.15 dm³ per kg cell-wall dry weight. Consequently, the total cell-wall cation exchange capacity (CEC) could be estimated based on the DFS volume, and amounted to approximately 1000 mol m^?3 negative charges. The results of Cd²⁺ -Rb⁺ exchange experiments indicated an apparent CEC value of about 350–450 mol m^?3 DFS, at external pH ～ 4. These data are in agreement with earlier reports on xylem wall CEC, and indicate the weak acid characteristics of the charge groups. The rational selectivity coefficient R^Cd_Rb, of the cell wall was shown to depend on external ion fractions and ionic strength, with a maximum R^Cd_Rb of 450 at ionic Cd²⁺ fraction near 0.3, based at the smallest experimental ionic strength of the external solution. The adsorption of Cd²⁺, applied at relatively high concentrations, was shown to be stimulated by simultaneous application of high Rb⁺ concentrations.     

Proton cellular influx as a probable mechanism of variation potential influence on photosynthesis in pea

Electrical signals (action potential and variation potential, VP) caused by environmental stimuli are known to induce various physiological responses in plants, including changes in photosynthesis; however, their functional mechanisms remain unclear. In this study, the influence of VP on photosynthesis in pea (Pisum sativum L.) was investigated and the proton participation in this process analysed. VP, induced by local heating, inactivated photosynthesis and activated respiration, with the initiation of the photosynthetic response connected with inactivation of the photosynthetic dark stage; however, direct VP influence on the light stage was also probable. VP generation was accompanied with pH increases in apoplasts (0.17–0.30 pH unit) and decreases in cytoplasm (0.18–0.60 pH unit), which probably reflected H⁺‐ATPase inactivation and H⁺ influx during this electrical event. Imitation of H⁺ influx using the protonophore carbonyl cyanide m‐chlorophenylhydrazone (CCCP) induced a photosynthetic response that was similar with a VP‐induced response. Experiments on chloroplast suspensions showed that decreased external pH also induced an analogous response and that its magnitude depended on the magnitude of pH change. Thus, the present results showed that proton cellular influx was the probable mechanism of VP's influence on photosynthesis in pea. Potential means of action for this influence are discussed.     

Ionic and osmotic equilibria of human red blood cells treated with nystatin

Human red blood cells have been incubated in the presence of nystatin, which allows Na and K, as well as Cl and pH to equilibrate rapidly when cell volume is set with external impermeant sucrose. The intracellular mean ionic activity coefficients, relative to values in the extracellular solution, for KCl and NaCl are 1.01 +/- 0.02 and 0.99 +/- 0.02 (SD, n = 10), respectively, and are independent of external pH, pH o, and of [sucrose]o. With nystatin the dependence of red cell volume on [sucrose]o deviates from ideal osmotic behavior by as much as a factor of three. A virial equation for the osmotic coefficient, phi, of human hemoglobin, Hb, accounts for the cell volumes, and is the same as that which describes Adair's measurements of phi Hb for Hb isolated from sheep and ox bloods. In the presence of nystatin the slope of the acid-base titration curve of the cells is independent of cell volume, implying that the charge on impermeant cellular solutes is independent of Hb concentration at constant pH. By modifying the Jacobs-stewart equations (1947. J. Cell. Comp. Physiol. 30: 79--103) with the osmotic coefficients of Hb and of salts, a nonideal thermodynamic model has been devised which predicts equilibrium Donnan ratios and red cell volume from the composition of the extracellular solution and from certain parameters of the cells. In addition to accounting for the dependence of cell volume on osmotic pressure, the model also describes accurately the dependence of Donnan ratios and cell volumes on pHo either in the presence or absence of nystatin.     

ζ-Potential and surface charge of Thermoplasma acidophila

The surface charge density and the ζ-potential of Thermoplasma acidophila was estimated from microscopic electrophoresis experiments. The cells moved towards the positive electrode. The mobility remained constant from pH 2 to 5, and increased for pH values higher than 6. The mobility at pH 6 decreased dramatically with increased external Ca²⁺ concentration. At pH 2 and an ionic strength similar to that of the growth medium, the ζ-potential was about 8 mV, negative relative to the bulk medium; the surface charge density was 1360esu/cm^-2 which corresponds to one elementary charge per 3500 A².